Expression Of Endoplasmic Reticulum Stress Markers Research Articles

The chemical chaperone 4-phenylbutyrate inhibits adipogenesis by modulating the unfolded protein response

Recent studies have shown a link between obesity and endoplasmic reticulum (ER) stress. Perturbations in ER homeostasis cause ER stress and activation of the unfolded protein response (UPR). Adipocyte differentiation contributes to weight gain, and we have shown that markers of ER stress/UPR activation, including GRP78, phospho-eIF2, and spliced XBP1, are upregulated during adipogenesis. Given these findings, the objective of this study was to determine whether attenuation of UPR activation by the chemical chaperone 4-phenylbutyrate (4-PBA) inhibits adipogenesis. Exposure of 3T3-L1 preadipocytes to 4-PBA in the presence of differentiation media decreased expression of ER stress markers. Concomitant with the suppression of UPR activation, 4-PBA resulted in attenuation of adipogenesis as measured by lipid accumulation and adiponectin secretion. Consistent with these in vitro findings, female C57BL/6 mice fed a high-fat diet supplemented with 4-PBA showed a significant reduction in weight gain and had reduced fat pad mass, as compared with the high-fat diet alone group. Furthermore, 4-PBA supplementation decreased GRP78 expression in the adipose tissue and lowered plasma triglyceride, glucose, leptin, and adiponectin levels without altering food intake. Taken together, these results suggest that UPR activation contributes to adipogenesis and that blocking its activation with 4-PBA prevents adipocyte differentiation and weight gain in mice.

Inhibition of endoplasm reticulum stress by ghrelin protects against ischemia/reperfusion injury in rat heart

Ghrelin is a multi-functional polypeptide with cardiovascular protective effects. We aimed to explore whether the cardioprotective effect of ghrelin is mediated by inhibiting myocardial endoplasmic reticulum stress (ERS). A Langendorff model of isolated rat heart was used with ischemia/reperfusion (I/R; 40/120 min). Cardiac function was monitored, and histomorphologic features, degree of myocardial injury, level of ERS markers, and number of apoptotic cardiomyocytes were determined. Compared with control group, the I/R group showed significantly decreased cardiac function, seriously damaged myocardial tissue, increased number of apoptotic cells, and overexpression of mRNA and protein of ERS markers. However, preadministration of ghrelin in vivo (10 −8 mol/kg, intraperitoneal injection, every 12 h, twice in all) greatly ameliorated the damaged heart function, attenuated myocardial injury and apoptosis, and decreased the expression of ERS markers: it decreased the mRNA and protein levels of glucose-regulated protein78 (GRP78) and C/EBP homologous protein (CHOP), with reduced caspase-12 protein expression. Furthermore, in vitro, ghrelin directly inhibited the myocardial ERS response induced by tunicamycin or dithiothreitol in rat cardiac tissue. Ghrelin could protect the heart against I/R injury, at least in part, through inhibiting myocardial ERS.

Amyloid formation in human IAPP transgenic mouse islets and pancreas, and human pancreas, is not associated with endoplasmic reticulum stress.

Supraphysiological levels of the amyloidogenic peptide human islet amyloid polypeptide have been associated with beta cell endoplasmic reticulum (ER) stress. However, in human type 2 diabetes, levels of human IAPP are equivalent or decreased relative to matched controls. Thus, we sought to investigate whether ER stress is induced during amyloidogenesis at physiological levels of human IAPP. Islets from human IAPP transgenic mice that develop amyloid, and non-transgenic mice that do not, were cultured for up to 7 days in 11.1, 16.7 and 33.3 mmol/l glucose. Pancreases from human IAPP transgenic and non-transgenic mice and humans with or without type 2 diabetes were also evaluated. Amyloid formation was determined histologically. ER stress was determined in islets by quantifying mRNA levels of Bip, Atf4 and Chop (also known as Ddit3) and alternate splicing of Xbp1 mRNA, or in pancreases by immunostaining for immunoglobulin heavy chain-binding protein (BIP), C/EBP homologous protein (CHOP) and X-box binding protein 1 (XBP1). Amyloid formation in human IAPP transgenic islets was associated with reduced beta cell area in a glucose- and time-dependent manner. However, amyloid formation was not associated with significant increases in expression of ER stress markers under any culture condition. Thapsigargin treatment, a positive control, did result in significant ER stress. Amyloid formation in vivo in pancreas samples from human IAPP transgenic mice or humans was not associated with upregulation of ER stress markers. Our data suggest that ER stress is not an obligatory pathway mediating the toxic effects of amyloid formation at physiological levels of human IAPP.

Autocrine regulation of mda -7/IL-24 mediates cancer-specific apoptosis

A noteworthy aspect of melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) as a cancer therapeutic is its ability to selectively kill cancer cells without harming normal cells. Intracellular MDA-7/IL-24 protein, generated from an adenovirus expressing mda-7/IL-24 (Ad.mda-7), induces cancer-specific apoptosis by inducing an endoplasmic reticulum (ER) stress response. Secreted MDA-7/IL-24 protein, generated from cells infected with Ad.mda-7, induces growth inhibition and apoptosis in surrounding noninfected cancer cells but not in normal cells, thus exerting an anti-tumor "bystander" effect. The present studies reveal a provocative finding that recombinant MDA-7/IL-24 protein can robustly induce expression of endogenous mda-7/IL-24, which generates the signaling events necessary for bystander killing. To evaluate the mechanism underlying this positive autocrine feedback loop, we show that MDA-7/IL-24 protein induces stabilization of its own mRNA without activating its promoter. Furthermore, this posttranscriptional effect depends on de novo protein synthesis. As a consequence of this autocrine feedback loop MDA-7/IL-24 protein induces sustained ER stress as evidenced by expression of ER stress markers (BiP/GRP78, GRP94, GADD153, and phospho-eIF2alpha) and reactive oxygen species production, indicating that both intracellular and secreted proteins activate similar signaling pathways to induce apoptosis. Thus, our results clarify the molecular mechanism by which secreted MDA-7/IL-24 protein (generated from Ad.mda-7-infected cells) exerts cancer-specific killing.

Involvement of hypoxia-triggered endoplasmic reticulum stress in outlet obstruction-induced apoptosis in the urinary bladder

In bladder outlet obstruction (BOO), mechanical stress and ischemia/hypoxia are implicated in structural and functional alterations of the urinary bladder. Because mechanical stress and hypoxia may trigger endoplasmic reticulum (ER) stress, we examined involvement of ER stress in the damage of the bladder caused by BOO. An experimental model of BOO was established in rats by complete ligature of the urethra for 24 h, and bladders were subjected to northern blot analysis and assessment of apoptosis. Isolated urinary bladders and bladder-derived smooth muscle cells (BSMCs) were also exposed to mechanical strain and hypoxia and used for analyses. To examine involvement of ER stress in the damage of the bladder, the effects of a chemical chaperone 4-phenylbutyrate (4-PBA) were evaluated in vitro and in vivo. Outlet obstruction for 24 h induced expression of ER stress markers, GRP78 and CCAAT/enhancer-binding protein-homologous protein (CHOP), in the bladder. It was associated with induction of markers for mechanical stress (cyclooxygenases 2) and hypoxia (vascular endothelial growth factor and glyceraldehyde-3-phosphate dehydrogenase). When isolated bladders and BSMCs were subjected to mechanical strain, induction of GRP78 and CHOP was not observed. In contrast, when BSMCs were exposed to hypoxic stress caused by CoCl2 or thenoyltrifluoroacetone (TTFA), substantial upregulation of GRP78 and CHOP was observed, suggesting involvement of hypoxia in the induction of ER stress. In the bladder subjected to BOO, the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells increased in the epithelial cells and BSMCs. Similarly, treatment with TTFA or CoCl2 induced apoptosis of BSMCs, and 4-PBA significantly attenuated ER stress and apoptosis triggered by these agents. Furthermore, in vivo administration with 4-PBA significantly reduced apoptosis in the bladder subjected to BOO. These results suggested that outlet obstruction caused ER stress via hypoxic stress in the bladder and that hypoxia-triggered ER stress may be involved in the induction of apoptosis in BOO.

Involvement of Selective Reactive Oxygen Species Upstream of Proapoptotic Branches of Unfolded Protein Response

Cadmium triggers apoptosis of LLC-PK1 cells through induction of endoplasmic reticulum (ER) stress. We found that cadmium caused generation of reactive oxygen species (ROS) and that cadmium-induced ER stress was inhibited by antioxidants. In contrast, suppression of ER stress did not attenuate cadmium-triggered oxidative stress, suggesting that ER stress occurs downstream of oxidative stress. Exposure of the cells to either O(2)(*), H(2)O(2), or ONOO(-) caused apoptosis, whereas ER stress was induced only by O(2)(*) or ONOO(-). Transfection with manganese superoxide dismutase significantly attenuated cadmium-induced ER stress and apoptosis, whereas pharmacological inhibition of ONOO(-) was ineffective. Interestingly, transfection with catalase attenuated cadmium-induced apoptosis without affecting the level of ER stress. O(2)(*) caused activation of the activating transcription factor 6-CCAAT/enhancer-binding protein-homologous protein (CHOP) and the inositol-requiring ER-to-nucleus signal kinase 1-X-box-binding protein 1 (XBP1) proapoptotic cascades, and overexpression of manganese superoxide dismutase attenuated cadmium-triggered induction of both pathways. Furthermore, phosphorylation of proapoptotic c-Jun N-terminal kinase by O(2)(*) or cadmium was suppressed by dominant-negative inhibition of XBP1. These data elucidated 1) cadmium caused ER stress via generation of ROS, 2) O(2)(*) was selectively involved in cadmium-triggered, ER stress-mediated apoptosis through activation of the activating transcription factor 6-CHOP and inositol-requiring ER-to-nucleus signal kinase 1-XBP1 pathways, and 3) phosphorylation of JNK was caused by O(2)(*)-triggered activation of XBP1.

Inhibition of Foxo1 Protects Pancreatic Islet β-Cells Against Fatty Acid and Endoplasmic Reticulum Stress–Induced Apoptosis

beta-Cells are particularly susceptible to fatty acid-induced apoptosis associated with decreased insulin receptor/phosphatidylinositol-3 kinase/Akt signaling and the activation of stress kinases. We examined the mechanism of fatty acid-induced apoptosis of mouse beta-cells especially as related to the role played by endoplasmic reticulum (ER) stress-induced Foxo1 activation and whether decreasing Foxo1 activity could enhance cell survival. Mouse insulinoma (MIN6) cells were administered with fatty acids, and the role of Foxo1 in mediating effects on signaling pathways and apoptosis was examined by measuring Foxo1 activity and using dominant-negative Foxo1. Increasing fatty acid concentrations (100-400 micromol/l palmitate or oleate) led to early Jun NH(2)-terminal kinase (JNK) activation that preceded induction of ER stress markers and apoptosis. Foxo1 activity was increased with fatty acid administration and by pharmacological inducers of ER stress, and this increase was prevented by JNK inhibition. Fatty acids induced nuclear localization of Foxo1 at 4 h when Akt activity was increased, indicating that FoxO1 activation was not mediated by JNK inhibition of Akt. In contrast, fatty acid administration for 24 h was associated with decreased insulin signaling. A dominant-negative Foxo1 adenovirus (Adv-DNFoxo) conferred cells with protection from ER stress and fatty acid-mediated apoptosis. Microarray analysis revealed that fatty acid induction of gene expression was in most cases reversed by Adv-DNFoxo, including the proapoptotic transcription factor CHOP (C/EBP [CCAAT/enhancer binding protein] homologous protein). Early induction of JNK and Foxo1 activation plays an important role in fatty acid-induced apoptosis. Expressing a dominant-negative allele of Foxo1 reduces expression of apoptotic and ER stress markers and promotes beta-cell survival from fatty acid and ER stress, identifying a potential therapeutic target for preserving beta-cells in type 2 diabetes.

Decreased Expression of Phospholipase C-.BETA.1 Protein in Endoplasmic Reticulum Stress-Loaded Neurons

The endoplasmic reticulum (ER) plays a critical role in the maintenance of intracellular homeostasis and its dysfunction is thought to lead to neuronal death, which results in neurodegenerative disorders. Since phospholipase C (PLC) isozymes are involved in maintenance of the intracellular Ca2+ concentration by regulating Ca2+ release from the ER, their expression might be affected by ER stress. Of these isozymes, PLC-beta 1 and -gamma 1, in particular, are known to protect cells from oxidative stress and thus alteration of their expression profile under ER stress-loaded conditions is interesting. Using primary cultured rat cortical neurons, we here examined whether expression of PLC-beta 1 and -gamma 1 was altered in ER stress-loaded neurons induced by tunicamycin (Tm). In ER stress-loaded neurons treated with Tm in the range of 0.03-3 microg/ml for 20 h, the viability of the neurons was decreased dose-dependently, the decrease being significant with 0.3 or more microg/ml, and expression of the representative ER stress markers, GRP78/BiP, and cleaved caspase-3 and -12, was increased after 24 h postincubation, confirming the induction of ER stress in the neurons. In the ER stress-loaded neurons obtained on Tm treatment, the expression level of PLC-beta 1 decreased dose-dependently. On the other hand, there was no difference in the PLC-gamma 1 protein expression level between control and ER stress-loaded neurons. Overall, we demonstrated that ER stress decreases the expression of PLC-beta 1, but not -gamma 1, in neurons.

The endoplasmic reticulum in pancreatic beta cells of type 2 diabetes patients

Pancreatic beta cells have highly developed endoplasmic reticulum (ER) due to their role in insulin secretion. Since ER stress has been associated with beta cell dysfunction, we studied several features of beta cell ER in human type 2 diabetes. Pancreatic samples and/or isolated islets from non-diabetic controls (ND) and type 2 diabetes patients were evaluated for insulin secretion, apoptosis (electron microscopy and ELISA), morphometric ER assessment (electron microscopy), and expression of ER stress markers in beta cell prepared by laser capture microdissection and in isolated islets. Insulin release was lower and beta cell apoptosis higher in type 2 diabetes than ND islets. ER density volume was significantly increased in type 2 diabetes beta cells. Expression of alpha-mannosidase (also known as mannosidase, alpha, class 1A, member 1) and UDP-glucose glycoprotein glucosyl transferase like 2 (UGCGL2), assessed by microarray and/or real-time reverse transcriptase polymerase chain reaction (RT-PCR), differed between ND and type 2 diabetes beta cells. Expression of immunoglobulin heavy chain binding protein (BiP, also known as heat shock 70 kDa protein 5 [glucose-regulated protein, 78 kDa] [HSPA5]), X-box binding protein 1 (XBP-1, also known as XBP1) and C/EBP homologous protein (CHOP, also known as damage-inducible transcript 3 [DDIT3]) was not higher in type 2 diabetes beta cell or isolated islets cultured at 5.5 mmol/l glucose (microarray and real-time RT-PCR) than in ND samples. When islets were cultured for 24 h at 11.1 mmol/l glucose, there was induction of BiP and XBP-1 in type 2 diabetes islets but not in ND islets. Beta cell in type 2 diabetes showed modest signs of ER stress when studied in pancreatic samples or isolated islets maintained at physiological glucose concentration. However, exposure to increased glucose levels induced ER stress markers in type 2 diabetes islet cells, which therefore may be more susceptible to ER stress induced by metabolic perturbations.

Atypical, bidirectional regulation of cadmium-induced apoptosis via distinct signaling of unfolded protein response

Cadmium is a widely distributed nephrotoxic metal that causes renal tubular injury. In this report, we investigated involvement of endoplasmic reticulum (ER) stress and individual unfolded protein responses in cadmium-initiated apoptosis of tubular epithelial cells. Cadmium chloride (CdCl(2)) induced expression of endogenous ER stress markers, GRP78, GRP94 and CHOP in vitro and in vivo, and subsequently caused cytological changes typical of apoptosis. Attenuation of ER stress by transfection with ER chaperone GRP78 or ORP150 suppressed CdCl(2)-triggered apoptosis. In response to CdCl(2), phosphorylation of RNA-dependent protein kinase-like ER kinase (PERK) and eukaryotic translation initiation factor 2alpha (eIF2alpha) was observed. Enhanced phosphorylation of eIF2alpha attenuated, whereas inhibition of eIF2alpha exacerbated CdCl(2)-induced apoptosis. Activating transcription factor 6 (ATF6) was also activated by CdCl(2) and blockade of this process suppressed induction of CHOP and thereby improved cell survival. CdCl(2) also triggered activation of the inositol-requiring ER-to-nucleus signal kinase 1 (IRE1)-X-box-binding protein 1 (XBP1) pathway and inhibition of XBP1 attenuated apoptosis independent of GRP78 and CHOP. c-Jun N-terminal kinase (JNK), another molecule downstream of IRE1, was also phosphorylated by CdCl(2) and its inhibition attenuated apoptosis. These results evidenced bidirectional regulation of apoptosis in cadmium-exposed cells. The ATF6 and IRE1 pathways cooperatively caused apoptosis via induction of CHOP, activation of XBP1 and phosphorylation of JNK, and the PERK-eIF2alpha pathway counteracted the proapoptotic processes.

Oxidative stress induces the endoplasmic reticulum stress and facilitates inclusion formation in cultured cells

The precise mechanism of formation and significance of Mallory bodies (MBs) are poorly understood. The endoplasmic reticulum (ER) is the organelle responsible for proper folding and elimination of unfolded proteins. Therefore, failure of this function increases defective proteins in the cell. We examined the effects of oxidative stress on induction of ER stress and keratin 8 and 18 (K8/18)-containing inclusion formation in cultured human hepatoma cells and hepatocytes by immunofluorescence and immunoblot analyses. Generation of H(2)O(2) was detected in glucose oxidase (GO)-treated cells by 2',7'-dichlorodihydrofluorescein diacetate and co-treatment with GO and acetyl-leucyl-leucyl-norleucinal (ALLN), a proteasome inhibitor, induced formation of extensive keratin inclusions that were inhibited by pre-treatment with N-acetyl-cysteine. These inclusions shared similar features with MBs by immunofluorescence analysis. Electron microscopy showed that these structures appeared near the nuclei, surrounded by filamentous structures. GO and ALLN upregulated the expression of ER stress markers, however, 4-phenylbutyrate, a chemical chaperone, reduced formation of inclusions and expression of the ER stress markers. The oxidative stress coupled with limited inhibition of the proteasome induces dysfunction of the ER and results in inclusion formation in cultured cells. This suggests that ER stress plays a role in MB formation in liver disease.

N-Glycosylation of MDA-7/IL-24 Is Dispensable for Tumor Cell–Specific Apoptosis and “Bystander” Antitumor Activity

Biochemical and genetic mutation-based analyses confirm that the MDA-7/IL-24 protein can induce transformed cell-specific apoptosis through a mechanism involving endoplasmic reticulum (ER) stress-associated pathways. Covalent modifications by N-linked glycans in the ER contribute to the conformational maturation and biological functions of many proteins. Because MDA-7/IL-24 is a glycosylated protein, we investigated the role of glycosylation in mediating the specific biological and "bystander" antitumor activities of this cytokine. An adenovirus vector expressing a nonsecreted and nonglycosylated version of MDA-7/IL-24 protein was generated via deletion of its signal peptide and point mutations of its three N-glycosylated sites. In this study, we showed that this intracellular nonglycosylated protein was as effective as wild-type MDA-7/IL-24 protein in inducing apoptosis in multiple tumor cell lines. Both constructs (a) displayed transformed cell specificity and localization to the ER compartment, (b) mediated apoptosis through JAK/STAT-independent and p38(MAPK)-dependent pathways, (c) induced sustained ER stress as evidenced by expression of ER stress markers (BiP/GRP78, GRP94, XBP-1, and eIF2alpha), and (d) generated proteins that physically interacted with BiP/GRP78. Additionally, an expression construct containing the mda-7/IL-24 signal peptide linked to the mutated nonglycosylated mda-7/IL-24 gene retained the ability to induce bystander antitumor activity. These studies reveal that MDA-7/IL-24 glycosylation is not mandatory for inducing cell death or bystander activities in different cancer cells, providing new insights into the mechanism by which MDA-7/IL-24 induces apoptosis and ER stress.

Bortezomib Inhibits PKR-Like Endoplasmic Reticulum (ER) Kinase and Induces Apoptosis via ER Stress in Human Pancreatic Cancer Cells

Bortezomib (Velcade, formerly known as PS-341) is a boronic acid dipeptide derivative that is a selective and potent inhibitor of the proteasome. We hypothesized that proteasome inhibition would lead to an accumulation of misfolded proteins in the cell resulting in endoplasmic reticulum (ER) stress. The ability of bortezomib to induce ER stress and the unfolded protein response was investigated in a human pancreatic cancer cell line, L3.6pl. Bortezomib increased expression of ER stress markers, CHOP and BiP, but inhibited PKR-like ER kinase and subsequent phosphorylation of eukaryotic initiation factor 2alpha (eif2alpha), both of which are key events in translational suppression. These effects resulted in an accumulation of ubiquitylated proteins leading to protein aggregation and proteotoxicity. Peptide inhibitor or small interfering RNA targeting ER-resident caspase-4 blocked DNA fragmentation, establishing a central role for caspase-4 in bortezomib-induced cell death. The translation inhibitor cycloheximide abrogated bortezomib-induced protein aggregation, caspase-4 processing, and all other characteristics of apoptosis. Because malignant cells have higher protein synthesis rates than normal cells, they may be more prone to protein aggregation and proteotoxicity and possess increased sensitivity to bortezomib-induced apoptosis. Taken together, the results show that bortezomib induces a unique type of ER stress compared with other ER stress agents characterized by an absence of eif2alpha phosphorylation, ubiquitylated protein accumulation, and proteotoxicity.