Showing papers on "Transcription Factor CHOP published in 2008"

Chop deletion reduces oxidative stress, improves β cell function, and promotes cell survival in multiple mouse models of diabetes

Abstract: The progression from insulin resistance to type 2 diabetes is caused by the failure of pancreatic β cells to produce sufficient levels of insulin to meet the metabolic demand. Recent studies indicate that nutrient fluctuations and insulin resistance increase proinsulin synthesis in β cells beyond the capacity for folding of nascent polypeptides within the endoplasmic reticulum (ER) lumen, thereby disrupting ER homeostasis and triggering the unfolded protein response (UPR). Chronic ER stress promotes apoptosis, at least in part through the UPR-induced transcription factor C/EBP homologous protein (CHOP). We assessed the effect of Chop deletion in multiple mouse models of type 2 diabetes and found that Chop–/– mice had improved glycemic control and expanded β cell mass in all conditions analyzed. In both genetic and diet-induced models of insulin resistance, CHOP deficiency improved β cell ultrastructure and promoted cell survival. In addition, we found that isolated islets from Chop–/– mice displayed increased expression of UPR and oxidative stress response genes and reduced levels of oxidative damage. These findings suggest that CHOP is a fundamental factor that links protein misfolding in the ER to oxidative stress and apoptosis in β cells under conditions of increased insulin demand.

Cytokine-Induced β-Cell Death Is Independent of Endoplasmic Reticulum Stress Signaling

Abstract: OBJECTIVE—Cytokines contribute to β-cell destruction in type 1 diabetes. Endoplasmic reticulum (ER) stress–mediated apoptosis has been proposed as a mechanism for β-cell death. We tested whether ER stress was necessary for cytokine-induced β-cell death and also whether ER stress gene activation was present in β-cells of the NOD mouse model of type 1 diabetes. RESEARCH DESIGN AND METHODS—INS-1 β-cells or rat islets were treated with the chemical chaperone phenyl butyric acid (PBA) and exposed or not to interleukin (IL)-1β and γ-interferon (IFN-γ). Small interfering RNA (siRNA) was used to silence C/EBP homologous protein (CHOP) expression in INS-1 β-cells. Additionally, the role of ER stress in lipid-induced cell death was assessed. RESULTS—Cytokines and palmitate triggered ER stress in β-cells as evidenced by increased phosphorylation of PKR-like ER kinase (PERK), eukaryotic initiation factor (EIF)2α, and Jun NH2-terminal kinase (JNK) and increased expression of activating transcription factor (ATF)4 and CHOP. PBA treatment attenuated ER stress, but JNK phosphorylation was reduced only in response to palmitate, not in response to cytokines. PBA had no effect on cytokine-induced cell death but was associated with protection against palmitate-induced cell death. Similarly, siRNA-mediated reduction in CHOP expression protected against palmitate- but not against cytokine-induced cell death. In NOD islets, mRNA levels of several ER stress genes were reduced (ATF4, BiP [binding protein], GRP94 [glucose regulated protein 94], p58, and XBP-1 [X-box binding protein 1] splicing) or unchanged (CHOP and Edem1 [ER degradation enhancer, mannosidase α–like 1]). CONCLUSIONS—While both cytokines and palmitate can induce ER stress, our results suggest that, in contrast to lipoapoptosis, the PERK-ATF4-CHOP ER stress–signaling pathway is not necessary for cytokine-induced β-cell death.

Overexpression of endoplasmic reticulum-resident chaperone attenuates cardiomyocyte death induced by proteasome inhibition

Abstract: Aims Proteasome inhibitors are a novel class of anticancer agents that induce tumour cell death via endoplasmic reticulum (ER) stress. Since ER stress is involved in the development of heart failure, we investigated the role of ER-initiated cardiomyocyte death by proteasome inhibition. Methods and results Rat neonatal cardiomyocytes were used in this study. Proteasome activity was assayed using proteasome peptidase substrates. Cell viability and apoptosis were measured by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide and flow cytometry, respectively. Western blot analysis, real-time polymerase chain reaction (PCR) and reverse transcriptional PCR were used to detect the expression of protein and messenger ribonucleic acid (RNA). The location of overexpressed glucose-regulated protein (GRP) 78 was observed by confocal fluorescence microscopy. Proteasome inhibition induced cardiomyocyte death and activated ER stress-induced transcriptional factor ATF6, but not XBP1 (X-box binding protein 1), without up-regulating ER chaperones. ER-initiated apoptosis signalling, including cytosine-cytosine-adenine-adenine-thymine enhancer-binding protein (C/EBP) homologous protein (CHOP), c-Jun-N-terminal kinase (JNK), and caspase-12, was activated by proteasome inhibition. Short interference RNA targeting CHOP, but not the blockage of caspase-12 or JNK pathway, attenuated cardiomyocyte death. Overexpression of GRP78 suppressed both CHOP expression and cardiomyocyte death by proteasome inhibition. Conclusion These findings demonstrate that proteasome inhibition induces ER-initiated cardiomyocyte death via CHOP-dependent pathways without compensatory up-regulation of ER chaperones. Supplement and/or pharmacological induction of GRP78 can attenuate cardiac damage by proteasome inhibition.

Coupling of Endoplasmic Reticulum Stress to CDDO-Me–Induced Up-regulation of Death Receptor 5 via a CHOP–Dependent Mechanism Involving JNK Activation

Abstract: The synthetic triterpenoid methyl-2-cyano-3,12-dioxoolean-1,9-dien-28-oate (CDDO-Me) is in phase I clinical trials as a novel cancer therapeutic agent. We previously showed that CDDO-Me induces c-Jun NH(2)-terminal kinase (JNK)-dependent death receptor 5 (DR5) expression and augments death receptor-induced apoptosis. The current study focused on addressing how CDDO-Me induces JNK-dependent DR5 expression. Analysis of DR5 promoter regions defines that the CCAAT/enhancer binding protein homologous protein (CHOP) binding site is responsible for CDDO-Me-induced transactivation of the DR5 gene. Consistently, CDDO-Me induced DR5 expression and parallel CHOP up-regulation. Blockade of CHOP up-regulation also abrogated CDDO-Me-induced DR5 expression. These results indicate that CDDO-Me induces CHOP-dependent DR5 up-regulation. Moreover, the JNK inhibitor SP600125 abrogated CHOP induction by CDDO-Me, suggesting a JNK-dependent CHOP up-regulation by CDDO-Me as well. Importantly, knockdown of CHOP attenuated CDDO-Me-induced apoptosis, showing that CHOP induction is involved in CDDO-Me-induced apoptosis. Additionally, CDDO-Me increased the levels of Bip, phosphorylated eukaryotic translation initiation factor 2alpha, inositol requiring kinase 1alpha, and activating transcription factor 4, all of which are featured changes during endoplasmic reticulum (ER) stress. Furthermore, salubrinal, an inhibitor of ER stress-induced apoptosis, inhibited JNK activation and up-regulation of CHOP and DR5 by CDDO-Me and protected cells from CDDO-Me-induced apoptosis. Thus, ER stress seems to be important for CDDO-Me-induced JNK activation, CHOP and DR5 up-regulation, and apoptosis. Collectively, we conclude that CDDO-Me triggers ER stress, leading to JNK-dependent, CHOP-mediated DR5 up-regulation and apoptosis.

Ethanol promotes endoplasmic reticulum stress‐induced neuronal death: Involvement of oxidative stress

Abstract: One of the most devastating effects of ethanol exposure during development is the loss of neurons in selected brain areas The underlying cellular/molecular mechanisms remain unclear The endoplasmic reticulum (ER) is involved in posttranslational protein processing and transport The accumulation of unfolded or misfolded proteins in the ER lumen triggers ER stress, which is characterized by translational attenuation, synthesis of ER chaperone proteins such as GRP78, and activation of transcription factors such as ATF4, ATF6, and CHOP Sustained ER stress ultimately leads to cell death ER stress response can be induced experimentally by treatment with tunicamycin and thapsigargin Using SH-SY5Y neuroblastoma cells and primary cerebellar granule neurons as in vitro models, we demonstrated that exposure to ethanol alone had little effect on the expression of markers for ER stress; however, ethanol drastically enhanced the expression of GRP78, CHOP, ATF4, ATF6, and phosphorylated PERK and elF2α when induced by tunicamycin and thapsigargin Consistently, ethanol promoted tunicamycin- and thapsigargin-induced cell death Ethanol rapidly caused oxidative stress in cultured neuronal cells; antioxidants blocked ethanol’s potentiation of ER stress and cell death, suggesting that the ethanol-promoted ER stress response is mediated by oxidative stress CHOP is a proapoptotic transcription factor We further demonstrated that CHOP played an important role in ethanol-promoted cell death Thus, the effect of ethanol may be mediated by the interaction between oxidative stress and ER stress

CHOP transcription factor mediates IL-8 signaling in cystic fibrosis bronchial epithelial cells.

Abstract: Interleukin (IL)-8 is a potent neutrophil chemoattractant that drives the inflammatory response in cystic fibrosis (CF). Traditional approaches to the pathophysiology of this inflammation have focused on targeting NF-κB–dependent signaling and therapy with glucocorticoids. We test the hypothesis that an alternative pathway, independent of NF-κB, operates through prostaglandin E2 (PGE-2) receptor EP-2 and stimulates IL-8 chemokine secretion. Using CF bronchial epithelial cells (IB3-1) in vitro, exogenous PGE-2 induces IL-8 release in a dose-dependent manner. These events are associated with elevation in the EP-2 receptors. Inhibition of cyclooxygenase (Cox)-2 with NS-398 was associated with reductions in Cox-2 (2-fold) and IL-6 (1.3-fold) mRNA transcripts, and in IL-8 and PGE-2 chemokine secretion. The inhibition of Cox-2 signaling led to down-regulation of the downstream C/EBP homologous protein (CHOP) transcription factor, resulting in a decrease in IL-8 activation. We confirmed the regulation of IL-8 promoter by CHOP in CF cells using the IL-8 reporter assay. We conclude that PGE-2 stimulates IL-8 production through the CHOP transcription factor in CF cells.

Shiga toxin 2 causes apoptosis in human brain microvascular endothelial cells via C/EBP homologous protein

Abstract: Shiga toxin 1 (Stx1) and Stx2 produced by Escherichia coli O157 are known to be cytotoxic to Vero and HeLa cells by inhibiting protein synthesis and by inducing apoptosis. In the present study, we have demonstrated that 10 ng/ml Stx2 induced DNA fragmentation in human brain microvascular endothelial cells (HBMEC), with cleavage activation of caspase-3, -6, -8, and -9. A microarray approach used to search for apoptotic potential signals in response to Stx2 revealed that Stx2 treatment induced a marked upregulation of C/EBP homologous protein (CHOP)/growth arrest and DNA damage-inducible protein 153 (GADD153). Increased CHOP expression was dependent on enzymatically active Stx1. Knockdown of CHOP mRNA reduced the activation of caspase-3 and prevented apoptotic cell death. These results suggest that Stx2-induced apoptosis is mediated by CHOP in HBMEC and involves activation of both the intrinsic and extrinsic pathways of apoptosis.

The role of CHOP messenger RNA expression in the link between oxidative stress and apoptosis.

Abstract: Low expression of antioxidant enzymes makes pancreatic beta-cells susceptible to cell damage by oxidative stress. Pancreatic beta-cell loss caused by endoplasmic reticulum stress is associated with the onset of diabetes mellitus. The present studies were undertaken to investigate a possible involvement of proapoptotic gene CHOP in pancreatic beta-cells damage by oxidative stress. The induction of CHOP messenger RNA and apoptosis were investigated in betaHC-9 cells after the oxidative stress by hydrogen peroxide and ribose. Latter was examined after the suppression of CHOP by small interfering RNA. For in vivo study, the pancreatic beta-cells were examined in CHOP-knockout (KO) mice after multiple low-dose streptozotocin (MLDS) administration. In betaHC-9 cells, both hydrogen peroxide and ribose obviously increased apoptotic cells, accompanied with enhanced CHOP messenger RNA expression. However, the number of apoptotic cells by those stimulations was significantly reduced by the addition of small interfering RNA against CHOP. In vivo study also showed that CHOP-KO mice were less susceptible to diabetes after MLDS administration. Although the oxidative stress marker level was similar to that of MLDS-treated wild type, the pancreatic beta-cell area was maintained in CHOP-KO mice. The present studies showed that CHOP should be important in pancreatic beta-cell injury by oxidative stress and indicate that CHOP may play a role in the development of pancreatic beta-cell damage on the onset of diabetes mellitus.

Rottlerin induces pro-apoptotic endoplasmic reticulum stress through the protein kinase C-δ-independent pathway in human colon cancer cells

Abstract: Rottlerin, a compound reported to be a PKC δ-selective inhibitor, has been shown to induce growth arrest or apoptosis of human cancer cell lines. In our study, rottlerin dose-dependently induced apoptotic cell death in colon carcinoma cells. Treatment of HT29 human colon carcinoma cells with rottlerin was found to induce a number of signature ER stress markers; phosphorylation of eukaryotic initiation factor-2α (eIF-2α), ER stress-specific XBP1 splicing, and up-regulation of glucose-regulated protein (GRP)-78 and CCAAT/enhancer-binding protein-homologous protein (CHOP). However, suppression of PKC δ expression by siRNA or overexpression of WT-PKC δ and DN-PKC δ did not abrogate the rottlerin-mediated induction of CHOP. These results suggest that rottlerin induces up-regulation of CHOP via PKC δ-independent pathway. Furthermore, down-regulation of CHOP expression using CHOP siRNA attenuated rottlerin-induced apoptosis. Taken together, the present study thus provides strong evidence to support an important role of ER stress response in mediating the rottlerin-induced apoptosis.

Many commercially available antibodies for detection of CHOP expression as a marker of endoplasmic reticulum stress fail specificity evaluation.

Abstract: Endoplasmic reticulum (ER) stress contributes to beta cell death in type 2 diabetes (T2DM). ER stress is characterized by increased level of ER stress markers such as C/EBP homologous protein (CHOP). Activation of CHOP leads to its translocation into the nucleus, where it induces cell death. We previously reported nuclear CHOP in pancreatic sections from T2DM, but not T1DM, and in human islet amyloid polypeptide (IAPP) transgenic rodent pancreatic sections. These studies underscore the importance of studying nuclear CHOP. We have observed inconsistency in the detection of CHOP antibodies reported in the literature and also in our own experiments. To investigate the specificity of CHOP antibodies, we first induced ER stress by tunicamycin in rat insulinoma (INS) cells and prepared nuclear and cytoplasmic fractions. Then we examined CHOP expression by Western blotting and immunocytochemistry using seven commercially available CHOP antibodies in INS cells and human IAPP (h-IAPP) transgenic rodent pancreatic tissue. These studies show that three commercially available CHOP antibodies out of seven tested were non-specific. In conclusion, we give recommendations for CHOP antibody selection and methods to verify CHOP antibody specificity. Also, we propose that the authors report the catalog and lot numbers of the CHOP antibodies used.

Molecular physiology of plasma cell differentiation

Abstract: Plasma cells are central to an effective immune response, being the sole producers of the antibodies, yet they can cause severe disease in autoimmunity and multiple myeloma. Therefore their differentiation and survival must be tightly regulated. In this work I investigated some of the mechanisms regulating plasma cell differentiation and life span. The differentiation of a long lived B cell to a short lived plasma cell entails a profound structural and functional metamorphosis finalized to the massive production of immunoglobulins (Ig). Exuberant Ig synthesis causes several types of stress in differentiating plasma cells. My work deals with the characterization of the C/EBP transcription factor CHOP in plasma cell differentiation. Comparing differentiation of B cells harvested from chop-l- mice to wt cells I found a mild phenotype, consisting in an increased accumulation of intracellular IgM aggregates and a decreased secretion of this antibody class, in vitro and in vivo. These findings reveal a novel role for CHOP in ensuring optimal functionality of the secretory pathway in the course of plasma cell differentiation. CHOP is involved in the differentiation of various cell types, where it interacts with other members of the C/EBP family favoring or impeding differentiation and it is an important factor in the ER stress response named unfolded protein response (UPR), in which it plays a pro-apoptotic role in most of the systems tested. I extended my investigation on the functions of CHOP in B cells by examining the resistance to ER stress-induced apoptosis in wt and in chop-l- cells. Surprisingly, I observed that in B cells CHOP expression in the UPR plays an anti-apoptotic function. Altogether my data suggest a cell-type specific role for CHOP in B cells and add information on the multi-faceted role of this transcription factor. Most plasma cells exhibit a short life span. The mechanisms at the basis of plasma cell apoptosis are still obscure. I propose that multiple forms of stress, linked to the massive antibody production, contribute to plasma cell death.