Levels Of C/EBP Homologous Protein Research Articles

Diazoxide attenuates hypothermic preservation-induced renal injury via down-regulation of CHOP and caspase-12

Successful clinical organ preservations are a prerequisite for organ transplantation. Diazoxide (DE), which shows a concentration-dependent selectivity for mitoK(+-)-ATP over plasma membrane K(+-)-ATP, displays protective effects during organ preservation. The current study investigated possible protective effects of DE on rat kidneys injured by hypothermic preservation. Forty-eight Sprague-Dawley rats were randomly divided into six groups (n = 8): Celsior groups with kidneys preserved in Celsior solution for 0, 24 and 48h and DE groups with kidneys preserved in DE (30μM) plus Celsior solution for 0, 24 and 48h. Superoxide dismutase (SOD) activity and the quantity of malonaldehyde (MDA) in the kidneys from each group were measured, and the levels of C/EBP homologous protein (CHOP) and caspase-12 were determined by immunohistochemistry staining and real-time reverse transcription quantitative polymerase chain reaction analysis. SOD activity was significantly higher and the quantity of MDA was significantly lower in the DE groups compared with the Celsior groups at both 24 and 48h (P < 0.05). The expressions of CHOP and caspase-12 were also lower in DE groups at 24 and 48h (P < 0.05). The present results demonstrate that DE exerts protective effects by attenuating oxidative stress injury through up-regulation of SOD activity and down-regulation of MDA quantity and by decreasing the cell apoptosis in kidneys by reducing the levels of CHOP and caspase-12 during hypothermic preservation.

T-cadherin attenuates the PERK branch of the unfolded protein response and protects vascular endothelial cells from endoplasmic reticulum stress-induced apoptosis

Endoplasmic reticulum (ER) stress activated by perturbations in ER homeostasis induces the unfolded protein response (UPR) with chaperon Grp78 as the key activator of UPR signalling. The aim of UPR is to restore normal ER function; however prolonged or severe ER stress triggers apoptosis of damaged cells to ensure protection of the whole organism. Recent findings support an association of ER stress-induced apoptosis of vascular cells with cardiovascular pathologies. T-cadherin (T-cad), an atypical glycosylphosphatidylinositol-anchored member of the cadherin superfamily is upregulated in atherosclerotic lesions. Here we investigate the ability of T-cad to influence UPR signalling and endothelial cell (EC) survival during ER stress. EC were treated with a variety of ER stress-inducing compounds (thapsigargin, dithiothereitol, brefeldin A, tunicamycin, A23187 or homocysteine) and induction of ER stress validated by increases in levels of UPR signalling molecules Grp78 (glucose-regulated protein of 78 kDa), phospho-eIF2α (phosphorylated eukaryotic initiation factor 2 α) and CHOP (C/EBP homologous protein). All compounds also increased T-cad mRNA and protein levels. Overexpression or silencing of T-cad in EC respectively attenuated or amplified the ER stress-induced increase in phospho-eIF2α, Grp78, CHOP and active caspases. Effects of T-cad-overexpression or T-cad-silencing on ER stress responses in EC were not affected by inclusion of either N-acetylcysteine (reactive oxygen species scavenger), LY294002 (phosphatidylinositol-3-kinase inhibitor) or SP6000125 (Jun N-terminal kinase inhibitor). The data suggest that upregulation of T-cad on EC during ER stress attenuates the activation of the proapoptotic PERK (PKR (double-stranded RNA-activated protein kinase)-like ER kinase) branch of the UPR cascade and thereby protects EC from ER stress-induced apoptosis.

Amyloid precursor protein promotes endoplasmic reticulum stress‐induced cell death via C/EBP homologous protein‐mediated pathway

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) is known to activate the ER, which is termed ER stress. Here, we demonstrated that amyloid precursor protein (APP) is a novel mediator of ER stress-induced apoptosis through the C/EBP homologous protein (CHOP) pathway. Expression of APP mRNA was elevated by tunicamycin- or dithiothreitol-induced ER stress. The levels of C83 and APP intracellular domain (AICD) fragments, which are cleaved from APP, were significantly increased under ER stress, although the protein level of full-length APP was decreased. Cellular viability was reduced in APP-over-expressing cells, which was attenuated by treatment with a gamma-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Cellular viability was also reduced in AICD-FLAG-over-expressing cells. The mRNA and protein levels of CHOP, an ER stress-responsive gene, were remarkably increased by APP over-expression, which was attenuated by treatment with DAPT. CHOP mRNA induction was also found in AICD-FLAG-over-expressing cells. Cell death and CHOP up-regulation by ER stress were attenuated by APP knockdown. Data obtained with a luciferase assay and chromatin immunoprecipitation assay indicated that AICD associates with the promoter region of the CHOP gene. In conclusion, ER stress-induced APP undergoes alpha- and gamma-secretase cleavage and subsequently induces CHOP-mediated cell death.

Effects of sarcoplasmic reticulum calcium ATPase 2a overexpression on endoplasmic reticulum stress in cardiomyocytes

To investigate the effects of sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) overexpression on the endoplasmic reticulum stress (ERS). Ventricular cardiomyocytes were obtained from a neonatal rat, cultured, and then randomly divided into 6 groups: normal control group; hypoxia group cultured in an airtight chamber gassed with 95% N(2)/5% CO2 at 37 degrees C for 72 hours so as to induce ERS; tunicamycin group treated with 10 microg/ml tunicamycin so as to induce ERS too; SERCA2a group transfected with recombinant adenovirus expressing the target gene SERCA2a (rAd-SERCA2a); SERCA2a + hypoxia group; and SERCA2a + tunicamycin group. Western blotting was used to detect the protein expression of SERCA2a, and glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP), both the ERS markers. Flow cytometry was used to detect the apoptosis of the cardiomyocytes, and trypan blue staining was used to detect the survival rate of the cardiomyocytes. (1) The GRP78 expression level of the hypoxia group was 5.1 times as high as that of the control group, and the CHOP expression level of the hypoxia group was 2.5 times as high as hat of the control group. The GRP78 expression level of the tunicamycin group was 4.9 times as high as that of the control group, and the CHOP expression level of the tunicamycin group was 3.1 times as high as hat of the control group. SERCA2a overexpression was found to relieve the expression of GRP78 induced by hypoxia and tunicamycin (49.1% and 50.4% decrease respectively), and to inhibit the activation of CHOP (52.7% and 66.1% decrease respectively). (2) In comparison with the hypoxia group, the protein expression levels of GRP78 and CHOP of the SERCA2a overexpression + hypoxia group were significantly lower by 49.1% and 52.7% respectively, the apoptotic rate was significantly lower by 66.0%, and the cardiomyocyte survival rate was significantly higher by 13.4% (all P < 0.05). Compared with the tunicamycin group, the protein expression levels of GRP78 and CHOP of the SERCA2a overexpression + tunicamycin group were significantly lower by 50.4% and 66.1% respectively, the apoptotic rate was significantly lower by 54.0%, and the cardiomyocyte survival rate was significantly higher by 6.7% (all P < 0.05). SERCA2a overexpression attenuates ERS induced by hypoxia or tunicamycin, and protects cardiomyocytes against ERS-mediated cellular injury.

Apolipoprotein E-deficient mice are more vulnerable to ER stress after transient forebrain ischemia

Apolipoprotein E-deficient (apoE −/−) mice have been shown to have increased vulnerability to neuronal damage induced by cerebral ischemia; however, the mechanism of this increased vulnerability remains unclear. In order to define the role of the apoE protein against ischemia-induced ER stress and cell death, experiments were performed to compare ER stress-associated chaperones and signal proteins in the hippocampus of apoE −/− mice to those of WT mice after being subjected to forebrain ischemia and reperfusion. Although neuronal loss in area CA1–CA3 of the hippocampus was observed 3 days after ischemia in both types of mice, the damage in apoE −/− mice was more severe. In apoE −/− mice, a more extensive increase in 78-kDa glucose-regulated protein (GRP78) was observed after the insult, whereas the level of GRP94 was not changed. The expression of both C/EBP homologous protein (CHOP) and caspase-12 was increased in the hippocampus in both WT and apoE −/− mice after ischemia. The increased levels of CHOP in apoE −/− mice were significantly higher than those in WT mice, whereas the levels of caspase-12 in the two were comparable. Furthermore, whereas the levels of c-Jun N-terminal kinase (JNK), p-JNK1 and p-JNK2 in WT mice were unchanged after ischemia, they were significantly increased in apoE −/− mice 24 h and 48 h after ischemia. These results suggest that increased vulnerability of the hippocampus to forebrain ischemia and reperfusion in apoE −/− mice is at least partly attributable to perturbed induction of an ER chaperone, GRP 94, and enhancement of the CHOP- and JNK-dependent apoptotic pathway in the hippocampus.

Amyloid β-protein potentiates tunicamycin-induced neuronal death in organotypic hippocampal slice cultures

We have assessed amyloid β protein (Aβ)–induced neurotoxicity, with and without added tunicamycin (TM), an inhibitor of N-glycosylation in the endoplasmic reticulum (ER), in rat organotypic hippocampal slice cultures (OHCs). In the rat OHCs cultured for 3 weeks, there was little neurotoxicity after treatment with Aβ 25-35 (25 μM) alone for 48 h. However, with TM alone, concentration-dependent neuronal death was observed at concentrations between 20 and 80 μg/mL. When amyloid-β protein was combined with tunicamycin (Aβ+TM), cell death was more acute than with TM alone. Western blot analysis revealed that calpain activity and the active forms of caspase-12 and caspase-3 was increased after exposure to Aβ+TM as compared with exposure to TM alone. In contrast, the levels of glucose regulated protein (GRP)94, GRP78 and C/EBP homologous protein (CHOP) were not changed in the presence of Aβ. Aβ potentiation of TM neurotoxicity was reversibly blocked by S-allyl- l-cysteine (SAC), an organosulfur compound purified from aged garlic extract, and the L-type calcium channel blocker, nifedipine, in a restricted neuronal area of the OHCs. Simultaneously applied SAC also reversed the increases in calpain activity and the active forms of caspase-12 and caspase-3 by Aβ+TM with no change in the increased levels of GRP94, GRP78 and CHOP. These data indicate that Aβ facilitates the calpain–caspase-12–caspase-3 pathway, thus potentiating TM-induced neuronal death in the hippocampus.

Protection of pancreatic β-cells by exendin-4 may involve the reduction of endoplasmic reticulum stress; in vivo and in vitro studies

The aim of this study was to investigate the in vivo and in vitro effects of exendin-4, a potent glucagon-like peptide 1 agonist, on the protection of the pancreatic beta-cells against their cell death. In in vivo experiments, we used beta-cell-specific calmodulin-overexpressing mice where massive apoptosis takes place in their beta-cells, and we examined the effects of chronic treatment with exendin-4. Chronic and s.c. administration of exendin-4 reduced hyperglycemia. The treatment caused significant increases of the insulin contents of the pancreas and islets, and retained the insulin-positive area. Dispersed transgenic islet cells lived only shortly, and several endoplasmic reticulum (ER) stress-related molecules such as immunoglobulin-binding protein (Bip), inositol-requiring enzyme-1alpha, X-box-binding protein-1 (XBP-1), RNA-activated protein kinase-like endoplasmic reticulum kinase, activating transcription factor-4, and C/EBP-homologous protein (CHOP) were more expressed in the transgenic islets. We also found that the spliced form of XBP-1, a marker of ER stress, was also increased in beta-cell-specific calmodulin-overexpressing transgenic islets. In the quantitative real-time PCR analyses, the expression levels of Bip and CHOP were reduced in the islets from the transgenic mice treated with exendin-4. These findings suggest that excess of ER stress occurs in the transgenic beta-cells, and the suppression of ER stress and resultant protection against cell death may be involved in the anti-diabetic effects of exendin-4.

CHOP (C/EBP homologous protein) and ASNS (asparagine synthetase) induction in cybrid cells harboring MELAS and NARP mitochondrial DNA mutations

Mitochondrial dysfunction caused by mutations in mitochondrial DNA (mtDNA) is related to a variety of diseases including MELAS and NARP syndromes. However, little is known about the intracellular responses induced by mtDNA mutations. In order to identify genes whose expression is altered as a result of the presence of mtDNA mutations, DNA microarray analysis was performed using human 143B osteosarcoma cells harboring 3243A > G [tRNA-Leu (UUR)] and 8993T > G [ATPase6 Leu156Arg] mtDNA mutations associated with MELAS and NARP syndromes (2SD and NARP3-1 cybrid cells), respectively. We found that mRNA and protein levels of ATF4, CHOP and ASNS were upregulated in 2SD and NARP3-1 cells as compared with parental cells. Reporter assays demonstrated that transcription of CHOP and ASNS genes was upregulated through the AARE (amino acid regulatory element) and NSRE-1 (nutrient-sensing response element-1) enhancer elements to which ATF4 binds, respectively. Furthermore, knockdown of ATF4 by RNA interference reduced CHOP and ASNS transcription in 2SD and NARP3-1 cells. These results suggest that the presence of mtDNA mutations elicits upregulation of CHOP and ASNS genes through the elevation of ATF4 expression and its binding to the AARE and NSRE-1, respectively.