Mechanical load-dependent cardiac ER stress in vitro and in vivo: effects of preload and afterload

Proteins are folded in the endoplasmic reticulum (ER). ER stress initially leads to compensatory upregulation of ER chaperones and later to apoptosis, but the contribution of biomechanical load vs. neurohumoral stress to myocardial ER stress is unknown. We show that the ER chaperones Grp78 and calreticulin (CRT) are upregulated by afterload, but not by preload in vitro and in vivo. Angiotensin II upregulated ER chaperones in unloaded muscle strips, but the angiotensin receptor-1 antagonist irbesartan did not significantly blunt the induction of ER chaperones by afterload. In monocrotaline-treated rats, Grp78 and CRT were upregulated in the afterloaded right ventricle, but not in the only neurohumorally stressed left ventricle. These findings suggest that afterload but not preload induces myocardial ER stress, largely independent of angiotensin II signaling.     

Endoplasmic reticulum stress contributed to inflammatory bowel disease by activating p38 MAPK pathway

Recent evidence suggests that endoplasmic reticulum (ER) stress plays a vital role in inflammatory bowel disease (IBD). Therefore, the aim of this study was to investigate the mechanism by which ER stress promotes inflammatory response in IBD. The expression of Gro-α, IL-8 and ER stress indicator Grp78 in colon tissues from patients with Crohn’s disease (CD) and colonic carcinoma was analyzed by immunohistochemistry staining. Colitis mouse model was established by the induction of trinitrobenzene sulphonic acid (TNBS), and the mice were treated with ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Then the body weight, colon length and colon inflammation were evaluated, and Grp78 and Gro-α in colon tissues were detected by immunohistochemistry. Epithelial cells of colon cancer HCT116 cells were treated with tunicamycin to induce ER stress. Grp78 was detected by Western blot, and chemokines were measured by PCR and ELISA. The expression levels of Grp78, Gro-α and IL-8 were significantly upregulated in intestinal tissues of CD patients. Mice with TNBS induced colitis had increased expression of Grp78 and Gro-α in colonic epithelia. TUDCA reduced the severity of TNBS-induced colitis. In HCT116 cells, tunicamycin increased the expression of Grp78, Gro-α and IL-8 in a concentration-dependent manner. Furthermore, p38 MAPK inhibitor significantly inhibited the upregulation of Gro-α and IL-8 induced by tunicamycin. In conclusion, ER stress promotes inflammatory response in IBD, and the effects may be mediated by the activation of p38 MAPK signaling pathway.Key words: Inflammatory bowel disease, endoplasmic reticulum stress, IL-8, Gro-α, p38 MAPK     

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 2α) 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.     

Endoplasmic reticulum stress-induced cysteine protease activation in cortical neurons: effect of an Alzheimer's disease-linked presenilin-1 knock-in mutation

Endoplasmic reticulum (ER) stress elicits protective responses of chaperone induction and translational suppression and, when unimpeded, leads to caspase-mediated apoptosis. Alzheimer's disease-linked mutations in presenilin-1 (PS-1) reportedly impair ER stress-mediated protective responses and enhance vulnerability to degeneration. We used cleavage site-specific antibodies to characterize the cysteine protease activation responses of primary mouse cortical neurons to ER stress and evaluate the influence of a PS-1 knock-in mutation on these and other stress responses. Two different ER stressors lead to processing of the ER-resident protease procaspase-12, activation of calpain, caspase-3, and caspase-6, and degradation of ER and non-ER protein substrates. Immunocytochemical localization of activated caspase-3 and a cleaved substrate of caspase-6 confirms that caspase activation extends into the cytosol and nucleus. ER stress-induced proteolysis is unchanged in cortical neurons derived from the PS-1 P264L knock-in mouse. Furthermore, the PS-1 genotype does not influence stress-induced increases in chaperones Grp78/BiP and Grp94 or apoptotic neurodegeneration. A similar lack of effect of the PS-1 P264L mutation on the activation of caspases and induction of chaperones is observed in fibroblasts. Finally, the PS-1 knock-in mutation does not alter activation of the protein kinase PKR-like ER kinase (PERK), a trigger for stress-induced translational suppression. These data demonstrate that ER stress in cortical neurons leads to activation of several cysteine proteases within diverse neuronal compartments and indicate that Alzheimer's disease-linked PS-1 mutations do not invariably alter the proteolytic, chaperone induction, translational suppression, and apoptotic responses to ER stress.     

Proteomic characterization of the striatum and midbrain treated with 6-hydroxydopamine: Alteration of 58-kDa glucose-regulated protein and C/EBP homologous protein

Abstract

The present study performed proteomic analysis of the midbrain and striatum of 6-hydroxydopamine (6-OHDA)-treated neonatal rats—a model of attention-deficit hyperactivity disorder (ADHD). Proteomic analysis revealed that a 58-kDa glucose-regulated protein (Grp58) was temporarily phosphorylated and its level was elevated by 6-OHDA. Furthermore, 6-OHDA increased the expression level of C/EBP homologous protein (CHOP), a mediator of endoplasmic reticulum (ER) stress response, in the midbrain and striatum. In vitro experiments using PC12 cells revealed that 6-OHDA or hydrogen peroxide could induce the elevation of Grp58 and CHOP. 6-OHDA could induce the elevation of Grp58 and CHOP in the presence of catalase, a hydrogen peroxide-removing enzyme, suggesting that the elevation of Grp58 and CHOP are induced by both hydrogen peroxide and p-quinone generated by 6-OHDA. Collectively, these findings suggest that ER stress involving the alteration of Grp58 and CHOP play a significant role in the induction of insults by 6-OHDA in vivo.     

The altered expression of glucose-regulated proteins 78 in different phase of streptozotocin-affected pancreatic beta-cells

Endoplasmic reticulum (ER) stress-mediated apoptosis plays an important role in the destruction of pancreatic beta-cells and contributes to the development of type 1 diabetes. The chaperone molecule, glucose-regulated proteins 78 (Grp78), is required to maintain ER function during toxic insults. In this study, we investigated the changes of Grp78 expression in different phases of streptozotocin (STZ)-affected beta-cells to explore the relationship between Grp78 and the response of beta-cells to ER stress. An insulinoma cell line (NIT-1) treated with STZ for different time periods and STZ-induced diabetic Balb/C mice at different time points were used as the model system. The level of Grp78 and C/EBP homologous protein (CHOP) mRNA were detected by real-time polymerase chain reaction and their protein by immunoblot. Apoptosis and necrosis was measured by flow cytometry. In addition, the changes of Grp78 protein in STZ-treated nondiabetic mice were also detected by immunoblot. Grp78 expression significantly increased in the early phase but decreased in the later phase of affected beta-cells, while CHOP was induced and apoptosis occurred along with the decrease of Grp78. Interestingly, the Grp78 protein of STZ-treated nondiabetic mice increased stably compared with that of the control. From the results, we can conclude that Grp78 may contribute to the response of beta-cells to ER stress, and more attention should be paid to Grp78 in the improvement of diabetes.     

ER stress contributes to ischemia-induced cardiomyocyte apoptosis

Myocardial ischemia is a severe stress condition that leads to loss of cardiomyocytes. The cell loss is attributed to apoptosis, although the exact mechanisms involved are only partially defined, which limits therapeutic opportunities. Here, we show caspase activation and apoptosis in neonatal rat cardiomyocyte cultures subjected to simulated ischemia by serum, glucose, and oxygen deprivation (SGO). Caspase activation was preceded by endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR), detected by the induction of Grp78, induction and splicing of XBP1, and phosphorylation of eukaryotic initiation factor 2-alpha (eIF2alpha). At a later time the ER stress response switched from UPR and cytoprotective response to a pro-apoptotic response as demonstrated by the upregulation of CHOP and processing of pro-caspase-12. Thus, we provide evidence that the ER can generate and propagate apoptotic signals in response to ischemic stress and this pathway is therefore a novel target for prevention of ischemia-mediated cardiomyocyte loss.     

The presence of extracellular matrix alters the chondrocyte response to endoplasmic reticulum stress

The objective of this study was to test the hypothesis that extracellular matrix (ECM) would alter the endoplasmic reticulum (ER) stress response of chondrocytes. Chondrocytes were isolated from calf knees and maintained in monolayer culture or suspended in collagen I to form spot cultures (SCs). Our laboratory has shown that bovine chondrocytes form cartilage with properties similar to native cartilage after 2-4 weeks in SCs. Monolayer cultures treated with ER stressors glucose withdrawal (-Glu), tunicamycin (TN), or thapsigargin (TG) up-regulated Grp78 and Gadd153, demonstrating a complete ER stress response. SCs were grown at specific times from 1 day to 6 weeks before treatment with ER stressors. Additionally, SCs grown for 1, 2, or 6 weeks were treated with increasing concentrations of TN or TG. Western blotting of SCs for Grp78 indicated that increased ECM accumulation results in delayed expression; however, Grp78 mRNA is up-regulated in response to ER stressors even after 6 weeks in culture. SCs treated with ER stressors did not up-regulate Gadd153, suggesting that the cells experienced ER stress but would not undergo apoptosis. In fact, SCs undergo apoptosis upon ER stress treatment after 0-1 day of growth; however, after 4 days and to 6 weeks, apoptosis in treated samples was not different than controls. Pro-survival molecules Bcl-2 and Bag-1 were up-regulated upon ER stress in SCs. These results suggest that presence of ECM confers protection from ER stressors. Future studies involving chondrocyte physiology should focus on responses in conditions more closely mimicking the in vivo cartilage environment.     

Metabolic syndrome and acute hyperglycemia are associated with endoplasmic reticulum stress in human mononuclear cells

Endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR) have been implicated in a number of complications associated with diabetes mellitus including micro‐ and macrovascular dysfunction. In this study we examine ER stress levels in blood cells isolated from human subjects with metabolic syndrome and in healthy controls. Total RNA and protein were isolated from leukocytes and the levels of specific ER stress markers were quantified by real‐time‐PCR and immunoblot analysis. Our results indicate that, compared to healthy controls, individuals with metabolic syndrome have elevated mRNA levels of genes indicative of ER stress; including spliced XBP‐1 (sXBP‐1), Grp78, and CHOP. Induced ER stress levels correlate with blood glucose but not plasma lipid concentration. Furthermore, in healthy individuals, a standard 75 g oral glucose challenge produced a significant elevation in spliced XBP‐1 (1.3 fold), Grp78 (2.0 fold), and calreticulin (3.5 fold) mRNA 60 min post challenge and a significant increase in Grp78 (2.0 fold), calreticulin (2.7 fold) protein levels 2 h postchallenge, relative to fasting levels. The UPR was also activated ex vivo, in human leukocytes cultured in the presence of 15 mmol/l glucose, supporting a specific role for glucose. The oral glucose challenge was associated with a significant increase in the expression of inflammatory cytokines, including interleukin (IL)‐1α/β, IL‐6, and IL‐8, that may result from ER stress. These findings suggest that there is an association between both acute and chronic dysglycemia and ER stress in humans.     

Depletion of oxidative and endoplasmic reticulum stress regulators in Pick disease

Both oxidative and endoplasmic reticulum (ER) stress is associated with multiple neurodegenerative, age-related diseases. The rare disorder Pick disease (PiD) shares some pathological hallmarks of other neurodegenerative diseases that may be related to oxidative stress. Importantly, activation of an ER stress response, which is also involved in aging, has not yet been investigated in PiD. In this study, we assessed the implication of ER stress associated with oxidative stress in PiD as a potential mechanism involved in its pathogenesis. Samples from morphologically affected frontal cortex and apparently pathologically preserved occipital cortex showed region-dependent increases in different protein oxidative damage pathways. The oxidative modifications targeted antioxidant enzymes, proteases, heat shock proteins, and synaptic proteins. These effects were associated with compromised proteasomal function and ER stress in frontal cortex samples. In addition, we observed a depletion in ER chaperones (glucose-regulated proteins Grp78/BiP and glucose-regulated protein 94) and differences in tissue content and distribution of nuclear factor-erythroid 2 p45-related respiratory 2, required for cell survival during the unfolded protein response. These results demonstrate increased region-specific protein oxidative damage in PiD, with proteasomal alteration and dysfunctional ER stress response. We suggest this was caused by complete and specific depletion of Grp78/BiP, contributing to the pathophysiology of this neurodegenerative disease.     

Grp94 acts as a mediator of curcumin‐induced antioxidant defence in myogenic cells

Curcumin is a non‐toxic polyphenol with pleiotropic activities and limited bioavailability. We investigated whether a brief exposure to low doses of curcumin would induce in the myogenic C2C12 cell line an endoplasmic reticulum (ER) stress response and protect against oxidative stress. A 3‐hr curcumin administration (5–10 μM) increased protein levels of the ER chaperone Grp94, without affecting those of Grp78, calreticulin and haeme‐oxygenase‐1 (HO‐1). Exposure of cells to hydrogen peroxide 24 hrs after the curcumin treatment decreased caspase‐12 activation, total protein oxidation and translocation of NF‐κB to the nucleus, compared with untreated cells. Grp94 overexpression, achieved by means of either stable or transient trasfection, induced comparable cytoprotective effects to hydrogen peroxide. The delayed cytoprotection induced by curcumin acted through Grp94, because the curcumin‐induced increase in Grp94 expression was hampered by either stable or transient transfection with antisense cDNA; in these latter cells, the extent of total protein oxidation, as well as the translocation of NF‐κB to the nucleus, and the percentage of apoptotic cells were comparable to those observed in both curcumin‐untreated wild‐type and empty vector transfected cells. Defining the mechanism(s) by which Grp94 exerts its antioxidant defence, the determination of cytosolic calcium levels in C2C12 cells by fura‐2 showed a significantly reduced amount of releasable calcium from intracellular stores, both in conditions of Grp94 overexpression and after curcumin pre‐treatment. Therefore, a brief exposure to curcumin induces a delayed cytoprotection against oxidative stress in myogenic cells by increasing Grp94 protein level, which acts as a regulator of calcium homeostasis.