Endoplasmic Reticulum Stress-induced Activation Research Articles

Death Receptor 5 Signaling Promotes Hepatocyte Lipoapoptosis

Nonalcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA), endoplasmic reticulum (ER) stress, and hepatocyte lipoapoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5) is significantly elevated in patients with nonalcoholic steatohepatitis, and steatotic hepatocytes demonstrate increased sensitivity to TRAIL-mediated cell death. Nonetheless, a role for TRAIL and/or DR5 in mediating lipoapoptotic pathways is unexplored. Here, we examined the contribution of DR5 death signaling to lipoapoptosis by free fatty acids. The toxic saturated free fatty acid palmitate induces an increase in DR5 mRNA and protein expression in Huh-7 human hepatoma cells leading to DR5 localization into lipid rafts, cell surface receptor clustering with subsequent recruitment of the initiator caspase-8, and ultimately cellular demise. Lipoapoptosis by palmitate was not inhibited by a soluble human recombinant DR5-Fc chimera protein suggesting that DR5 cytotoxic signaling is ligand-independent. Hepatocytes from murine TRAIL receptor knock-out mice (DR(-/-)) displayed reduced palmitate-mediated lipotoxicity. Likewise, knockdown of DR5 or caspase-8 expression by shRNA technology attenuated palmitate-induced Bax activation and apoptosis in Huh-7 cells, without altering induction of ER stress markers. Similar observations were verified in other cell models. Finally, knockdown of CHOP, an ER stress-mediated transcription factor, reduced DR5 up-regulation and DR5-mediated caspase-8 activation upon palmitate treatment. Collectively, these results suggest that ER stress-induced CHOP activation by palmitate transcriptionally up-regulates DR5, likely resulting in ligand-independent cytotoxic signaling by this death receptor.

Carbon monoxide, a reaction product of heme oxygenase-1, suppresses the expression of C-reactive protein by endoplasmic reticulum stress through modulation of the unfolded protein response

The expression of C-reactive protein (CRP) rises rapidly in response to inflammation. The endoplasmic reticulum (ER) stress has been reported to cause CRP expression. Carbon monoxide (CO), a reaction product of heme oxygenase, exerts anti-inflammatory effects. In this study, we aimed to examine the role of CO in modulating ER stress-induced CRP expression. In HepG2 cells, ER stress triggered by tunicamycin, thapsigargin and homocysteine markedly induced CRP expression and the activation of protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring transmembrane kinase/endonuclease 1α (IRE1α), activating transcription factor 6 (ATF6), and hepatocyte-specific cyclic AMP response element binding protein H (CREBH). A CO-releasing molecule (CORM) inhibited ER stress-induced CRP expression. While CORM attenuated ER stress-induced activation of IRE1α, ATF6 and CREBH, it augmented PERK activation, which was associated with its inhibition of CRP expression. CORM also inhibited CRP expression in response to the pro-inflammatory cytokine IL-6 that was found to induce ER stress response in HepG2 cells. Moreover, in mice treated with the ER stress inducer tunicamycin, CORM administration reduced serum levels of CRP and the expression of CRP mRNA in the liver. Collectively, our findings suggest that CO may attenuate ER stress-induced CRP expression through modulation of the unfolded protein response.

ER Stress‐Induced SREBP‐2 Activation Contributes to Lipid Accumulation in Tubular Nephrotoxicity

Disruption of protein folding in the endoplasmic reticulum (ER) leads to ER stress and activation of the unfolded protein response (UPR). Tubular damage caused by nephrotoxic drugs is associated with ER stress. Since ER stress is known to induce lipid dysregulation through the activation of sterol regulatory element binding proteins (SREBPs), we hypothesized that lipid accumulation observed in proximal tubules affected by nephrotoxic drugs is a consequence of ER stress‐induced SREBP activation. Mice injected with tunicamycin display vacuolization and ER stress in proximal tubules. The vacuoles contain lipids that co‐localize with increased SREBP‐2 expression and markers of ER stress. In kidney biopsies from cyclosporine A (CsA)‐treated patients we show by immunohistochemistry the co‐localization of SREBP‐2 and ER stress markers in the damaged tubules. Treatment of HK2 renal cells with tunicamycin, thapsigargin or CsA caused UPR activation and increased lipid content. SREBP‐2 activation was shown by the enhanced sterol‐regulatory element‐GFP fluorescence, increased mRNA of downstream genes, and the presence of the mature form of SREBP‐2 on Western blot. These outcomes were blocked by AEBSF, an inhibitor of SREBP‐2 processing. We conclude that lipid accumulation in diseased kidney is associated with SREBP‐2 activation by ER stress. Supported by Heart and Stroke Foundation grant NA6024.

Novel insights in the interplay between inflammation and metabolic diseases: A role for the pathogen sensing kinase PKR

COMMENTARY ON: Double-stranded RNA-dependent protein kinase links pathogen sensing with stress and metabolic homeostasis. Nakamura T, Furuhashi M, Li P, Cao H, Tuncman G, Sonenberg N, Gorgun CZ, Hotamisligil GS. Cell 140, 338–348, February 5, 2010. Copyright 2010. Abstract reprinted with permission from Elsevier . http://www.ncbi.nlm.nih.gov/pubmed/20144759 Abstract: As chronic inflammation is a hallmark of obesity, pathways that integrate nutrient- and pathogen sensing pathways are of great interest in understanding the mechanisms of insulin resistance, type 2 diabetes, and other chronic metabolic pathologies. Here, Nakamura et al. provide evidence that double-stranded RNA-dependent protein kinase (PKR) can respond to nutrient signals as well as endoplasmic reticulum (ER) stress and coordinate the activity of other critical inflammatory kinases such as the c-Jun N-terminal kinase (JNK) to regulate insulin action and metabolism. PKR also directly targets and modifies insulin receptor substrate and hence integrates nutrients and insulin action with a defined pathogen response system. Dietary and genetic obesity features marked activation of PKR in adipose and liver tissues and absence of PKR alleviates metabolic deterioration due to nutrient or energy excess in mice. These findings demonstrate PKR as a critical component of an inflammatory complex that responds to nutrients and organelle dysfunction .

Endoplasmic Reticulum Stress-activated C/EBP Homologous Protein Enhances Nuclear Factor-κB Signals via Repression of Peroxisome Proliferator-activated Receptor γ

Endoplasmic reticulum (ER) stress is a causative factor of inflammatory bowel diseases. ER stress mediators, including CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP), are elevated in intestinal epithelia from patients with inflammatory bowel diseases. The present study arose from the question of how chemical ER stress and CHOP protein were associated with nuclear factor-κB (NF-κB)-mediated epithelial inflammatory response. In a human intestinal epithelial cell culture model, chemical ER stresses induced proinflammatory cytokine interleukin-8 (IL-8) expression and the nuclear translocation of CHOP protein. CHOP was positively involved in ER-activated IL-8 production and was negatively associated with expression of peroxisome proliferator-activated receptor γ (PPARγ). ER stress-induced IL-8 production was enhanced by NF-κB activation that was negatively regulated by PPARγ. Mechanistically, ER stress-induced CHOP suppressed PPARγ transcription by sequestering C/EBPβ and limiting availability of C/EBPβ binding to the PPARγ promoter. Due to the CHOP-mediated regulation of PPARγ action, ER stress can enhance proinflammatory NF-κB activation and maintain an increased level of IL-8 production in human intestinal epithelial cells. In contrast, PPARγ was a counteracting regulator of gut inflammatory response through attenuation of NF-κB activation. The collective results support the view that balances between CHOP and PPARγ are crucial for epithelial homeostasis, and disruption of these balances in mucosal ER stress can etiologically affect the progress of human inflammatory bowel diseases.

ASK1 Overexpression Accelerates Paraquat-Induced Autophagy via Endoplasmic Reticulum Stress

Apoptosis signal-regulating kinase 1 (ASK1) is activated by various types of stress, including, endoplasmic reticulum (ER) stress. ER stress-induced ASK1 activation could play an important role both in neuronal apoptosis and an autophagic response in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease. The mechanism by which ASK1 executes apoptosis and/or autophagy under ER stress is still unclear. We have addressed this question using SH-SY5Y cells overexpressing wild-type (WT) ASK1. We show an important autophagic response and an acceleration of the paraquat (PQ)-induced autophagy with hallmarks as accumulation of autophagic vacuoles, activation of beclin-1, accumulation of LC3 II, p62 degradation, and mammalian target of rapamycin dephosphorylation. Inhibition of autophagy caused an exacerbation of the apoptosis induced by WT ASK1 overexpression with or without PQ. These data support the idea that the autophagic response could have a protector role. We found also an increase in the phosphorylation of the proteins such as IRE1 and eIF2α in response to both the overexpression of WT ASK1 and pesticide exposure. These data suggest that the WT ASK1 overexpression-induced autophagy is an event that occurs in parallel with ER stress activation. The importance of ER stress in the autophagy induced by ASK1 and/or PQ was confirmed with salubrinal, a selective inhibitor of eIF2α dephosphorylation. In conclusion, we report that PQ induces an early ER stress response that is correlated with the activation of autophagy as a protective response, which is accelerated in cells that overexpress WT ASK1. However, when the toxic stimuli remain, the cell eventually succumbs to apoptosis.

A unique modulator of endoplasmic reticulum stress‐signalling pathways: the novel pharmacological properties of amiloride in glial cells

Stress on the endoplasmic reticulum (ER) can trigger rescuer responses such as the unfolded protein response (UPR). However, pharmacological modulators of these ER-regulated stress responses are not well understood. In the present study, we found that amiloride, a potassium-sparing diuretic, has unique properties relating to such stress. We treated mouse primary cultured glial cells with amiloride, in the absence and presence of the ER stress-inducing reagents tunicamycin (Tm) or dithiothreitol, and measured UPR and ER stress-induced cell death. IRE1alpha phosphorylation, eIF2alpha phosphorylation, X-box binding protein 1 (XBP1) splicing, glucose regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) expression by reverse transcription-polymerase chain reaction and Western blotting were used to assess UPR and lactate dehydrogenase activity was determined to measure ER stress-induced cell death. Amiloride completely inhibited ER stress-induced activation of IRE1alpha, an ER-localized stress sensor protein, splicing of XBP1, and subsequent expression of GRP78 at the mRNA and protein levels. ER stress induces the phosphorylation of eIF2alpha, leading to the expression of CHOP or an attenuation of translation in cells. Surprisingly, treatment with amiloride alone markedly promoted the phosphorylation but actually inhibited ER stress-induced CHOP expression. Finally, we found that amiloride (200 microM) synergistically enhanced ER stress-induced cell death, which was mediated through caspases. On the other hand, a low dose of amiloride (20 microM) significantly prevented Tm-induced cell death. These results suggest that amiloride can modulate UPR. They also suggest amiloride to be an important pharmacological agent and provide basic information for understanding and preventing ER stress-related diseases.

Endoplasmic Reticulum Stress-Induced Activation of Activating Transcription Factor 6 Decreases cAMP-Stimulated Hepatic Gluconeogenesis via Inhibition of CREB

The expression of genes encoding key hepatic gluconeogenic enzymes, including phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), is regulated at the transcriptional level by a network of transcription factors and cofactors, including cAMP response element-binding protein (CREB). It has been suggested that increased endoplasmic reticulum (ER) stress in the liver impairs hepatic glucose metabolism. However, the direct effect of ER stress on hepatic gluconeogenesis is still not clear. Here, we investigated whether ER stress influences hepatic gluconeogenesis and whether this process is mediated by activating transcription factor 6 (ATF6) through the inhibition of cAMP-mediated activation of CREB. A cAMP stimulant, forskolin, and 8-bromoadenosine-cAMP increased PEPCK and G6Pase mRNA expression in H4IIE rat hepatoma cells, and ER stress induced by tunicamycin or thapsigargin decreased the expression of these genes in forskolin or 8-bromoadenosine-cAMP-treated cells. In a transient transfection study, ATF6 inhibited the PEPCK and G6Pase promoters. Also, adenovirus-mediated overexpression of ATF6 in H4IIE cells decreased forskolin-stimulated PEPCK and G6Pase gene expression. Moreover, the inhibition of endogenous ATF6 expression by small interfering RNAs restored the ER stress-induced suppression of PEPCK and G6Pase gene expression. Transient transfection of ATF6 inhibited transactivation by CREB on the PEPCK and G6Pase promoters, and a gel shift assay showed that Ad-ATF6 inhibits forskolin-stimulated CREB DNA-binding activity. Finally, we found that expression of ATF6 decreased fasting-induced PEPCK, G6Pase mRNA expression, and blood glucose levels in mice. Taken together, these data extend our understanding of ER stress and the regulation of liver gluconeogenesis by ATF6.

O-GlcNAc signaling attenuates ER stress-induced cardiomyocyte death

We previously demonstrated that the O-linked beta-N-acetylglucosamine (O-GlcNAc) posttranslational modification confers cardioprotection at least partially through mitochondrial-dependent mechanisms, but it remained unclear if O-GlcNAc signaling interfered with other mechanisms of cell death. Because ischemia/hypoxia causes endoplasmic reticulum (ER) stress, we ascertained whether O-GlcNAc signaling could attenuate ER stress-induced cell death per se. Before induction of ER stress (with tunicamycin or brefeldin A), we adenovirally overexpressed O-GlcNAc transferase (AdOGT) or pharmacologically inhibited O-GlcNAcase [via O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate] to augment O-GlcNAc levels or adenovirally overexpressed O-GlcNAcase to reduce O-GlcNAc levels. AdOGT significantly (P < 0.05) attenuated the activation of the maladaptive arm of the unfolded protein response [according to C/EBP homologous protein (CHOP) activation] and cardiomyocyte death (reflected by percent propidium iodide positivity). Moreover, pharmacological inhibition of O-GlcNAcase significantly (P < 0.05) mitigated ER stress-induced CHOP activation and cardiac myocyte death. Interestingly, overexpression of GCA did not alter ER stress markers but exacerbated brefeldin A-induced cardiomyocyte death. We conclude that enhanced O-GlcNAc signaling represents a partially proadaptive response to reduce ER stress-induced cell death. These results provide new insights into a possible interaction between O-GlcNAc signaling and ER stress and may partially explain a mechanism of O-GlcNAc-mediated cardioprotection.

Endoplasmic reticulum stress induces autophagy in renal proximal tubular cells

Autophagy, an intracellular self-degradation system conserved throughout eukaryotes, plays an important role in a variety of biological processes, including cell death, development, cancer, defence against infection and neurodegeneration. However, little information about autophagy in renal tubular cells is available. We investigated the relationship of autophagy with endoplasmic reticulum (ER) stress in proximal tubular cells. Immortalized rat proximal tubular cells were exposed to the classic ER stress inducers tunicamycin or brefeldin A. Autophagy was detected mainly by immunoblot analysis of LC3, a widely used marker of autophagy, and also by immunofluorescent cytochemistry of LC3 and electron microscopy. Biological significance of the phenomenon was studied using bafilomycin A1, an inhibitor of autophagosome degradation. Signal transduction pathways following ER stress were also investigated using inhibitors of the MAPK pathway. Both ER stress inducers significantly increased LC3-II as a marker of autophagy in immunoblot analysis. Immunocytochemistry of LC3 and electron microscopy also showed activation of autophagy by ER stress. Inhibition by bafilomycin A1 showed that autophagy following ER stress fulfilled its intrinsic function, namely degradation of cytoplasmic components. Further, use of the MEK 1/2 inhibitor U0126, which inhibits ER stress-induced autophagy induction and ERK activation, showed that ERK, a MAPK family member, was necessary to the induction of autophagy. For the first time, we demonstrate the induction of autophagy following ER stress in renal tubules, and clarify its mechanism. These findings serve as the foundation for further investigation into autophagy in renal diseases.

Chemical Biology Investigation of Cell Death Pathways Activated by Endoplasmic Reticulum Stress Reveals Cytoprotective Modulators of ASK1

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) is caused by many disease-relevant conditions, inducing conserved signaling events collectively known as the unfolded protein response. When ER stress is excessive or prolonged, cell death (usually occurring by apoptosis) is triggered. We undertook a chemical biology approach for investigating mechanisms of ER stress-induced cell death. Using a cell-based high throughput screening assay to identify compounds that rescued a neuronal cell line from thapsigargin-induced cell death, we identified benzodiazepinones that selectively inhibit cell death caused by inducers of ER stress (thapsigargin and tunicamycin) but not by inducers of extrinsic (tumor necrosis factor) or intrinsic (mitochondrial) cell death pathways. The compounds displayed activity in several cell lines and primary cultured neurons. Mechanism of action studies revealed that these compounds inhibit ER stress-induced activation of p38 MAPK and kinases responsible for c-Jun phosphorylation. Active benzodiazepinones suppressed cell death at the level of apoptotic signal kinase-1 (ASK1) within the IRE1 pathway but without directly inhibiting the kinase activity of ASK1 or >400 other kinases tested. Rather, active compounds enhanced phosphorylation of serine 967 of ASK1, promoting ASK1 binding to 14-3-3, an event associated with suppression of ASK1 function. Reducing ASK1 protein expression using small interfering RNA also protected cells from ER stress-induced apoptosis, confirming the importance of this protein kinase. Taken together, these findings demonstrate an essential role for ASK1 in cell death induced by ER stress. The compounds identified may prove useful for revealing endogenous mechanisms that regulate inhibitory phosphorylation of ASK1.

PERK-dependent regulation of IAP translation during ER stress

Exposure of cells to Endoplasmic Reticulum (ER) stress leads to activation of phosphatidylinositol 3-kinase (PI3K)–Akt signaling pathway and transcriptional induction of the inhibitor of apoptosis family of proteins. One of the proximal effectors of the ER stress response, the PKR-like ER kinase (PERK), leads to cellular adaptation to stress by multiple mechanisms, including attenuation of protein synthesis, and transcriptional induction of pro-survival genes. While PERK activity leads to cellular adaptation to ER stress, we now demonstrate that PERK activity also inhibits the ER stress-induced apoptotic program through induction of cellular inhibitor of apoptosis (cIAP1 and cIAP2) proteins. This induction of IAPs occurs through both transcriptional and translational responses that are PERK-dependent. Reintroduction of cIAP1 or cIAP2 expression into PERK−/− MEFs during ER stress delays the early onset of ER stress-induced caspase activation and apoptosis observed in these cells. Furthermore, we demonstrate that activation of the PI3K-Akt pathway by ER stress is dependent on PERK, suggesting additional ways in which PERK activity protects cells from ER stress-induced apoptosis.

Opposing roles of prion protein in oxidative stress- and ER stress-induced apoptotic signaling

Although the prion protein is abundantly expressed in the CNS, its biological functions remain unclear. To determine the endogenous function of the cellular prion protein (PrP c), we compared the effects of oxidative stress and endoplasmic reticulum (ER) stress inducers on apoptotic signaling in PrP c-expressing and PrP ko (knockout) neural cells. H 2O 2, brefeldin A (BFA), and tunicamycin (TUN) induced increases in caspase-9 and caspase-3, PKCδ proteolytic activation, and DNA fragmentation in PrP c and PrP ko cells. Interestingly, ER stress-induced activation of caspases, PKCδ, and apoptosis was significantly exacerbated in PrP c cells, whereas H 2O 2-induced proapoptotic changes were suppressed in PrP c compared to PrP ko cells. Additionally, caspase-12 and caspase-8 were activated only in the BFA and TUN treatments. Inhibitors of caspase-9, caspase-3, and PKCδ significantly blocked H 2O 2-, BFA-, and TUN-induced apoptosis, whereas the caspase-8 inhibitor attenuated only BFA- and TUN-induced cell death, and the antioxidant MnTBAP blocked only H 2O 2-induced apoptosis. Overexpression of the kinase-inactive PKCδ K376R or the cleavage site-resistant PKCδ D327A mutant suppressed both ER and oxidative stress-induced apoptosis. Thus, PrP c plays a proapoptotic role during ER stress and an antiapoptotic role during oxidative stress-induced cell death. Together, these results suggest that cellular PrP enhances the susceptibility of neural cells to impairment of protein processing and trafficking, but decreases the vulnerability to oxidative insults, and that PKCδ is a key downstream mediator of cellular stress-induced neuronal apoptosis.

ALS-linked mutant SOD1 induces ER stress- and ASK1-dependent motor neuron death by targeting Derlin-1

Mutation in Cu/Zn-superoxide dismutase (SOD1) is a cause of familial amyotrophic lateral sclerosis (ALS). Mutant SOD1 protein (SOD1(mut)) induces motor neuron death, although the molecular mechanism of SOD1(mut)-induced cell death remains controversial. Here we show that SOD1(mut) specifically interacted with Derlin-1, a component of endoplasmic reticulum (ER)-associated degradation (ERAD) machinery and triggered ER stress through dysfunction of ERAD. SOD1(mut)-induced ER stress activated the apoptosis signal-regulating kinase 1 (ASK1)-dependent cell death pathway. Perturbation of binding between SOD1(mut) and Derlin-1 by Derlin-1-derived oligopeptide suppressed SOD1(mut)-induced ER stress, ASK1 activation, and motor neuron death. Moreover, deletion of ASK1 mitigated the motor neuron loss and extended the life span of SOD1(mut) transgenic mice. These findings demonstrate that ER stress-induced ASK1 activation, which is triggered by the specific interaction of Derlin-1 with SOD1(mut), is crucial for disease progression of familial ALS.

AIP1 Is Critical in Transducing IRE1-mediated Endoplasmic Reticulum Stress Response

We have previously shown that ASK1-interacting protein 1 (AIP1) transduces tumor necrosis factor-induced ASK1-JNK signaling. Because endoplasmic reticulum (ER) stress activates ASK1-JNK signaling cascade, we investigated the role of AIP1 in ER stress-induced signaling. We created AIP1-deficient mice (AIP1-KO) from which mouse embryonic fibroblasts and vascular endothelial cells were isolated. AIP1-KO cells show dramatic reductions in ER stress-induced, but not oxidative stress-induced, ASK1-JNK activation and cell apoptosis. The ER stress-induced IRE1-JNK/XBP-1 axis, but not the PERK-CHOP1 axis, is blunted in AIP1-KO cells. ER stress induced formation of an AIP1-IRE1 complex, and the PH domain of AIP1 is critical for the IRE1 interaction. Furthermore, reconstitution of AIP1-KO cells with AIP1 wild type, not an AIP1 mutant with a deletion of the PH domain (AIP1-DeltaPH), restores ER stress-induced IRE1-JNK/XBP-1 signaling. AIP1-IRE1 association facilitates IRE1 dimerization, a critical step for activation of IRE1 signaling. More importantly, AIP1-KO mice show impaired ER stress-induced IRE1-dependent signaling in vivo. We conclude that AIP1 is essential for transducing the IRE1-mediated ER stress response.

Endoplasmic Reticulum Stress-Induced Activation of Activating Transcription Factor 6 Decreases Insulin Gene Expression via Up-Regulation of Orphan Nuclear Receptor Small Heterodimer Partner

The highly developed endoplasmic reticulum (ER) structure of pancreatic beta-cells is a key factor in beta-cell function. Here we examined whether ER stress-induced activation of activating transcription factor (ATF)-6 impairs insulin gene expression via up-regulation of the orphan nuclear receptor small heterodimer partner (SHP; NR0B2), which has been shown to play a role in beta-cell dysfunction. We examined whether ER stress decreases insulin gene expression, and this process is mediated by ATF6. A small interfering RNA that targeted SHP was used to determine whether the effect of ATF6 on insulin gene expression is mediated by SHP. We also measured the expression level of ATF6 in pancreatic islets in Otsuka Long Evans Tokushima Fatty rats, a rodent model of type 2 diabetes. High glucose concentration (30 mmol/liter glucose) increased ER stress in INS-1 cells. ER stress induced by tunicamycin, thapsigargin, or dithiotreitol decreased insulin gene transcription. ATF6 inhibited insulin promoter activity, whereas X-box binding protein-1 and ATF4 did not. Adenovirus-mediated overexpression of active form of ATF6 in INS-1 cells impaired insulin gene expression and secretion. ATF6 also down-regulated pancreatic duodenal homeobox factor-1 and RIPE3b1/MafA gene expression and repressed the cooperative action of pancreatic duodenal homeobox factor-1, RIPE3b1/MafA, and beta-cell E box transactivator 2 in stimulating insulin transcription. The ATF6-induced suppression of insulin gene expression was associated with up-regulation of SHP gene expression. Finally, we found that expression of ATF6 was increased in the pancreatic islets of diabetic Otsuka Long Evans Tokushima Fatty rats, compared with their lean, nondiabetic counterparts, Long-Evans Tokushima Otsuka rats. Collectively, this study shows that ER stress-induced activation of ATF6 plays an important role in the development of beta-cell dysfunction.

Inhibition of MEK Sensitizes Human Melanoma Cells to Endoplasmic Reticulum Stress-Induced Apoptosis

Past studies have shown that activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK is a common cause for resistance of melanoma cells to death receptor-mediated or mitochondria-mediated apoptosis. We report in this study that inhibition of the MEK/ERK pathway also sensitizes melanoma cells to endoplasmic reticulum (ER) stress-induced apoptosis, and this is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperon glucose-regulated protein 78 (GRP78) expression. Treatment with the ER stress inducer tunicamycin or thapsigargin did not induce significant apoptosis in the majority of melanoma cell lines, but resistance to these agents was reversed by the MEK inhibitor U0126 or MEK1 small interfering RNA (siRNA). Induction of apoptosis by ER stress when MEK was inhibited was caspase dependent with caspase-4, caspase-9, and caspase-3 being involved. Caspase-4 seemed to be the apical caspase in that caspase-4 activation occurred before activation of caspase-9 and caspase-3 and that inhibition of caspase-4 by a specific inhibitor or siRNA blocked activation of caspase-9 and caspase-3, whereas inhibition of caspase-9 or caspase-3 did not inhibit caspase-4 activation. Moreover, overexpression of Bcl-2 inhibited activation of caspase-9 and caspase-3 but had minimal effect on caspase-4 activation. Inhibition of MEK/ERK also resulted in down-regulation of GRP78, which was physically associated with caspase-4, before and after treatment with tunicamycin or thapsigargin. In addition, siRNA knockdown of GRP78 increased ER stress-induced caspase-4 activation and apoptosis. Taken together, these results seem to have important implications for new treatment strategies in melanoma by combinations of agents that induce ER stress and inhibitors of the MEK/ERK pathway.

Akt up- and down-regulation in response to endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of CNS diseases such as Alzheimer's disease, Parkinson's disease, and cerebral ischemia. In the present study, we found that Akt activation is regulated dually by ER stress in primary cultured glial cells. We observed that Akt activation was increased by short-term exposure to ER stress but was down-regulated by long-term exposure to ER stress. ER stress-induced Akt activation was mediated through phosphatidylinositol 3-kinase (PI3K) because the PI3K inhibitors, LY294002 and wortmannin, inhibited Akt activation. Moreover, Akt was localized in the ER, as assessed by immunohistochemistry, and ER stress increased microsomally localized Akt activation. These results suggest that Akt plays an important role in stress conditions, which impair ER function.

Endoplasmic reticulum stress causes the activation of sterol regulatory element binding protein-2

Background Sterol regulatory element binding protein-2 (SREBP-2) is a membrane-bound transcription factor that upon proteolytic processing can activate the expression of genes involved in cholesterol biosynthesis and uptake. We as well as others have demonstrated that the accumulation of misfolded proteins within the endoplasmic reticulum (ER), a condition known as ER stress, can dysregulate lipid metabolism by activating the SREBPs. The purpose of this study was to determine the mechanism by which ER stress induces SREBP-2 activation. Methods and results HeLa and MCF7 cells were treated with ER stress-inducing agents to determine the effect of ER stress on SREBP-2 cleavage and subsequent cholesterol accumulation. Cells treated with thapsigargin (Tg) exhibit proteolytic cleavage of SREBP-2. Proteolytic cleavage of SREBP-2 induced by Tg occurred independently of caspase activation and was inhibited by the site-1 protease inhibitor AEBSF, suggesting that Tg-induced SREBP-2 cleavage occurs through the conventional site-1/-2 pathway. Treatment of HeLa cells with Tg also led to the accumulation of free cholesterol as measured by Filipin staining. Conclusions These results imply that ER stress-induced SREBP-2 activation occurs through the conventional pathway that normally regulates SREBP in accordance with intracellular sterol concentration.

Albumin induces endoplasmic reticulum stress and apoptosis in renal proximal tubular cells

Chronic proteinuria appears to be a key factor in tubulointerstitial damage. Recent studies have emphasized a pathogenic role of endoplasmic reticulum (ER) stress which is induced by the accumulation of misfolded proteins in ER, extracellular stress, etc. In the present study, we investigated ER stress and ER stress-induced apoptosis in proximal tubular cells (PTCs). Immortalized rat PTCs (IRPTCs) were cultured with bovine serum albumin (BSA). The viability of IRPTCs decreased proportionately with BSA overload in a time-dependent manner. Quantitative real-time polymerase chain reaction analysis revealed that 40 mg/ml BSA increases mRNA of ER stress markers by 7.7- and 4.6-fold (glucose-regulated protein 78 (GRP78) and oxygen-regulated protein 150 (ORP150), respectively) as compared to control. The increased expression of ORP150 and GRP78 in IRPTCs with albumin overload was detected by Western blot and immunofluorescence study. These in vitro observations were supported by in vivo studies, which demonstrated that ER stress proteins were upregulated at PTCs in experimental proteinuric rats. Furthermore, increased ER stress-induced apoptosis and activation of caspase-12 were observed in IRPTCs with albumin overload and kidneys of experimental proteinuric rats. We confirmed that apoptotic cell death was attenuated by co-incubation with caspase-3 inhibitor or calpain inhibitors. These results indicate that the ER stress-induced apoptosis pathway contributed to the insult of tubular cells by proteinuria. In conclusion, renal tubular cells exposed to high protein load suffer from ER stress. ER stress may subsequently lead to tubular damage by activation of caspase-12.