Lipid aldehyde-mediated cross-linking of apolipoprotein B-100 inhibits secretion from HepG2 cells

Hepatic oxidative stress and lipid peroxidation are common features of several prevalent disease states, including alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD), a common component of the metabolic syndrome. These conditions are characterized in part by excessive accumulation of lipids within hepatocytes, which can lead to autocatalytic degradation of cellular lipids giving rise to electrophilic end products of lipid peroxidation. The pathobiology of reactive lipid aldehydes remains poorly understood. We therefore sought to investigate the effects of 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE) on the transport and secretion of very low-density lipoprotein using HepG2 cells as a model hepatocyte system. Physiologically relevant concentrations of 4-HNE and 4-ONE rapidly disrupted cellular microtubules in a concentration-dependent manner. Interestingly, 4-ONE reduced apolipoprotein B-100 (ApoB) secretion while 4-HNE did not significantly impair secretion. Both 4-HNE and 4-ONE formed adducts with ApoB protein, but 4-HNE adducts were detectable as mono-adducts, while 4-ONE adducts were present as protein–protein cross-links. These results demonstrate that reactive aldehydes generated by lipid peroxidation can differ in their biological effects, and that these differences can be mechanistically explained by the structures of the protein adducts formed.     

La3+ binds to BiP/GRP78 and induces unfolded protein response in HepG2 cells

The effects of La3+ on the unfolded protein response signaling pathways were investigated in human hepatoblastoma HepG2 cells. Our data showed that La3+ could induce unfolded protein response in HepG2 cells, including a significant increase of BiP/GRP78 level, which is an important ER residential chaperone and an ER stress hallmark, in a concentration and time-dependent manner, UPR transducer IRE1 phosphorylation and splicing activation IRE1 downstream substrate XBP1 mRNA. By using La3+-affinity chromatography, the possible cellular target of La3+ leading to UPR events was shown to be the ER residential chaperone BiP/GRP78. BiP/GRP78 was shown to be a La3+ binding protein and the interaction of La3+ with BiP/GRP78 resulted in dissociation of BiP-IRE1 complexes. La3+ induced dissociation of the BiP/GRP78-IRE1 complex was in a time and concentration manner. The apparent dissociation constant was estimated to be 4 nM. In addition, La3+ was observed to slightly stimulate the production of cellular ROS and cause alteration of intracellular Ca2+, indicating the possible involvement of ROS and Ca2+ alteration in La3+ induced UPR. The present work provides a new perspective for understanding the biological and toxicological effects of La3+.     

The antitumor natural compound falcarindiol promotes cancer cell death by inducing endoplasmic reticulum stress

Falcarindiol (FAD) is a natural polyyne with various beneficial biological activities. We show here that FAD preferentially kills colon cancer cells but not normal colon epithelial cells. Furthermore, FAD inhibits tumor growth in a xenograft tumor model and exhibits strong synergistic killing of cancer cells with 5-fluorouracil, an approved cancer chemotherapeutic drug. We demonstrate that FAD-induced cell death is mediated by induction of endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). Decreasing the level of ER stress, either by overexpressing the ER chaperone protein glucose-regulated protein 78 (GRP78) or by knockout of components of the UPR pathway, reduces FAD-induced apoptosis. In contrast, increasing the level of ER stress by knocking down GRP78 potentiates FAD-induced apoptosis. Finally, FAD-induced ER stress and apoptosis is correlated with the accumulation of ubiquitinated proteins, suggesting that FAD functions at least in part by interfering with proteasome function, leading to the accumulation of unfolded protein and induction of ER stress. Consistent with this, inhibition of protein synthesis by cycloheximide significantly decreases the accumulation of ubiquitinated proteins and blocks FAD-induced ER stress and cell death. Taken together, our study shows that FAD is a potential new anticancer agent that exerts its activity through inducing ER stress and apoptosis.     

Presenilin-1 mutations downregulate the signalling pathway of the unfolded-protein response

Missense mutations in the human presenilin-1 (PS1) gene, which is found on chromosome 14, cause early-onset familial Alzheimer's disease (FAD). FAD-linked PS1 variants alter proteolytic processing of the amyloid precursor protein and cause an increase in vulnerability to apoptosis induced by various cell stresses. However, the mechanisms responsible for these phenomena are not clear. Here we report that mutations in PS1 affect the unfolded-protein response (UPR), which responds to the increased amount of unfolded proteins that accumulate in the endoplasmic reticulum (ER) under conditions that cause ER stress. PS1 mutations also lead to decreased expression of GRP78/Bip, a molecular chaperone, present in the ER, that can enable protein folding. Interestingly, GRP78 levels are reduced in the brains of Alzheimer's disease patients. The downregulation of UPR signalling by PS1 mutations is caused by disturbed function of IRE1, which is the proximal sensor of conditions in the ER lumen. Overexpression of GRP78 in neuroblastoma cells bearing PS1 mutants almost completely restores resistance to ER stress to the level of cells expressing wild-type PS1. These results show that mutations in PS1 may increase vulnerability to ER stress by altering the UPR signalling pathway.     

Exploring the Conserved Role of MANF in the Unfolded Protein Response in Drosophila melanogaster

Disturbances in the homeostasis of endoplasmic reticulum (ER) referred to as ER stress is involved in a variety of human diseases. ER stress activates unfolded protein response (UPR), a cellular mechanism the purpose of which is to restore ER homeostasis. Previous studies show that Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is an important novel component in the regulation of UPR. In vertebrates, MANF is upregulated by ER stress and protects cells against ER stress-induced cell death. Biochemical studies have revealed an interaction between mammalian MANF and GRP78, the major ER chaperone promoting protein folding. In this study we discovered that the upregulation of MANF expression in response to drug-induced ER stress is conserved between Drosophila and mammals. Additionally, by using a genetic in vivo approach we found genetic interactions between Drosophila Manf and genes encoding for Drosophila homologues of GRP78, PERK and XBP1, the key components of UPR. Our data suggest a role for Manf in the regulation of Drosophila UPR.     

Localization of GRP78 to mitochondria under the unfolded protein response

The ubiquitously expressed molecular chaperone GRP78 (78 kDa glucose-regulated protein) generally localizes to the ER (endoplasmic reticulum). GRP78 is specifically induced in cells under the UPR (unfolded protein response), which can be elicited by treatments with calcium ionophore A23187 and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor TG (thapsigargin). By using confocal microscopy, we have demonstrated that GRP78 was concentrated in the perinuclear region and co-localized with the ER marker proteins, calnexin and PDI (protein disulphide-isomerase), in cells under normal growth conditions. However, treatments with A23187 and TG led to diminish its ER targeting, resulting in redirection into a cytoplasmic vesicular pattern, and overlapping with the mitochondrial marker MitoTracker. Cellular fractionation and protease digestion of isolated mitochondria from ER-stressed cells suggested that a significant portion of GRP78 is localized to the mitochondria and is protease-resistant. Localizations of GRP78 in ER and mitochondria were confirmed by using immunoelectron microscopy. In ER-stressed cells, GRP78 mainly localized within the mitochondria and decorated the mitochondrial membrane compartment. Submitochondrial fractionation studies indicated further that the mitochondria-resided GRP78 is mainly located in the intermembrane space, inner membrane and matrix, but is not associated with the outer membrane. Furthermore, radioactive labelling followed by subcellular fractionation showed that a significant portion of the newly synthesized GRP78 is localized to the mitochondria in cells under UPR. Taken together, our results indicate that, at least under certain circumstances, the ER-resided chaperone GRP78 can be retargeted to mitochondria and thereby may be involved in correlating UPR signalling between these two organelles.     

Luteolin and Apigenin Attenuate 4-Hydroxy-2-Nonenal-Mediated Cell Death through Modulation of UPR,Nrf2-ARE and MAPK Pathways in PC12 Cells

Luteolin and apigenin are dietary flavones and exhibit a broad spectrum of biological activities including antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) has been implicated as a causative agent in the development of neurodegenerative disorders. This study investigates the cytoprotective effects of luteolin and apigenin against 4-HNE-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Both flavones restored cell viability and repressed caspase-3 and PARP-1 activation in 4-HNE-treated cells. Luteolin also mitigated 4-HNE-mediated LC3 conversion and reactive oxygen species (ROS) production. Luteolin and apigenin up-regulated 4-HNE-mediated unfolded protein response (UPR), leading to an increase in endoplasmic reticulum chaperone GRP78 and decrease in the expression of UPR-targeted pro-apoptotic genes. They also induced the expression of Nrf2-targeted HO-1 and xCT in the absence of 4-HNE, but counteracted their expression in the presence of 4-HNE. Moreover, we found that JNK and p38 MAPK inhibitors significantly antagonized the increase in cell viability induced by luteolin and apigenin. Consistently, enhanced phosphorylation of JNK and p38 MAPK was observed in luteolin- and apigenin-treated cells. In conclusion, this result shows that luteolin and apigenin activate MAPK and Nrf2 signaling, which elicit adaptive cellular stress response pathways, restore 4-HNE-induced ER homeostasis and inhibit cytotoxicity. Luteolin exerts a stronger cytoprotective effect than apigenin possibly due to its higher MAPK, Nrf2 and UPR activation, and ROS scavenging activity.     

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-eIF2α, 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.     

HSP105 interacts with GRP78 and GSK3 and promotes ER stress-induced caspase-3 activation

Stress of the endoplasmic reticulum (ER stress) is caused by the accumulation of misfolded proteins, which occurs in many neurodegenerative diseases. ER stress can lead to adaptive responses or apoptosis, both of which follow activation of the unfolded protein response (UPR). Heat shock proteins (HSP) support the folding and function of many proteins, and are important components of the ER stress response, but little is known about the role of one of the major large HSPs, HSP105. We identified several new partners of HSP105, including glycogen synthase kinase-3 (GSK3), a promoter of ER stress-induced apoptosis, and GRP78, a key component of the UPR. Knockdown of HSP105 did not alter UPR signaling after ER stress, but blocked caspase-3 activation after ER stress. In contrast, caspase-3 activation induced by genotoxic stress was unaffected by knockdown of HSP105, suggesting ER stress-specificity in the apoptotic action of HSP105. However, knockdown of HSP105 did not alter cell survival after ER stress, but instead diverted signaling to a caspase-3-independent cell death pathway, indicating that HSP105 is necessary for apoptotic signaling after UPR activation by ER stress. Thus, HSP105 appears to chaperone the responses to ER stress through its interactions with GRP78 and GSK3, and without HSP105 cell death following ER stress proceeds by a non-caspase-3-dependent process.     

Protective role of Gipie, a Girdin family protein, in endoplasmic reticulum stress responses in endothelial cells

Continued exposure of endothelial cells to mechanical/shear stress elicits the unfolded protein response (UPR), which enhances intracellular homeostasis and protect cells against the accumulation of improperly folded proteins. Cells commit to apoptosis when subjected to continuous and high endoplasmic reticulum (ER) stress unless homeostasis is maintained. It is unknown how endothelial cells differentially regulate the UPR. Here we show that a novel Girdin family protein, Gipie (78 kDa glucose-regulated protein [GRP78]-interacting protein induced by ER stress), is expressed in endothelial cells, where it interacts with GRP78, a master regulator of the UPR. Gipie stabilizes the interaction between GRP78 and the ER stress sensor inositol-requiring protein 1 (IRE1) at the ER, leading to the attenuation of IRE1-induced c-Jun N-terminal kinase (JNK) activation. Gipie expression is induced upon ER stress and suppresses the IRE1-JNK pathway and ER stress-induced apoptosis. Furthermore we found that Gipie expression is up-regulated in the neointima of carotid arteries after balloon injury in a rat model that is known to result in the induction of the UPR. Thus our data indicate that Gipie/GRP78 interaction controls the IRE1-JNK signaling pathway. That interaction appears to protect endothelial cells against ER stress-induced apoptosis in pathological contexts such as atherosclerosis and vascular endothelial dysfunction.     

Endoplasmic reticulum chaperone protein GRP78 protects cells from apoptosis induced by topoisomerase inhibitors: role of ATP binding site in suppression of caspase-7 activation

A large number of correlative studies have established that the activation of the unfolded protein response (UPR) alters the cell's sensitivity to chemotherapeutic agents. Although the induction of the glucose-regulated proteins (GRPs) is commonly used as an indicator for the UPR, the direct role of the GRPs in conferring resistance to DNA damaging agents has not been proven. We report here that without the use of endoplasmic reticulum (ER) stress inducers, specific overexpression of GRP78 results in reduced apoptosis and higher colony survival when challenged with topoisomerase II inhibitors, etoposide and doxorubicin, and topoisomerase I inhibitor, camptothecin. While investigating the mechanism for the GRP78 protective effect against etoposide-induced cell death, we discovered that in contrast to the UPR, GRP78 overexpression does not result in G1 arrest or depletion of topoisomerase II. Caspase-7, an executor caspase that is associated with the ER, is activated by etoposide. We show here that specific expression of GRP78 blocks caspase-7 activation by etoposide both in vivo and in vitro, and this effect can be reversed by addition of dATP in a cell-free system. Recently, it was reported that ectopically expressed GRP78 and caspases-7 and -12 form a complex, thus coupling ER stress to the cell death program. However, the mechanism of how GRP78, a presumably ER lumen protein, can regulate cytosolic effectors of apoptosis is not known. Here we provide evidence that a subpopulation of GRP78 can exist as an ER transmembrane protein, as well as co-localize with caspase-7, as confirmed by fluorescence microscopy. Co-immunoprecipitation studies further reveal endogenous GRP78 constitutively associates with procaspase-7 but not with procaspase-3. Lastly, a GRP78 mutant deleted of its ATP binding domain fails to bind procaspase-7 and loses its protective effect against etoposide-induced apoptosis.     

FAD-linked presenilin-1 mutants impede translation regulation under ER stress

FAD mutations in presenilin-1 (PS1) cause attenuation of the induction of the endoplasmic reticulum (ER)-resident chaperone GRP78/BiP under ER stress, due to disturbed function of IRE1, the sensor for accumulation of unfolded protein in the ER lumen. PERK, an ER-resident transmembrane protein kinase, is also a sensor for the unfolded protein response (UPR), causing phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit translation initiation. Here, we report that the FAD mutant PS1 disturbs the UPR by attenuating both the activation of PERK and the phosphorylation of eIF2alpha. Consistent with the results of a disturbed UPR, inhibition of protein synthesis under ER stress was impaired in cells expressing PS1 mutants. These results suggest that mutant PS1 impedes general translational attenuation regulated by PERK and eIF2alpha, resulting in an increased load of newly synthesized proteins into the ER and subsequently increasing vulnerability to ER stress.     

Lead-induced endoplasmic reticulum (ER) stress responses in the nervous system

Lead (Pb) poisoning continues to be a significant health risk because of its pervasiveness in the environment, its known neurotoxic effects in children, and potential endogenous exposure from Pb deposited in bone. New information about mechanisms by which Pb enters cells and its organelle targets within cells are briefly reviewed. Toxic effects of Pb on the endoplasmic reticulum (ER) are considered in detail, based on recent evidence that Pb induces the expression of the gene for 78-kD glucose-regulated protein (GRP78) and other ER stress genes. GRP78 is a molecular chaperone that binds transiently to proteins traversing through the ER and facilitates their folding, assembly, and transport. Models are presented for the induction of ER stress by Pb in astrocytes, the major cell type of the central nervous system, in which Pb accumulates. A key feature of the models is disruption of GRP78 function by direct Pb binding. Possible pathways by which Pb-bound GRP78 stimulates the unfolded protein response (UPR) in the ER are discussed, specifically transduction by IRE1/ATF6 and/or IRE1/JNK. The effect of Pb binding to GRP78 in the ER is expected to be a key component for understanding mechanisms of Pb-induced ER stress gene expression.     

MPTP-induced reactive oxygen species promote cell death through a gradual activation of caspase-3 without expression of GRP78/Bip as a preventive measure against ER stress in PC12 cells

Glucose-regulated protein 78 (GRP78)/Immunoglobulin binding protein (Bip) is a chaperone which functions to protect cells from endoplasmic reticulum (ER) stress. GRP78/Bip is expressed following ER stress induced by thapsigargin, tunicamycin or chemical factors. However, the mechanism of progression of ER stress against stress factors is still obscure. We examined whether reactive oxygen species (ROS) were involved in GRP78/Bip expression and caspase-3 activity was induced in PC12 cells using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to produce ROS. We report that PC12 cells lost viability in the presence of MPTP for 24 hours as a partial effect of ROS. We also show that N-acetyl-L-cysteine diminished the MPTP-induced apoptosis with expunction of ROS. Furthermore, we observed that GRP78/Bip was not up-regulated and the caspase-3 activity was increased in the presence of MPTP. These results suggest that insubstantial ROS do not contribute to the ER stress-mediated cell death while caspase-3 is involved in ROS-promoted cell death in MPTP-treated cells.