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.     

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.     

Anticancer compound Oplopantriol A kills cancer cells through inducing ER stress and BH3 proteins Bim and Noxa

Oplopantriol-A (OPT) is a natural polyyne from Oplopanax horridus. We show here that OPT preferentially kills cancer cells and inhibits tumor growth. We demonstrate that OPT-induced cancer cell death is mediated by excessive endoplasmic reticulum (ER) stress. Decreasing the level of ER stress either by inactivating components of the unfolded protein response (UPR) pathway or by expression of ER chaperone protein glucose-regulated protein 78 (GRP78) decreases OPT-induced cell death. We show that OPT induces the accumulation of ubiquitinated proteins and the stabilization of unstable proteins, suggesting that OPT functions, at least in part, through interfering with the ubiquitin/proteasome pathway. In support of this, inhibition of protein synthesis significantly decreased the accumulation of ubiquitinated proteins, which is correlated with significantly decreased OPT-induced ER stress and cell death. Finally, we show that OPT treatment significantly induced the expression of BH3-only proteins, Noxa and Bim. Knockdown of both Noxa and Bim significantly blocked OPT-induced cell death. Taken together, our results suggest that OPT is a potential new anticancer agent that induces cancer cell death through inducing ER stress and BH3 proteins Noxa and Bim.     

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.     

Inhibition of Akt potentiates 2-DG-induced apoptosis via downregulation of UPR in acute lymphoblastic leukemia

The ability to pair the regulation of metabolism and cellular energetics with oncogenes and tumor suppressor genes provides cancer cells with a growth and survival advantage over normal cells. We investigated the mechanism of cell death induced by 2-deoxy-D-glucose (2-DG), a sugar analog with dual activity of inhibiting glycolysis and N-linked glycosylation, in acute lymphoblastic leukemia (ALL). We found that, unlike most other cancer phenotypes in which 2-DG only inhibits cell proliferation under normoxic conditions, ALL lymphoblasts undergo apoptosis. Bp-ALL cell lines and primary cells exhibited sensitivity to 2-DG, whereas T-ALL cells were relatively resistant, revealing phenotypic differences within ALL subtypes. Cotreatment with D-mannose, a sugar essential for N-linked glycosylation, rescues 2-DG-treated ALL cells, indicating that inhibition of N-linked glycosylation and induction of ER stress and the unfolded protein response (UPR) is the predominant mechanism of 2-DG's cytotoxicity in ALL. 2-DG-treated ALL cells exhibit upregulation of P-AMPK, P-Akt, and induction of ER stress/UPR markers (IRE1α, GRP78, P-eIF2α, and CHOP), which correlate with PARP cleavage and apoptosis. In addition, we find that pharmacologic and genetic Akt inhibition upregulates P-AMPK, downregulates UPR, and sensitizes ALL cells to remarkably low doses of 2-DG (0.5 mmol/L), inducing 85% cell death and overcoming the relative resistance of T-ALL. In contrast, AMPK knockdown rescues ALL cells by upregulating the prosurvival UPR signaling. Therefore, 2-DG induces ALL cell death under normoxia by inducing ER stress, and AKT and AMPK, traditionally thought to operate predominantly on the glycolytic pathway, differentially regulate UPR activity to determine cell death or survival.     

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.     

Role of the unfolded protein response in cell death

Unfolded protein response (UPR) is an important genomic response to endoplasmic reticulum (ER) stress. The ER chaperones, GRP78 and Gadd153, play critical roles in cell survival or cell death as part of the UPR, which is regulated by three signaling pathways: PERK/ATF4, IRE1/XBP1 and ATF6. During the UPR, accumulated unfolded protein is either correctly refolded, or unsuccessfully refolded and degraded by the ubiquitin-proteasome pathway. When the unfolded protein exceeds a threshold, damaged cells are committed to cell death, which is mediated by ATF4 and ATF6, as well as activation of the JNK/AP-1/Gadd153-signaling pathway. Gadd153 suppresses activation of Bcl-2 and NF-κB. UPR-mediated cell survival or cell death is regulated by the balance of GRP78 and Gadd153 expression, which is coregulated by NF-κB in accordance with the magnitude of ER stress. Less susceptibility to cell death upon activation of the UPR may contribute to tumor progression and drug resistance of solid tumors.     

Endoplasmic reticulum stress induced by oxidative stress in decidual cells: a possible mechanism of early pregnancy loss

Early pregnancy loss (EPL) is one of the most common complications of human reproduction. Combined with our previous proteomic studies on villous and decidual tissues of EPL, we found that alterations of the proteins involved in oxidative stress (OS), unfolded protein response (UPR) and proteolysis presented a complex and dynamic interaction at the maternal-fetal interface. In the present study, we developed a cell model of OS using normal decidual cells to examine cell viability and expression levels of proteins related to endoplasmic reticulum stress (ER stress) and UPR. We found that glucose regulated protein 78 (GRP 78) and ubiquitinated proteins were significantly up-regulated in hydrogen peroxide (H(2)O(2)) treated decidual cells in a dose-dependent manner. Excessive OS could influence proper function of UPR by decreasing VCP in decidual cells, thereby leading to cell damage as well as inhibition of cell growth and activation of apoptosis. Furthermore, when pretreated with MG 132, a pharmacological inhibition of the proteasome, the H(2)O(2) treated decidual cells became less viable and could not up-regulate the expression level of GRP 78 to resolve the protein-folding defects, which indicating that malfunction of UPR in decidual cells might aggravate the inhibitory effect of OS in decidual cells. The present results reveal that abnormal protein profiles associated with OS induced ER stress and malfunction of UPR might be involved in the development of EPL, and OS and ER stress are potential targets for pregnant care and prognosis in normal pregnancy and its disorders.     

Phloroglucinol derivative MCPP induces cell apoptosis in human colon cancer

This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (3,6-bis(3-chlorophenylacetyl)phloroglucinol; MCPP) in human colon cancer cells. MCPP induced cell death and antiproliferation in three human colon cancer, HCT-116, SW480, and Caco-2 cells, but not in primary human dermal fibroblast cells. MCPP-induced concentration-dependent apoptotic cell death in colon cancer cells was measured by fluorescence-activated cell sorter (FACS) analysis. Treatment of HCT-116 human colon cancer cells with MCPP was found to induce a number of signature endoplasmic reticulum (ER) stress markers; and up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose-regulated protein (GRP)-78, phosphorylation of eukaryotic initiation factor-2α (eIF-2α), suggesting the induction of ER stress. MCPP also increased GSK3α/β(Tyr270/216) phosphorylation and reduced GSK3α/β(Ser21/9) phosphorylation time-dependently. Transfection of cells with GRP78 or CHOP siRNA, or treatment of GSK3 inhibitor SB216163 reduced MCPP-mediated cell apoptosis. Treatment of MCPP also increased caspase-7, caspase-9, and caspase-3 activity. The inhibition of caspase activity by z-DEVE-FMK or z-VAD-FMK significantly reduced MCPP-induced apoptosis. Furthermore, treatment of GSK3 inhibitor SB216763 also dramatically reversed MCPP-induced GRP and CHOP up-regulation, and pro-caspase-3 and pro-caspase-9 degradation. Taken together, the present study provides evidences to support that GRP78 and CHOP expression, and GSK3α/β activation in mediating the MCPP-induced human colon cancer cell apoptosis.     

Hyperoxia augments ER-stress-induced cell death independent of BiP loss

Cytotoxic reactive oxygen species are constantly formed as a by-product of aerobic respiration and are thought to contribute to aging and disease. Cells respond to oxidative stress by activating various pathways, whose balance is important for adaptation or induction of cell death. Our lab recently reported that BiP (GRP78), a proposed negative regulator of the unfolded protein response (UPR), declines during hyperoxia, a model of chronic oxidative stress. Here, we investigate whether exposure to hyperoxia, and consequent loss of BiP, activates the UPR or sensitizes cells to ER stress. Evidence is provided that hyperoxia does not activate the three ER stress receptors IRE1, PERK, and ATF6. Although hyperoxia alone did not activate the UPR, it sensitized cells to tunicamycin-induced cell death. Conversely, overexpression of BiP did not block hyperoxia-induced ROS production or increased sensitivity to tunicamycin. These findings demonstrate that hyperoxia and loss of BiP alone are insufficient to activate the UPR. However, hyperoxia can sensitize cells to toxicity from unfolded proteins, implying that chronic ROS, such as that seen throughout aging, could augment the UPR and, moreover, suggesting that the therapeutic use of hyperoxia may be detrimental for lung diseases associated with ER stress.     

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.     

Attenuation of unfolded protein response and apoptosis by mReg2 induced GRP78 in mouse insulinoma cells

Murine regenerating (mReg) genes have been implicated in preserving islet cell biology. Expanding on our previous work showing that overexpression of mReg2 protects MIN6 insulinoma cells against streptozotocin-induced apoptosis, we now demonstrate that mReg2 induces glucose-regulated peptide 78 (GRP78) expression via the Akt–mTORC1 axis and protects MIN6 cells against ER stress induced by thapsigargin and glucolipotoxicity. Activation of mTORC1 activity results from both mReg2-induced increased mTOR phosphorylation as well as increased expression of Raptor and GβL. Inhibition of Akt and mTORC1 blunted the ability of mReg2 to induce GRP78 and attenuate unfolded protein response (UPR). Knockdown of GRP78 sensitized the cells overexpressing mReg2 to UPR without affecting its ability to activate Akt–mTORC1 signaling. Induced expression of mReg2 may protect insulin producing cells from ER stress in diabetes.