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.     

Involvement of androgen receptor and glucose-regulated protein 78 kDa in human hepatocarcinogenesis

Previous studies demonstrated that androgen receptor (AR) is expressed in human hepatocellular carcinoma (HCC), one of the male-dominant diseases. Glucose-regulated protein 78 kDa (GRP78/Bip), which has a role in cancer development, is one of the androgen response genes in prostate cell lines. The aim of this study was to investigate the impact of AR on endoplasmic reticulum (ER)-stress signaling in human hepatoma. AR and GRP78 expressions were examined in human liver tissue panels. Human hepatoma cells stably expressing short hairpin RNA targeting AR and cells over-expressing AR were generated. The expressions of ER-stress molecules and AR were measured by real-time RT-PCR and Western blotting. The effect of AR on ER-stress responsive gene expression was examined by reporter assay. Strong positive correlation between AR mRNA and GRP78 mRNA was observed in stage I/II-HCCs. AR enhanced ER-stress responsive element activities and GRP78 expression, and regulated ER-stress response in hepatocytes. Sorafenib strongly induced significant apoptosis in HepG2 cells by the inhibition of AR and inhibition of the downstream GRP78. AR seems a co-regulator of GRP78 especially in earlier-stage HCC. AR plays a critical role in controlling ER-stress, providing new therapeutic options against HCC.     

Overexpression of GRP78 mitigates stress induction of glucose regulated proteins and blocks secretion of selective proteins in Chinese hamster ovary cells.

GRP78 is a resident protein of the endoplasmic reticulum (ER) and a member of the glucose regulated protein (GRP) family. Many secretion incompetent proteins are found in stable association with GRP78 and are retained in the ER. Some proteins which are destined for secretion transiently associate with GRP78. To further increase our understanding of the role of GRP78 in secretion, we have stably overexpressed GRP78 in Chinese hamster ovary (CHO) cells and examined the effect on protein secretion and the stress response. GRP78 overexpressing cells treated with tunicamycin or A23187 exhibited a reduced induction of endogenous GRP78 and GRP94 mRNAs compared to wild-type CHO cells. This suggests that GRP78 overexpression either alleviates the stress or is directly involved in signaling stress-induced expression of GRPs. Transient expression of secreted proteins was used to measure secretion efficiency in the GRP78 overexpressing cells. Secretion of von Willebrand factor and a mutant form of factor VIII, two proteins which transiently associate with GRP78, was reduced by GRP78 overexpression. In contrast, secretion of M-CSF, which was not detected in association with GRP78, was unaffected. This indicates that elevated levels of GRP78 may increase stable association and decrease the secretion efficiency of proteins which normally transiently associate with GRP78. These results indicate that one function of GRP78 is selective protein retention in the ER.     

GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis

The chaperone glucose-regulated protein, 78/immunoglobulin binding protein (GRP78/Bip), protects cells from cytotoxicity induced by DNA damage or endoplasmic reticulum (ER) stress. In this study, we showed that GRP78 is a major inducible protein in human non-small cell lung cancer H460 cells treated with ER stress inducers, including A23187 and thapsigargin. AEBSF, an inhibitor of serine protease, diminished GRP78 induction, enhanced mitochondrial permeability, and augmented apoptosis in H460 cells during ER stress. Simultaneously, AEBSF promoted Raf-1 degradation and suppressed phosphorylation of Raf-1 at Ser338 and/or Tyr340 during ER stress. Coimmunoprecipitation assays and subcellular fractionations showed that GRP78 associated and colocalized with Raf-1 on the outer membrane of mitochondria, respectively. While treatment of cells with ER stress inducers inactivated BAD by phosphorylation at Ser75, a Raf-1 phosphorylation site; AEBSF attenuated phosphorylation of BAD, leading to cytochrome c release from mitochondria. Additionally, overexpression of GRP78 and/or Raf-1 protected cells from ER stress-induced apoptosis. Taken together, our results indicate that GRP78 may stabilize Raf-1 to maintain mitochondrial permeability and thus protect cells from ER stress-induced apoptosis.     

Lysozyme Mutants Accumulate in Cells while Associated at their N-terminal Alpha-domain with the Endoplasmic Reticulum Chaperone GRP78/BiP

Amyloidogenic human lysozyme variants deposit in cells and cause systemic amyloidosis. We recently observed that such lysozymes accumulate in the endoplasmic reticulum (ER) with the ER chaperone GRP78/BiP, accompanying the ER stress response. Here we investigated the region of lysozyme that is critical to its association with GRP78/BiP. In addition to the above-mentioned variants of lysozyme, we constructed lysozyme truncation or substitution mutants. These were co-expressed with GRP78/BiP (tagged with FLAG) in cultured human embryonic kidney cells, which were analyzed by western blotting and immunocytochemistry using anti-lysozyme and anti-FLAG antibodies. The amyloidogenic variants were confirmed to be strongly associated with GRP78/BiP as revealed by the co-immunoprecipitation assay, whereas N-terminal mutants pruned of 1-41 or 1-51 residues were found not to be associated with the chaperone. Single amino acid substitutions for the leucine array along the α-helices in the N-terminal region resulted in wild-type lysozyme remaining attached to GRP78/BiP. These mutations also tended to show lowered secretion ability. We conclude that the N-terminal α-helices region of the lysozyme is pivotal for its strong adhesion to GRP78/BiP. We suspect that wild-type lysozyme interacts with the GRP at this region as a step in the proper folding monitored by the ER chaperone.     

Overexpression of the 78-kDa glucose-regulated protein/immunoglobulin-binding protein (GRP78/BiP) inhibits tissue factor procoagulant activity

Previous studies have demonstrated that overexpression of GRP78/BiP, an endoplasmic reticulum (ER)-resident molecular chaperone, in mammalian cells inhibits the secretion of specific coagulation factors. However, the effects of GRP78/BiP on activation of the coagulation cascade leading to thrombin generation are not known. In this study, we examined whether GRP78/BiP overexpression mediates cell surface thrombin generation in a human bladder cancer cell line T24/83 having prothrombotic characteristics. We report here that cells overexpressing GRP78/BiP exhibited significant decreases in cell surface-mediated thrombin generation, prothrombin consumption and the formation of thrombin-inhibitor complexes, compared with wild-type or vector-transfected cells. This effect was attributed to the ability of GRP78/BiP to inhibit cell surface tissue factor (TF) procoagulant activity (PCA) because conversion of factor X to Xa and factor VII to VIIa were significantly lower on the surface of GRP78/BiP-overexpressing cells. The additional findings that (i) cell surface factor Xa generation was inhibited in the absence of factor VIIa and (ii) TF PCA was inhibited by a neutralizing antibody to human TF suggests that thrombin generation is mediated exclusively by TF. GRP78/BiP overexpression did not decrease cell surface levels of TF, suggesting that the inhibition in TF PCA does not result from retention of TF in the ER by GRP78/BiP. The additional observations that both adenovirus-mediated and stable GRP78/BiP overexpression attenuated TF PCA stimulated by ionomycin or hydrogen peroxide suggest that GRP78/BiP indirectly alters TF PCA through a mechanism involving cellular Ca(2+) and/or oxidative stress. Similar results were also observed in human aortic smooth muscle cells transfected with the GRP78/BiP adenovirus. Taken together, these findings demonstrate that overexpression of GRP78/BiP decreases thrombin generation by inhibiting cell surface TF PCA, thereby suppressing the prothrombotic potential of cells.     

Thapsigargin down-regulates protein levels of GRP78/BiP in INS-1E cells

Pancreatic β-cells have a well-developed endoplasmic reticulum (ER) and express large amounts of chaperones and protein disulfide isomerases (PDI) to meet the high demand for synthesis of proteins. We have observed an unexpected decrease in chaperone protein level in the β-cell model INS-1E after exposure to the ER stress inducing agent thapsigargin. As these cells are a commonly used model for primary β-cells and has been shown to be vulnerable to ER stress, we hypothesize these cells are incapable of mounting a chaperone defense upon activation of ER stress. To investigate the chaperone expression during an ER stress response, induced by thapsigargin in INS-1E cells, we used quantitative mass spectrometry based proteomics. The results displayed a decrease of GRP78/BiP, PDIA3 and PDIA6. Decrease of GRP78/BiP was verified by Western blot and occurred in parallel with enhanced levels of p-eIF2α and CHOP. In contrast to INS-1E cells, GRP78/BiP was not decreased in MIN6 cell or rat and mouse islets after thapsigargin exposure. Investigation of the decreased protein levels of GRP78/BiP indicates that this is not a consequence of reduced mRNA expression. Rather the reduction results from the combined effect of reduced protein synthesis and enhanced proteosomal degradation and possibly also degradation via autophagy. Induction of ER stress with thapsigargin leads to lower protein levels of GRP78/BiP, PDIA3 and PDIA6 in INS-1E cells which may contribute to the susceptibility of ER stress in this β-cell model.     

Glucose regulated protein 78: A critical link between tumor microenvironment and cancer hallmarks

Glucose regulated protein 78 (GRP78) has long been recognized as a molecular chaperone in the endoplasmic reticulum (ER) and can be induced by the ER stress response. Besides its location in the ER, GRP78 has been found to be present in cell plasma membrane, cytoplasm, mitochondria, nucleus as well as cellular secretions. GRP78 is implicated in tumor cell proliferation, apoptosis resistance, immune escape, metastasis and angiogenesis, and its elevated expression usually correlates with a variety of tumor microenvironmental stresses, including hypoxia, glucose deprivation, lactic acidosis and inflammatory response. GRP78 protein acts as a centrally located sensor of stress, which feels and adapts to the alteration in the tumor microenvironment. This article reviews the potential contributions of GRP78 to the acquisition of cancer hallmarks based on intervening in stress responses caused by tumor niche alterations. The paper also introduces several potential GRP78 relevant targeted therapies.     

Betanodavirus up-regulates chaperone GRP78 via ER stress: roles of GRP78 in viral replication and host mitochondria-mediated cell death

Whether viral pathogens that induce ER stress responses benefit the host or the virus remains controversial. In this study we show that betanodavirus induced ER stress responses up-regulate GRP78, which regulates the viral replication and host cellular mitochondrial-mediated cell death. Betanodavirus (redspotted grouper nervous necrosis virus, RGNNV) infection resulted in the following increased ER stress responses in fish GF-1 grouper fin cells: (1) IRE-1 and ATF-6 sensors at 48 h post-infection (p.i.) that up-regulated chaperone protein GRP78; (2) activation of caspase-12; and (3) PERK phosphorylation and down-regulation of Bcl-2. Analyses of GRP78 functions during viral replication using either loss-of-function or gain-of-function approaches showed that GRP78 over-expression also enhanced viral replication and induced cell death. Then, we found that zfGRP78 localization gradually increased in mitochondria after RGNNV infection by EGFP tagging approach. Furthermore, zfGRP78 can interact with viral RNA-dependent RNA polymerase (RdRp) by using immunofluorescent and immunoprecipitation assays. Finally, we found that blocking GRP78-mediated ER signals can reduce the viral death factors protein α and protein B2 expression and decrease the Bcl-2 down-regulation mediated mitochondria-dependent cell death, which also enhances host cellular viability. Taken together, our results suggest that RGNNV infection and expression can trigger ER stress responses, which up-regulate the chaperone GRP78 at early replication stage. Then, GRP78 can interact with RdRp that may enhance the viral replication for increasing viral death factors’ expressions at middle-late replication stage, which can enhance mitochondrial-mediated cell death pathway and viral spreading. These results may provide new insights into the mechanism of ER stress-mediated cell death in RNA viruses.     

ATM blocks tunicamycin-induced endoplasmic reticulum stress

Endoplasmic reticulum stress (ER-stress) is associated with ataxia telangiectasia mutated (ATM) gene. We present here conclusive data showing that ATM blocks ER-stress induced by tunicamycin or ionizing radiation (IR). X-box protein-1 (XBP-1) splicing, GRP78 expression and caspase-12 activation were increased by tunicamycin or IR in Atm-deficient AT5BIVA fibroblasts. Activation of caspase-12 and caspase-3 by tunicamycin was significantly reduced in cells transfected with wild-type Atm (AT5BIVA/wtATM). Atm knockdown by siRNA, however, noticeably elevated ER-stress and chemosensitivity to tunicamycin. In summary, we present substantial data demonstrating that ATM blocks the ER stress signaling associated with cancer cell proliferation.     

The interaction of S100A16 and GRP78 actives endoplasmic reticulum stress-mediated through the IRE1α/XBP1 pathway in renal tubulointerstitial fibrosis

Recent studies have indicated that the development of acute and chronic kidney disease including renal fibrosis is associated with endoplasmic reticulum (ER) stress. S100 calcium-binding protein 16 (S100A16) as a novel member of the S100 family is involved in kidney disease; however, few studies have examined fibrotic kidneys for a relationship between S100A16 and ER stress. In our previous study, we identified GRP78 as a protein partner of S100A16 in HK-2 cells. Here, we confirmed a physical interaction between GRP78 and S100A16 in HK-2 cells and a markedly increased expression of GRP78 in the kidneys of unilateral ureteral occlusion mice. S100A16 overexpression in HK-2 cells by infection with Lenti-S100A16 also induced upregulation of ER stress markers, including GRP78, p-IRE1α, and XBP1s. Immunofluorescence staining demonstrated that the interaction between S100A16 and GRP78 predominantly occurred in the ER of control HK-2 cells. By contrast, HK-2 cells overexpressing S100A16 showed colocalization of S100A16 and GRP78 mainly in the cytoplasm. Pretreatment with BAPTA-AM, a calcium chelator, blunted the upregulation of renal fibrosis genes and ER stress markers induced by S100A16 overexpression in HK-2 cells and suppressed the cytoplasmic colocalization of GRP78 and S100A16. Co-immunoprecipitation studies suggested a competitive binding between S100A16 and IRE1α with GRP78 in HK-2 cells. Taken together, our findings demonstrate a significant increase in S100A16 expression in the cytoplasm following renal injury. GRP78 then moves into the cytoplasm and binds with S100A16 to promote the release of IRE1α. The subsequent phosphorylation of IRE1α then leads to XBP1 splicing that activates ER stress.Subject terms: Stress signalling, Experimental models of disease     

Coupling endoplasmic reticulum stress to the cell death program: role of the ER chaperone GRP78

Alterations in Ca(2+) homeostasis and accumulation of unfolded proteins in the endoplasmic reticulum (ER) lead to an ER stress response. Prolonged ER stress may lead to cell death. Glucose-regulated protein (GRP) 78 (Bip) is an ER lumen protein whose expression is induced during ER stress. GRP78 is involved in polypeptide translocation across the ER membrane, and also acts as an apoptotic regulator by protecting the host cell against ER stress-induced cell death, although the mechanism by which GRP78 exerts its cytoprotective effect is not understood. The present study was carried out to determine whether one of the mechanisms of cell death inhibition by GRP78 involves inhibition of caspase activation. Our studies indicate that treatment of cells with ER stress inducers causes GRP78 to redistribute from the ER lumen with subpopulations existing in the cytosol and as an ER transmembrane protein. GRP78 inhibits cytochrome c-mediated caspase activation in a cell-free system, and expression of GRP78 blocks both caspase activation and caspase-mediated cell death. GRP78 forms a complex with caspase-7 and -12 and prevents release of caspase-12 from the ER. Addition of (d)ATP dissociates this complex and may facilitate movement of caspase-12 into the cytoplasm to set in motion the cytosolic component of the ER stress-induced apoptotic cascade. These results define a novel protective role for GRP78 in preventing ER stress-induced cell death.     

Identification of GRP78 as a molecular target of medicarpin in osteoblast cells by proteomics

Osteogenic activity was identified in medicarpin (Med), a natural pterocarpan. Further, it was decided to study the differentially regulated protein expression during osteoblast differentiation in the presence of Med. Using 2D proteomic approach, we found that Med treatment to osteoblasts significantly downregulated GRP78, an ER chaperone with anti-apoptotic properties which also controls the activation of unfolded protein response signaling, a pro-survival strategy for normal ER functioning. However, severe stress leads to triggering of apoptotic responses and signaling switches to pro-apoptotic. In order to elucidate the effect of Med downregulation of GRP78, osteoblasts were transfected with SiGRP78 or SiGRP78+ Med or Med alone. It was seen that mRNA and protein levels of ER stress markers like GRP78, ATF-4, and CHOP were decreased in all the three groups with maximum reduction in SiGRP78+ Med group. Med targets GRP78 by inhibiting mitochondrial-mediated apoptosis which is evident by reduced levels of cytochrome c, caspase-3, Bax/BCL2 ratio, and enhanced expression of survivin. Finally, Annexin-PI staining of apoptotic cells revealed that MED inhibition of GRP78 leads to reduced osteoblast apoptosis and increased osteoblast survival. Altogether, our data show that Med inhibits ER stress-induced apoptosis and promotes osteoblast cell survival by targeting GRP78.

     

Decreased protein and mRNA expression of ER stress proteins GRP78 and GRP94 in HepG2 cells over-expressing CYP2E1

CYP2E1 causes oxidative stress mediated cell death; the latter is one mechanism for endoplasmic reticulum (ER) stress in the cell. Unfolded proteins accumulate during ER stress and ER resident proteins GRP78 and GRP94 protect cells against ER dysfunction. We examined the possible role of GRP78 and GRP94 as protective factors against CYP2E1-mediated toxicity in HepG2 cells expressing CYP2E1 (E47 cells). E47 cells expressed high levels of CYP2E1 protein and catalytic activity which is associated with increased ROS generation, lipid peroxidation and the elevated presence of ubiquinated and aggregated proteins as compared to control HepG2 C34 cells which do not express CYP2E1. The mRNA and protein expression of GRP78 and GRP94 were decreased in E47 cells compared to the C34 cells, which may explain the accumulation of ubiquinated and aggregated proteins. Expression of these GRP proteins was induced with the ER stress agent thapsigargin in E47 cells, and E47 cells were more resistant to the toxicity caused by thapsigargin and calcimycin, possibly due to this upregulation and also because of the high expression of GSH and antioxidant enzymes in E47 cells. Antioxidants such as trolox and N-acetylcysteine increased GRP78 and GRP94 levels in the E47 cells, suggesting that CYP2E1- derived oxidant stress was responsible for down regulation of these GRPs in the E47 cells. Thapsigargin mediated toxicity was decreased in cells treated with the antioxidant trolox indicating a role for oxidative stress in this toxicity. These results suggest that CYP2E1 mediated oxidative stress downregulates the expression of GRP proteins in HepG2 cells and oxidative stress is an important mechanism in causing ER dysfunction in these cells.