Bip/GRP78-induced production of cytokines and uptake of amyloid-beta(1-42) peptide in microglia

In the brains of Alzheimer's disease (AD) patients, fibrillar amyloid-beta peptides (Abeta) are markedly accumulated and the microglia associate with the amyloid plaques. However, the regulation of Abeta clearance is still unclear. In the present study, we examined the effect of a chaperone protein BiP/GRP78 on the microglial function. Exogenous addition of recombinant BiP/GRP78 induced the production of cytokines such as interleukin-6 and tumor necrosis factor-alpha, but heat treatment of this protein abolished the activity. Although Abeta(1-42) did not induce cytokine production, it was taken up by the microglia. In addition, the amount of Abeta(1-42) uptake and the number of microglia that phagocytosed Abeta(1-42) were markedly increased by BiP/GRP78. Exogenous BiP/GRP78 also translocated to the endoplasmic reticulum (ER). These results suggest that BiP/GRP78 stimulates Abeta clearance in the microglia, and that dysfunction in the ER may cause the accumulation of extracellular Abeta(1-42).     

Endoplasmic reticulum chaperones inhibit the production of amyloid-beta peptides

Abeta (amyloid-beta peptides) generated by proteolysis of APP (beta-amyloid precursor protein), play an important role in the pathogenesis of AD (Alzheimer's disease). ER (endoplasmic reticulum) chaperones, such as GRP78 (glucose-regulated protein 78), make a major contribution to protein quality control in the ER. In the present study, we examined the effect of overexpression of various ER chaperones on the production of Abeta in cultured cells, which produce a mutant type of APP (APPsw). Overexpression of GRP78 or inhibition of its basal expression, decreased and increased respectively the level of Abeta40 and Abeta42 in conditioned medium. Co-expression of GRP78's co-chaperones ERdj3 or ERdj4 stimulated this inhibitory effect of GRP78. In the case of the other ER chaperones, overexpression of some (150 kDa oxygen-regulated protein and calnexin) but not others (GRP94 and calreticulin) suppressed the production of Abeta. These results indicate that certain ER chaperones are effective suppressors of Abeta production and that non-toxic inducers of ER chaperones may be therapeutically beneficial for AD treatment. GRP78 was co-immunoprecipitated with APP and overexpression of GRP78 inhibited the maturation of APP, suggesting that GRP78 binds directly to APP and inhibits its maturation, resulting in suppression of the proteolysis of APP. On the other hand, overproduction of APPsw or addition of synthetic Abeta42 caused up-regulation of the mRNA of various ER chaperones in cells. Furthermore, in the cortex and hippocampus of transgenic mice expressing APPsw, the mRNA of some ER chaperones was up-regulated in comparison with wild-type mice. We consider that this up-regulation is a cellular protective response against Abeta.     

Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP

Disturbance of endoplasmic reticulum (ER) homeostasis causes ER stress and leads to activation of the unfolded protein response, which reduces the stress and promotes cell survival at the early stage of stress, or triggers cell death and apoptosis when homeostasis is not restored under prolonged ER stress. Here, we report that Cab45S, a member of the CREC family, inhibits ER stress-induced apoptosis. Depletion of Cab45S increases inositol-requiring kinase 1 (IRE1) activity, thus producing more spliced forms of X-box-binding protein 1 mRNA at the early stage of stress and leads to phosphorylation of c-Jun N-terminal kinase, which finally induces apoptosis. Furthermore, we find that Cab45S specifically interacts with 78-kDa glucose-regulated protein/immunoglobulin heavy chain binding protein (GRP78/BiP) on its nucleotide-binding domain. Cab45S enhances GRP78/BiP protein level and stabilizes the interaction of GRP78/BiP with IRE1 to inhibit ER stress-induced IRE1 activation and apoptosis. Together, Cab45S, a novel regulator of GRP78/BiP, suppresses ER stress-induced IRE1 activation and apoptosis by binding to and elevating GRP78/BiP, and has a role in the inhibition of ER stress-induced apoptosis.     

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.     

The role of endoplasmic reticulum‐related BiP/GRP78 in interferon gamma‐induced persistent Chlamydia pneumoniae infection

Direct interaction of Chlamydiae with the endoplasmic reticulum (ER) is essential in intracellular productive infection. However, little is known about the interplay between Chlamydiae and the ER under cellular stress conditions that are observed in interferon gamma (IFN‐γ) induced chlamydial persistent infection. ER stress responses are centrally regulated by the unfolded protein response (UPR) under the control of the ER chaperone BiP/GRP78 to maintain cellular homeostasis. In this study, we could show that the ER directly contacted with productive and IFN‐γ‐induced persistent inclusions of Chlamydia pneumoniae (Cpn). BiP/GRP78 induction was observed in the early phase but not in the late phase of IFN‐γ‐induced persistent infection. Enhanced BiP/GRP78 expression in the early phase of IFN‐γ‐induced persistent Cpn infection was accompanied by phosphorylation of the eukaryotic initiation factor‐2α (eIF2α) and down‐regulation of the vesicle‐associated membrane protein‐associated protein B. Loss of BiP/GRP78 function resulted in enhanced phosphorylation of eIF2α and increased host cell apoptosis. In contrast, enhanced BiP/GRP78 expression in IFN‐γ‐induced persistent Cpn infection attenuated phosphorylation of eIF2α upon an exogenous ER stress inducer. In conclusion, ER‐related BiP/GRP78 plays a key role to restore cells from stress conditions that are observed in the early phase of IFN‐γ‐induced persistent infection.     

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.     

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.     

Intracellularly generated amyloid-beta peptide counteracts the antiapoptotic function of its precursor protein and primes proapoptotic pathways for activation by other insults in neuroblastoma cells

Most mutations in amyloid precursor proteins (APPs) linked to early onset familial Alzheimer's disease (FAD) increase the production of amyloid-beta peptides ending at residue 42 (Abeta42), which are released from APP by beta- and gamma-secretase cleavage. Stably transfected cells expressing wild-type human APP (APP(WT)) were more resistant to apoptosis-inducing treatments than cells expressing FAD-mutant human APP (APP(FAD)). Preventing Abeta42 production with an M596I mutation (beta-), which blocks beta-secretase cleavage of APP, or by treatment with a gamma-secretase inhibitor increased the resistance of APP(FAD)-expressing cells to apoptosis. Exposing hAPP(FAD/beta-) cells to exogenous Abeta42 or conditioned medium from Abeta42-producing APP(FAD) cells did not diminish their resistance to apoptosis. Preventing APP from entering the distal secretory pathway, where most Abeta peptides are generated, by retaining APP in the endoplasmic reticulum (ER)/intermediate compartment (IC) increased the resistance of APP(FAD)-expressing cells to apoptosis and did not alter the resistance of APP(WT)-expressing cells. p53-mediated gene transactivation after apoptosis-inducing treatments was much stronger in APP(FAD) cells than in hAPP(WT) or hAPP(FAD/beta-) cells. In contrast, upon induction of ER stress, cells expressing APP(FAD), hAPP(FAD/beta-), or APP(WT) had comparable levels of glucose-regulated protein-78 mRNA, an unfolded protein response indicator. We conclude that Abeta, especially intracellular Abeta, counteracts the antiapoptotic function of its precursor protein and predisposes cells to p53-mediated, and possibly other, proapoptotic pathways.     

Expression of beta-amyloid precursor protein-CD3gamma chimeras to demonstrate the selective generation of amyloid beta(1-40) and amyloid beta(1-42) peptides within secretory and endocytic compartments.

Amyloid beta-protein (Abeta) is the main constituent of amyloid fibrils found in senile plaques and cerebral vessels in Alzheimer's disease (AD) and is derived by proteolysis from the beta-amyloid precursor protein (APP). We have analyzed the amyloidogenic processing of APP using chimeric proteins stably transfected in Chinese hamster ovary cells. The extracellular and transmembrane domains of APP were fused to the cytoplasmic region derived from the CD3 gamma chain of the T cell antigen receptor (CD3gamma). CD3gamma contains an endoplasmic reticulum (ER) retention motif (RKK), in the absence of which the protein is targeted to lysosomes without going through the cell surface (Letourneur, F., and Klausner, R.D. (1992) Cell 69, 1143-1157). We used the wild-type sequence of CD3gamma to create an APP chimera predicted to remain in the ER (gammaAPP(ER)). Deletion of the RKK motif at the C terminus directed the protein directly to the lysosomes (gammaAPP(LYS)). A third chimera was created by removing both lysosomal targeting signals in addition to RKK (gammaAPP(DeltaDelta)). This last construct does not contain known targeting signals and consequently accumulates at the cell surface. We show by immunofluorescence and by biochemical methods that all three APP chimeras localize to the predicted compartments within the cell, thus providing a useful model to study the processing of APP. We found that Abeta(1-40) is generated in the early secretory and endocytic pathways, whereas Abeta(1-42) is made mainly in the secretory pathway. More importantly, we provide evidence that, unlike in neuronal models, both ER/intermediate compartment- and endocytic-derived Abeta forms can enter the secretable pool. Finally, we directly demonstrate that lysosomal processing is not involved in the generation or secretion of either Abeta(1-40) or Abeta(1-42).     

Aluminium exposure induces Alzheimer's disease-like histopathological alterations in mouse brain

Aluminium (Al) is a neurotoxic metal and Al exposure may be a factor in the aetiology of various neurodegenerative diseases such as Alzheimer's disease (AD). The major pathohistological findings in the AD brain are the presence of neuritic plaques containing beta-amyloid (Abeta) which may interfere with neuronal communication. Moreover, it has been observed that GRP78, a stress-response protein induced by conditions that adversely affect endoplasmic reticulum (ER) function, is reduced in the brain of AD patients. In this study, we investigated the correlation between the expression of Abeta and GRP78 in the brain cortex of mice chronically treated with aluminium sulphate. Chronic exposure over 12 months to aluminium sulphate in drinking water resulted in deposition of Abeta similar to that seen in congophilic amyloid angiopathy (CAA) in humans and a reduction in neuronal expression of GRP78 similar to what has previously been observed in Alzheimer's disease. So, we hypothesise that chronic Al administration is responsible for oxidative cell damage that interferes with ER functions inducing Abeta accumulation and neurodegenerative damage.     

Overexpressing GRP78 influences Ca2+ handling and function of mitochondria in astrocytes after ischemia-like stress

Ca(2+) transfer from endoplasmic reticulum (ER) to mitochondria at contact sites between the organelles can induce mitochondrial dysfunction and programmed cell death after stress. The ER-localized chaperone glucose-regulated protein 78kDa (GRP78/BiP) protects neurons against excitotoxicity and apoptosis. Here we show that overexpressing GRP78 protects astrocytes against ischemic injury, reduces net flux of Ca(2+) from ER to mitochondria, increases Ca(2+) uptake capacity in isolated mitochondria, reduces free radical production, and preserves respiratory activity and mitochondrial membrane potential after stress. We conclude that GRP78 influences ER-mitochondrial Ca(2+) crosstalk to maintain mitochondrial function and protect astrocytes from ischemic injury.     

Protective effect of catechin in type I Gaucher disease cells by reducing endoplasmic reticulum stress

Gaucher disease (GD) is the most common lysosomal storage disorder (LSD) and is divided into three phenotypes, I, II, and III. Type I is the most prevalent form and has its onset in adulthood. The degree of endoplasmic reticulum (ER) stress is one of the factors that determine GD severity. It has recently been reported that antioxidants reduce ER stress and apoptosis by scavenging the oxidants that cause oxidative stress. For this report, we investigated the possibility that catechin can act on type I GD patient cells to alleviate the pathogenic conditions of GD. We treated GD cells with catechin and examined the expression level of GRP78/BiP (an ER stress marker) by western blots and fluorescence microscopy, the proliferation rate of GD cells, and scratch-induced wound healing activity. Our results show that catechin reduces the expression level of GRP78/BiP, leads to cell proliferation rates of GD cells similar levels to normal cells, and improves wound healing activity. We conclude that catechin protects against ER stress in GD cells and catechin-mediated reductions in ER stress may be associated with enhanced cell survival.     

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+.     

Endoplasmic reticulum stress promotes amyloid-beta peptides production in RGC-5 cells

Endoplasmic reticulum (ER) stress has been implicated in various neurodegenerative diseases, including Alzheimer’s disease. We have previously observed amyloid production in the retina of the Tg2576 transgenic mouse model of Alzheimer’s disease. In this study, we used tunicamycin-induced ER stress in RGC-5 cells, a cell line identical to the photoreceptor cell line 661W, to investigate the effect of ER stress on production of amyloid-beta (Abeta) peptides. We found that the mRNA level of amyloid-beta precursor protein (APP) remained stable, while the protein level of amyloid-beta precursor protein (APP) was decreased, the amyloid-beta precursor protein cleaving enzymes beta-site APP-cleaving enzyme 1 and presenilin 1 were upregulated, Abeta_1–40 and Abeta_1–42 production were increased, and reactive oxygen species production and apoptosis markers were elevated following induction of ER stress. The protein level of Abeta degradation enzymes, neprilysin, endothelin-converting enzyme 1, and endothelin-converting enzyme 2 remained unchanged during the prolonged ER stress, showing that the generation of Abeta did not result from reduction of proteolysis by these enzymes. Inclusion of group II caspase inhibitor, Z-DEVD-FMK, increased the ER stress mediated Abeta production, suggesting that they are generated by a caspase-independent mechanism. Our findings provided evidence of a role of ER stress in Abeta peptide overproduction and apoptotic pathway activation in RGC-5 cells.     

Presenilin-1-mediated retention of APP derivatives in early biosynthetic compartments

Processing of the amyloid precursor protein (APP) leads to the production of amyloid-beta (Abeta), the major component of extracellular plaques in the brains of Alzheimer's disease (AD) patients. Presenilin-1 (PS-1) plays a key role in the final step of Abeta formation, the gamma-secretase cleavage. Previously, we showed that PS-1 is retained in pre-Golgi compartments by incorporation into COPI-coated membranes of the vesicular tubular clusters (VTCs) between endoplasmic reticulum (ER) and Golgi complex. Here, we show that PS-1 also mediates the retention of the beta-cleavage-derived APP-C-terminal fragment (CTFbeta) and/or Abeta in pre-Golgi membranes. Overexpression of PS-1 increased the percentage of CTFbeta and/or Abeta in VTCs as well as their distribution to COPI-coated VTC membranes. By contrast, overexpression of the dominant-negative aspartate mutant PS-1(D257A) or PS-knockout decreased incorporation of these APP derivatives into COPI-coated membranes. Sorting of APP derivatives to COPI-coated VTC membranes was not depending on the APP cytosolic tail. In post-Golgi compartments, PS-1 expression enhanced the association of full-length APP/APPs with endosomal compartments at the expense of plasma membrane-bound APP. We conclude that PS-1, in addition to its role in gamma-secretase cleavage, is also required for the subcellular routing of APP and its derivatives. Malfunctioning of PS-1 in this role may have important consequences for the progress of AD.     

A Peptidic Unconjugated GRP78/BiP Ligand Modulates the Unfolded Protein Response and Induces Prostate Cancer Cell Death

The molecular chaperone GRP78/BiP is a key regulator of protein folding in the endoplasmic reticulum, and it plays a pivotal role in cancer cell survival and chemoresistance. Inhibition of its function has therefore been an important strategy for inhibiting tumor cell growth in cancer therapy. Previous efforts to achieve this goal have used peptides that bind to GRP78/BiP conjugated to pro-drugs or cell-death-inducing sequences. Here, we describe a peptide that induces prostate tumor cell death without the need of any conjugating sequences. This peptide is a sequence derived from the cochaperone Bag-1. We have shown that this sequence interacts with and inhibits the refolding activity of GRP78/BiP. Furthermore, we have demonstrated that it modulates the unfolded protein response in ER stress resulting in PARP and caspase-4 cleavage. Prostate cancer cells stably expressing this peptide showed reduced growth and increased apoptosis in in vivo xenograft tumor models. Amino acid substitutions that destroyed binding of the Bag-1 peptide to GRP78/BiP or downregulation of the expression of GRP78 compromised the inhibitory effect of this peptide. This sequence therefore represents a candidate lead peptide for anti-tumor therapy.