Overexpression of GRP78 protects glial cells from endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress induces apoptotic cell death by causing the accumulation of structurally abnormal proteins. The 78-kDa glucose-regulated protein (GRP78) is an ER chaperone that regulates protein folding in the ER and has been suggested to contribute to cell survival. Using the rat C6 glioma cell line and flow cytometry, we assessed GRP78 expression following tunicamycin- and glutamate-induced ER stress. The results showed that GRP78 expression is upregulated following ER stress and has protective effects on injured glial cells. Annexin V and propidium iodide labeling revealed cells transiently expressing GRP78 prior to injury were protected against high-concentrations of tunicamycin and glutamate within 72 h. Our findings support the hypothesis that GRP78 inhibits cell death associated with ER stress.     

GRP78 expression beyond cellular stress: A biomarker for tumor manipulation

Physiological stress takes place in the endoplasmic reticulum (ER) of cells where activation and up-regulation of genes and proteins are primarily induced to enhance pro-survival mechanisms such as the unfolded protein response (UPR). A dominant protein in the UPR response is the heat shock GRP78 protein. Although GRP78 is primarily located in the ER, under certain conditions it is transported to the cell surface, where it acts as a receptor inducing pathways of cell signaling such as proliferation or apoptosis. In the prolonged chronic stress transportation of the GRP78 from the ER to the cell membrane is a major event where in addition to the presentation of the GRP78 as a receptor to various ligands, it also marks the cells that will proceed to apoptotic pathways. In the normal cell that under stress acquires cell surface GRP78 and in the tumor cell that already presents cell surface GRP78, cell surface GRP78 is an apoptotic flag. The internalization of GRP78 from the cell surface in normal cells by ligands such as peptides will enhance cell survival and alleviate cardiovascular ischemic diseases. The absence of cell surface GRP78 in the tumor cells portends proliferative and metastatic tumors. Pharmacological induction of cell surface GRP78 will induce the process of apoptosis and might be used as a therapeutic modality for cancer treatment.     

Expression of autophagy and ER stress-related proteins in primary salivary adenoid cystic carcinoma

Autophagy is the endogenous cellular pathway that facilitates cellular survival by maintaining energy homeostasis and macromolecular synthesis during cellular stress and nutrient deprivation. Endoplasmic reticulum (ER) stress is the process in which disruption of these physiological functions leads to an accumulation of unfolded proteins and induces the unfolded protein response (UPR). ER stress and autophagy are involved in human cancer. We investigated the expression of autophagic proteins (LC3 and beclin 1) and ER stress-related protein (GRP78) in head and neck adenoid cystic carcinoma tissue. Tissue samples from 79 cases of head and neck adenoid cystic carcinoma tissue were utilized for immunohistochemistry. LC3 expression was significantly correlated with lymph node involvement (P = .016) and TNM (P = .021). Beclin 1 expression was significantly correlated with the histological growth pattern (P = .002), the histological grade (P = .000), and longer survival (P = .000). GRP78 expression was significantly correlated with the histological growth pattern (P = .019), the histological grade (P = .019), and longer survival (P = .001). LC3 expression was positively correlated with beclin 1 expression (P = .000); LC3 and beclin 1 expressions were positively correlated with GRP78 expression respectively (P = .035) (P = .008). Our study describes the expression of LC3, beclin 1, and GRP78 in adenoid cystic carcinoma and its relationship with clinicopathologic factors and overall survival. These results suggest that LC3, beclin 1, and GRP78 may play an important role in the tumorigenesis of adenoid cystic carcinoma, and that beclin 1 and GRP78 may serve as new prognostic indicators for the outcome of patients with adenoid cystic carcinoma.     

Involvement of endoplasmic reticulum stress in myofibroblastic differentiation of lung fibroblasts

Stress that impairs endoplasmic reticulum (ER) function leads to an accumulation of unfolded or misfolded proteins in the ER (ER stress) and triggers the unfolded protein response (UPR). Recent studies suggest that ER stress is involved in idiopathic pulmonary fibrosis (IPF). The present study was undertaken to determine the role of ER stress on myofibroblastic differentiation of fibroblasts. Fibroblasts in fibroblastic foci of IPF showed immunoreactivity for GRP78. To determine the role of ER stress on α-smooth muscle actin (α-SMA) and collagen type I expression in fibroblasts, mouse and human lung fibroblasts were treated with TGF-β1, and expression of ER stress-related proteins, α-SMA, and collagen type I was analyzed by Western blotting. TGF-β1 significantly increased expression of GRP78, XBP-1, and ATF6α, which was accompanied by increases in α-SMA and collagen type I expression in mouse and human fibroblasts. A chemical chaperone, 4-PBA, suppressed TGF-β1-induced UPR and α-SMA and collagen type I induction. We also showed that TGF-β1-induced UPR was mediated through the reactive oxygen species generation. Our study provides the first evidence implicating the UPR in myofibroblastic differentiation during fibrosis. These findings of the role of ER stress and chemical chaperones in pulmonary fibrosis may improve our understanding of the pathogenesis of IPF.     

Endoplasmic reticulum stress and unfolded protein response in inclusion body myositis muscle

Proteins in the endoplasmic reticulum (ER) require an efficient system of molecular chaperones whose role is to assure their proper folding and to prevent accumulation of unfolded proteins. The response of cells to accumulation of unfolded proteins in the ER is termed "unfolded protein response" (UPR). UPR is a functional mechanism by which cells attempt to protect themselves against ER stress, resulting from the accumulation of the unfolded/misfolded proteins. Because intracellular inclusions, containing either amyloid-beta (Abeta) or phosphorylated tau, are the characteristic feature of sporadic inclusion body myositis (s-IBM) muscle biopsies, we studied expression and immunolocalization of five ER chaperones, calnexin, calreticulin, GRP94, BiP/GRP78, and ERp72, in s-IBM and control muscle biopsies. Physical interaction of the ER chaperones with amyloid-beta precursor protein (AbetaPP) was studied by a combined immunoprecipitation/immunoblotting technique in s-IBM and control muscle biopsies, and in AbetaPP-overexpressing cultured human muscle fibers. In all s-IBM muscle biopsies, all five of the ER chaperones were immunodetected in the form of inclusions that co-localized with amyloid-beta. By immunoblotting, expression of ER chaperones was greatly increased as compared to the controls. By immunoprecipitation/immunoblotting experiments, ER chaperones co-immunoprecipitated with AbetaPP. Our studies provide evidence of the UPR in s-IBM muscle and demonstrate for the first time that the ER chaperones calnexin, calreticulin, GRP94, BiP/GRP78, and ERp72 physically associate with AbetaPP in s-IBM muscle, suggesting their playing a role in AbetaPP folding and processing.     

PKR-like endoplasmic reticulum kinase (PERK) activation following brain ischemia is independent of unfolded nascent proteins

Transient global brain ischemia results in an immediate inhibition of protein translation upon reperfusion. During early brain reperfusion protein synthesis is inhibited by alpha subunit of eukaryotic initiation factor 2 (eIF2α) phosphorylation by the PKR-like endoplasmic reticulum kinase (PERK). Normally, PERK is held in an inactive, monomeric state by the binding of the endoplasmic reticulum (ER) chaperone GRP78 to the lumenal end of PERK. The prevailing view is that ER stress leads to the accumulation of unfolded proteins in the ER lumen. GRP78 dissociates from PERK to bind these accumulated unfolded proteins, leading to PERK activation, phosphorylation of eIF2α, and inhibition of translation. To determine if an increase in unfolded nascent proteins following transient brain ischemia contributes to PERK activation, protein synthesis was blocked by intracerebral injection of anisomycin prior to induction of ischemia. Anisomycin inhibited protein synthesis by over 99% and reduced newly synthesized proteins in the ER to ∼20% of controls. With an ER nearly devoid of newly synthesized proteins, PERK was still activated and was able to phosphorylate eIF2α in CA1 neurons during reperfusion. These data strongly argue that PERK activation is independent of the large increase in unfolded nascent proteins within the ER following transient global brain ischemia.     

The novel white spot syndrome virus-induced gene,PmERP15, encodes an ER stress-responsive protein in black tiger shrimp,Penaeus monodon

By microarray screening, we identified a white spot syndrome virus (WSSV)-strongly induced novel gene in gills of Penaeus monodon. The gene, PmERP15, encodes a putative transmembrane protein of 15 kDa, which only showed some degree of similarity (54–59%) to several unknown insect proteins, but had no hits to shrimp proteins. RT-PCR showed that PmERP15 was highly expressed in the hemocytes, heart and lymphoid organs, and that WSSV-induced strong expression of PmERP15 was evident in all tissues examined. Western blot analysis likewise showed that WSSV strongly up-regulated PmERP15 protein levels. In WSSV-infected hemocytes, immunofluorescence staining showed that PmERP15 protein was colocalized with an ER enzyme, protein disulfide isomerase, and in Sf9 insect cells, PmERP15-EGFP fusion protein colocalized with ER -Tracker™ Red dye as well. GRP78, an ER stress marker, was found to be up-regulated in WSSV-infected P. monodon, and both PmERP15 and GRP78 were up-regulated in shrimp injected with ER stress inducers tunicamycin and dithiothreitol. Silencing experiments showed that although PmERP15 dsRNA-injected shrimp succumbed to WSSV infection more rapidly, the WSSV copy number had no significant changes. These results suggest that PmERP15 is an ER stress-induced, ER resident protein, and its induction in WSSV-infected shrimp is caused by the ER stress triggered by WSSV infection. Furthermore, although PmERP15 has no role in WSSV multiplication, its presence is essential for the survival of WSSV-infected shrimp.     

The functional role of stress proteins in ER stress mediated cell death

The endoplasmic reticulum (ER) is an intracellular organelle involved in biosynthesis and the secretory pathway. This organelle has many resident proteins including biosynthetic enzymes and secretory proteins. Recent studies have suggested that dysfunction of the ER or secretory pathway is involved in the pathogenesis of various human diseases. Some stresses acting on the ER, which are designated ER stress, induce the accumulation of unfolded/misfolded proteins in the ER, leading to cell death. Misfolded proteins are retained until they form their native conformation or returned to the cytosol for degradation by the proteasome. Among the ER-resident proteins, molecular chaperones prevent aggregation of proteins within the ER, and orchestrate the ER quality control systems. We have reported the roles of novel stress proteins, namely 150-kDa oxygen-regulated protein, 94-kDa glucose-regulated protein and RA410. These proteins are induced significantly by hypoxia or oxidative stress and have cytoprotective effects under these conditions. These findings suggest that hypoxia and oxidative stress target the ER and secretory pathway, resulting in ER stress, and that these proteins exert cytoprotective effects in various diseases associated with ER stress.     

Tyrosine Phosphorylated Proteins in Synovial Cells of Rheumatoid Arthritis

Two of the key events in the pathogenesis of rheumatoid arthritis are the synovial cell proliferation and lymphocyte infiltration into the synovium. The resulting synovitis is longlasting and leads to destructive arthritis, which is a hallmark of rheumatoid arthritis. Accumulating evidence suggests that one of the key biochemical events in the altered cell function of RA is phosphorylation of the tyrosine residues of proteins.

In this paper we review the cellular components participating in the chronic inflammation of RA joints. We present the results of analyzing tyrosine phosphorylated proteins of synovial cells from RA patients and discuss a possible pathogenic role of non-receptor tyrosine kinase in RA.     

Role of endoplasmic reticulum calcium disequilibria in the mechanism of homocysteine-induced ER stress

Our laboratory demonstrated that hyperhomocysteinemia accelerates atherosclerosis in mouse models through ER stress and activation of the unfolded protein response (UPR). In this study, we tested the hypothesis that homocysteine-induced ER stress may arise from ER-Ca(2+) disequilibria. We found that homocysteine-induced cytosolic Ca(2+) transients in T24/83 cells and human aortic smooth muscle cells (HASMCs). These calcium effects occurred at concentrations of homocysteine in the external medium (1-5 mM) that increase intracellular homocysteine in these cell types. Prolonged homocysteine treatment (5 h) at these exogenous concentrations reduced ER-Ca(2+) emptying evoked by thapsigargin. However, these homocysteine-induced effects on ER-Ca(2+) emptying were of a much smaller magnitude than those evoked by A23187 or thapsigargin (ER stressors known to induce ER stress through ER-Ca(2+) depletion). T24/83 cells stably overexpressing the Ca(2+)-binding ER chaperone GRP78 showed diminished cytosolic Ca(2+) transients induced by homocysteine and reduced ER-Ca(2+) emptying evoked by thapsigargin. Prevention of the homocysteine-induced UPR by cycloheximide pretreatment normalized GRP78 expression and ER-Ca(2+) emptying evoked by thapsigargin. These results are inconsistent with a mechanism of ER stress induction by homocysteine through ER-Ca(2+) depletion.     

Expression of GRP78 predicts taxane-based therapeutic resistance and recurrence of human gastric cancer

Cancer cells adapt to chronic stress in the tumor microenvironment by inducing the expression of glucose-regulated protein 78 (GRP78), a major endoplasmic reticulum chaperone with Ca^2 +-binding and antiapoptotic properties. The effect in and potential role of its expression in progression of and prognosis for gastric cancer (GC) are unclear. In the present study, we investigated the clinical value of GRP78 expression in judgment of the severity of and prognosis for GC in a retrospective cohort study of 160 patients who underwent D2 radical gastrectomy and adjuvant chemotherapy. GRP78 expression was detected using immunohistochemistry. The relationships of GRP78 expression with age, sex, differentiation, invasion depth, disease stage, lymph node metastasis, and time to recurrence (TTR) were analyzed. The GRP78 expression was higher in tumors from patients with deep tumor infiltration, with poor differentiation, at late disease stages, and with lymph node metastasis than that in tumors from patients without. Also, GRP78 positivity was associated with short TTR (hazard ratio [HR], 1.75; 95% confidence interval [CI], 1.07–4.85; P = 0.041). Subgroup analysis revealed that the HR in the GRP78-high group increased significantly in patients who did not receive taxane-containing regimens (HR, 2.21; 95% CI, 1.23–7.36; P = 0.038). In contrast, in the patients who received taxane-based chemotherapy, the association between GRP78 positivity and increased risk of recurrence was not statistically significant (HR, 1.16; 95% CI, 0.81–2.98; P = 0.111). In the patients with GRP78 expression, those who underwent taxane-containing chemotherapy had longer median TTRs than did those who did not undergo this treatment (P = 0.017). Downregulation of GRP78 expression markedly inhibited proliferation of the GC cells at the G1 phase, whereas GRP78 overexpression promoted cell-cycle progression. These findings suggest that GRP78 overexpression promotes GC cells proliferation and is an independent indicator of poor prognosis for GC.     

Major histocompatibility class one molecule associates with glucose regulated protein (GRP) 78 on the cell surface

Glucose regulated protein 78 (GRP78) is a member of the heat shock protein (hsp) 70 family. It is an endoplasmic reticulum (ER) chaperone, whose function is generally thought to be limited to the structural maturation of nascent glycoproteins. However, recent observations have shown that ER chaperones, such as GRP78, display peptide-binding activity. These peptide-binding activities along with the observation that heat shock proteins associated with peptides can elicit antigen-specific CTL responses suggest additional roles for these proteins. In this study we provide evidence that GRP78 is not only resident in the ER, but also exists on the cell surface. Furthermore, using biochemical and imaging studies we have found that GRP78 associates with MHC class I on the cell surface. Its presence on the cell surface is not dependent on MHC class I expression. In the absence of MHC class I its cell surface expression is upregulated.     

Thiamine deficiency induces endoplasmic reticulum stress in neurons

Thiamine (vitamin B1) deficiency (TD) causes region selective neuronal loss in the brain; it has been used to model neurodegeneration that accompanies mild impairment of oxidative metabolism. The mechanisms for TD-induced neurodegeneration remain incompletely elucidated. Inhibition of protein glycosylation, perturbation of calcium homeostasis and reduction of disulfide bonds provoke the accumulation of unfolded proteins in the endoplasmic reticulum (ER), and cause ER stress. Recently, ER stress has been implicated in a number of neurodegenerative models. We demonstrated here that TD up-regulated several markers of ER stress, such as glucose-regulated protein (GRP) 78, growth arrest and DNA-damage inducible protein or C/EBP-homologus protein (GADD153/Chop), phosphorylation of eIF2alpha and cleavage of caspase-12 in the cerebellum and the thalamus of mice. Furthermore, ultrastructural analysis by electron microscopic study revealed an abnormality in ER structure. To establish an in vitro model of TD in neurons, we treated cultured cerebellar granule neurons (CGNs) with amprolium, a potent inhibitor of thiamine transport. Exposure to amprolium caused apoptosis and the generation of reactive oxygen species in CGNs. Similar to the observation in vivo, TD up-regulated markers for ER stress. Treatment of a selective inhibitor of caspase-12 significantly alleviated amprolium-induced death of CGNs. Thus, ER stress may play a role in TD-induced brain damage.