GRP78 mRNA Expression Research Articles

Effects of edaravone on the expression of GRP78, Caspase-12, and neuron apoptosis in juvenile rat hippocampus after status convulsive

To observe the expression of GRP78 (glucose regulated protein, GRP78), Caspase-12 and the change of neuron apoptosis in the juvenile rat hippocampus after status convulsive (SC), and to explore the effect of edaravone on them. One hundred and ninety-five juvenile male Sprague-Dawley (SD) rats were randomly divided into normal saline control group (NS group), status convulsive group (SC group) and edaravone treatment group (ED group). Each group was further divided into five subgroups in different executed time points after SC. The rats in status convulsive group were kindled into epilepsy by lithium-pilocarpine method. Expression of GRP78 mRNA and caspase-12 mRNA was detected with reverse transcription-polymerase chain reaction (RT-PCR) method. Expressions of GRP78 and caspase-12 protein were detected with immunohistochemical methods. The neuron apoptosis was observed by TdT-mediated dUTP nick end labeling (TUNEL). (1) Measured by immunohistochemistry the value of OD of GRP78 (0.1480 ± 0.0164, 0.1682 ± 0.0114, and 0.1540 ± 0.0102, respectively, 12 h - 48 h points) and caspase-12 (0.1325 ± 0.0165, 0.1794 ± 0.0213, 0.1525 ± 0.0423, and 0.1309 ± 0.0199, respectively, 12 h-72 h points) positive cells in the SC group increased, there was a significant difference compared with NS group (GRP78: 0.1214 ± 0.0147, 0.1272 ± 0.0177, and 0.1260 ± 0.0157, respectively, 12 h-72 h points. Caspase-12: 0.1050 ± 0.0121, 0.1041 ± 0.0151, 0.1058 ± 0.0222, and 0.1036 ± 0.0186, respectively, 12 h - 72 h points) (P < 0.01, or P < 0.05). By ED intervention GRP78 (0.1550 ± 0.0131, 0.1886 ± 0.0154, and 0.1721 ± 0.0151, respectively, 12 h - 48 h points) positive cells value of the OD increased as compared with SC group (P < 0.01, or P < 0.05). and caspase-12 (0.1211 ± 0.0184, 0.1545 ± 0.0205, and 0.1085 ± 0.0219, respectively, 12 h, 24 h and 72 h points) positive cells value of the A decreased as compared with SC group (P < 0.01, or P < 0.05). (2) Measured by RT-PCR, the expression of GRP78 mRNA and caspase-12 mRNA trend was similar to protein. (3) The TUNEL positive cells in hippocampus CA(1) of SC group (11.41 ± 2.37) were more than that of NS group after the SC 12 h (P < 0.01), reached its highest level at 48 h (28.78 ± 5.11), after the intervention with edaravone (8.98 ± 2.22, 13.09 ± 2.54 and 20.57 ± 4.89, respectively, 12 h-48 h points), TUNEL positive cells showed a significant drop in SC group at 12 h-48 h time points (P < 0.01, or P < 0.05), but still significantly higher than that of the NS group (6.22 ± 1.50, 6.57 ± 1.61 and 6.72 ± 1.14, respectively) (P < 0.01, or P < 0.05), at the 4 h time point (NS group 6.29 ± 1.49, SC group 6.61 ± 1.71, ED group 5.75 ± 1.41) among the three groups, no significant difference in TUNEL positive cells was found (P = 0.759). The expression of GRP78 and caspase-12 increased after SC. Edaravone increased expression of GRP78 and decreased expression of caspase-12 in hippocampus rat with pilocarpine-induced seizures, reduced the number of neuronal apoptosis. These results suggest that edaravone may have protective effect against the hippocampal damage caused by status convulsive.

Expression and clinical significance of glucose regulated proteins GRP78 and GRP94 in human colon cancer

ObjectiveTo investigate the expression of glucose regulated proteins GRP78 and GRP94 in human colon cancer.MethodsTissues of resected primary colon cancer, colon adenoma and normal tissue were investigated. Protein expression was detected with immunohistochemical staining. mRNA expression levels of GRP78 and GRP94 were determined by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) after mRNA extraction.ResultsThe expression of GRP94 and GRP78 was significantly higher in colon cancer when compared to those in colon adenoma and normal tissue (P<0.01). GRP94 mRNA and protein expression was found to be in close relationship with the grade of differentiation, Dukes stages, lymph node involvement and remote metastasis in colon cancer (P<0.01), but no relationship with gender and age (P>0.05). GRP78 mRNA and protein expression increased with cancer progression along the normal tissue-adenoma-cancer sequence, but showed no association with grade of differentiation, Dukes stages, lymph node involvement, remote metastasis, gender and age (P>0.05). The mRNA expression of GRP78 and GRP94 was consistent with the proteins (P<0.01), but there is no correlation between overexpression of GRP78 and GRP94 (P>0.05), and the patients with both strong GRP78 and GRP94 protein expression did not show advanced tumor stages (P>0.05).ConclusionOverexpression of GRP78 and GRP94 was found in colon cancer. Overexpression of GRP94 was closely related to cellular differentiation, Dukes stages, invasion and metastasis.

Upregulation of GRP78 in Renal Cell Carcinoma and Its Significance

To investigate the expression of GRP78 in human renal cell carcinoma (RCC) and its significance. We studied RNA and tissue section of a tumor and adjacent nontumorous renal tissues obtained from radical nephrectomy specimens of 42 patients and RCC cell lines. We used reverse transcriptase-PCR and immunohistochemistry to detect the GRP78 mRNA and protein expression, respectively. Reverse transcriptase-PCR revealed that GRP78 mRNA is positively expressed in RCC cell lines (786-0, OS-RC-2, and Caki-1); the GRP78 mRNA expression in the RCC and adjacent nontumorous renal tissues was 0.88 +/- 0.34 and 0.44 +/- 0.15, respectively (P < .001). The GRP78 protein was found in RCC cell lines. Immunohistochemistry results also showed that the level of GRP78 protein expression of RCC tissues was significantly higher than that of the adjacent nontumorous renal tissues (P < .001). The high levels of GRP78 mRNA and protein expression were related to the large tumor size and high clinical stage (P < .001) but not to sex, age, and cell differentiate (P > .05). To our knowledge, the present study is the first to report the upregulated expression of GRP78 and that it is possibly involved in pathogenesis of RCC.

P03: A novel marker, alpha2-macroglobulin and GRP78, characteristic of rat hepatocellular preneoplastic and neoplastic lesions undetectable by Glutathione S-transferase placental form

Previously, we reported that alpha 2 -macroglobulin (alpha 2 M) was candidate for a novel marker characteristic for rat hepatocellular preneoplastic and neoplastic lesions, undetectable by well established cytochemical markers “Glutathione S-transferase placental form (GST-P)”. (Sukata et al. 2004 Am. J. Pathol.) In this study, we examined whether 78-kDa glucose-regulated protein (GRP78), which is a molecular chaperone and a member of the HSP70 family of heat shock proteins and is known as a receptor of alpha 2 M, also became a candidate for a new marker of these lesions. GST-P-negative hepatocellular altered foci (HAF), hepatocellular adenoma (HCA), and hepatocellular carcinoma (HCC) were generated by two initiation-promotion models with N-nitrosodiethylamine (DEN) and peroxisome proliferators, Wy-14,643 and clofibrate. Total RNAs isolated from laser-microdissected GST-P-negative HAF (amphophilic cell foci) and adjacent normal tissues were applied to microarray analysis. As a result, microarray analysis revealed that GRP78 mRNA expression was not increased in all GST-P negative lesions, regardless of overexpression of alpha 2 M mRNA. Immunohistochemical staining for GRP78 could be detectable in GST-P-negative HAF in the same way as alpha 2 M. GRP78-positive reaction was also detectable in HCC. Furthermore, GRP78 was recognized not only in HCC of rats, but also in HCC of mice. These results suggested that GRP78 was also candidate for a novel marker characteristic for GST-P negative rat hepatocellular preneoplastic and neoplastic lesions. More interestingly, since GRP78 was recognized in the mouse HCC, it is suggested that GRP78 has a possibility that it can become a liver carcinogenic marker beyond a species.

Expression of two endoplasmic reticulum stress markers, GRP78 and GADD153, in rat retinal detachment model and its implication

To investigate the expression of endoplasmic reticulum (ER) stress-related genes, glucose-regulated protein 78 (GRP78) and growth arrest DNA damage-inducible gene 153 (GADD153)/CPEBP homologous protein (CHOP), in rat retinal detachment (RD) model. At various time points after RD, the apoptosis of retinal cells was detected by TdT-mediated fluorescein-16-dUTP nick-end labelling (TUNEL) assay; GRP78 and GADD153 mRNA levels were detected by reverse transcription (RT)-PCR; proteins were detected by western blotting analysis; protein distributions in the retinal cells were observed by immunofluorescence using laser-scanning confocal microscope. After RD, the apoptosis was peaked on 2-4 d and then dropped down. The GRP78 mRNA and GADD153 mRNA levels in RD groups on 0.5, 1, 2, and 4 d were all significantly higher than those in the control group (P<0.05). The expression of GRP78 mRNA peaked on 1-2 d after RD. Expression of GRP78 protein was significantly higher than that in the normal control group on 0.5, 1, 2, 4, 8, 16, and 32 d after RD (P<0.05). The expression of GRP78 protein was observed in all the layers of retina in the RD groups, and peaked on 8, 16, and 32 d. The expression of GADD153 protein, mostly in photoreceptor layers, was significantly higher than that in the control group on 0.5, 1, 2, and 4 d after RD (P<0.05). ER stress-related markers, GRP78 and GADD153, are elevated after RD. The elevation of GADD153 is in parallel with the post-RD apoptosis of retinal cells, suggesting that ER stress-mediated death is likely to be activated after RD and involved in post-RD vision loss.

Expression and clinical significance of Glucose Regulated Proteins GRP78 (BiP) and GRP94 (GP96) in human adenocarcinomas of the esophagus

BackgroundGlucose regulated proteins (GRPs) are main regulators of cellular homeostasis due to their role as molecular chaperones. Moreover, the functions of GRPs suggest that they also may play important roles in cancer biology. In this study we investigated the glucose regulated proteins GRP78 (BiP) and GRP94 (GP96) in a series of human esophageal adenocarcinomas to determine their implications in cancer progression and prognosis.MethodsFormalin-fixed, paraffin-embedded tissues of primary resected esophageal (Barrett) adenocarcinomas (n = 137) and corresponding normal tissue were investigated. mRNA-gene expression levels of GRP78 and GRP94 were determined by quantitative real-time RT-PCR after mRNA extraction. Protein expression analysis was performed with immunohistochemical staining of the cases, assembled on a tissue micorarray. The results were correlated with pathologic features (pT, pN, G) and overall survival.ResultsGRP78 and GRP94 mRNA were expressed in all tumors. The relative gene expression of GRP78 was significantly higher in early cancers (pT1m and pT1sm) as compared to more advanced stages (pT2 and pT3) and normal tissue (p = 0.031). Highly differentiated tumors showed also higher GRP78 mRNA levels compared to moderate and low differentiated tumors (p = 0.035). In addition, patients with higher GRP78 levels tended to show a survival benefit (p = 0.07). GRP94 mRNA-levels showed no association to pathological features or clinical outcome.GRP78 and GRP94 protein expression was detectable by immunohistochemistry in all tumors. There was a significant correlation between a strong GRP78 protein expression and early tumor stages (pT1m and pT1sm, p = 0.038). For GRP94 low to moderate protein expression was significantly associated with earlier tumor stage (p = 0.001) and less lymph node involvement (p = 0.036). Interestingly, the patients with combined strong GRP78 and GRP94 protein expression exclusively showed either early (pT1m or pT1sm) or advanced (pT3) tumor stages and no pT2 stage (p = 0.031).ConclusionWe could demonstrate an association of GRP78 and GRP94 mRNA and protein expression with tumor stage and behaviour in esophageal adenocarcinomas. Increased expression of GRP78 may be responsible for controlling local tumor growth in early tumor stages, while high expression of GRP78 and GRP94 in advanced stages may be dependent from other factors like cellular stress reactions due to glucose deprivation, hypoxia or the hosts' immune response.

Endoplasmic reticulum stress induced by oxidative stress in retinal pigment epithelial cells

Induction of glucose-regulated protein (GRP)-78 in the endoplasmic reticulum (ER) is a protective mechanism cells use to adapt to ER stress. We evaluated the expression of GRP-78 and its regulation by an oxidant tert-butyl hydroperoxide (tBH) in human retinal pigment epithelium (RPE) cells. We used a carboxy-H2-DCFDA staining method to detect tBH-induced accumulation of reactive oxygen species (ROS) in RPE cells, and analyzed the expression of GRP-78 in normal human fetal and adult retinas and in cultured human RPE cells by immunohistochemistry. The effects of tBH (10-100 microM) on GRP-78 and on growth arrest and DNA damage inducible genes 153 (GADD153) protein and mRNA expression were studied using Western blot and real-time polymerase chain reaction. Sections of fetal retinas were negative for GRP-78. Adult retinas showed moderate cytoplasmic GRP-78 staining in the RPE and choroid. tBH-induced ROS accumulation in RPE cells showed partial colocalization with the ER. GRP-78 and GADD153 mRNA and protein expression in cultured RPE cells were significantly upregulated by treatment with tBH. tBH increases oxidative stress, increases accumulation of ROS in the ER, and upregulates expression of GRP-78 and GADD153. This supports the connection between oxidative stress and ER stress, and suggests that GRP-78 may serve a protective role in the RPE response to oxidative stress.

Involvement of Selective Reactive Oxygen Species Upstream of Proapoptotic Branches of Unfolded Protein Response

Cadmium triggers apoptosis of LLC-PK1 cells through induction of endoplasmic reticulum (ER) stress. We found that cadmium caused generation of reactive oxygen species (ROS) and that cadmium-induced ER stress was inhibited by antioxidants. In contrast, suppression of ER stress did not attenuate cadmium-triggered oxidative stress, suggesting that ER stress occurs downstream of oxidative stress. Exposure of the cells to either O(2)(*), H(2)O(2), or ONOO(-) caused apoptosis, whereas ER stress was induced only by O(2)(*) or ONOO(-). Transfection with manganese superoxide dismutase significantly attenuated cadmium-induced ER stress and apoptosis, whereas pharmacological inhibition of ONOO(-) was ineffective. Interestingly, transfection with catalase attenuated cadmium-induced apoptosis without affecting the level of ER stress. O(2)(*) caused activation of the activating transcription factor 6-CCAAT/enhancer-binding protein-homologous protein (CHOP) and the inositol-requiring ER-to-nucleus signal kinase 1-X-box-binding protein 1 (XBP1) proapoptotic cascades, and overexpression of manganese superoxide dismutase attenuated cadmium-triggered induction of both pathways. Furthermore, phosphorylation of proapoptotic c-Jun N-terminal kinase by O(2)(*) or cadmium was suppressed by dominant-negative inhibition of XBP1. These data elucidated 1) cadmium caused ER stress via generation of ROS, 2) O(2)(*) was selectively involved in cadmium-triggered, ER stress-mediated apoptosis through activation of the activating transcription factor 6-CHOP and inositol-requiring ER-to-nucleus signal kinase 1-XBP1 pathways, and 3) phosphorylation of JNK was caused by O(2)(*)-triggered activation of XBP1.

Insulin induces chaperone and CHOP gene expressions in adipocytes

Adipocyte secretes bioactive proteins called adipocytokines, and biosynthesis of secretory proteins requires molecular chaperones and folding enzymes in endoplasmic reticulum (ER). ER chaperones are known to be induced by unfolded protein response (UPR) and growth factors, however, it has not been determined how ER chaperones expression is regulated in adipocytes. Here we show that insulin treatment induced GRP78 and ERO1L mRNA levels in 3T3-L1 adipocytes. Insulin also upregulated CHOP mRNA levels, but did not induce phosphorylation of eIF2α. Pretreatment with insulin protected 3T3-L1 adipocytes against thapsigargin-mediated phosphorylation of eIF2α but did not against DTT-mediated one. In vivo mice study showed that GRP78 and CHOP expressions were regulated by feeding conditions. These results suggest that insulin signaling is important to induce mRNA expressions of GRP78 and CHOP, and may have a protective role against UPR.

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.

THE HERBAL PRESCRIPTION YOUKONGDAN MODULATES RODENT MEMORY, ISCHEMIC DAMAGE AND CORTICAL mRNA GENE EXPRESSION

The effects of the herbal prescription youkongdan (YKD) on memory performance of rodents following cerebral ischemia/reperfusion and scopolamine administration were measured and compared to the age-related changes in mRNA expression induced by dietary supplementation of YKD. Following ischemia, YKD decreased neuronal cell loss in the CA1 region of rat hippocampus by 89% relative to controls. YKD improved the water maze performance of both ischemic and scopolamine-treated animals. Dietary administration of YKD resulted in significant modulation of Egr1, Grp78, Hsp86, SOD1, and αB crystallin mRNA expression and a trend toward increased exploratory behavior in older mice.

Increased GADD153 gene expression during iron chelation-induced apoptosis in Jurkat T-lymphocytes

Depriving cells of iron likely stresses them and can result in cell death. To examine the potential relationship between this form of stress and cell death, Jurkat T-lymphocytes were made iron-deficient by exposing them to the iron chelator, deferoxamine (DFO). Such treatment produced evidence of apoptosis, including cell shrinkage, membrane blebbing, chromatin condensation and fragmentation, and also formation of apoptotic bodies. Additionally, proteolytic cleavage of poly(ADP-ribose)polymerase was detected, suggesting involvement of caspases in initiating apoptosis. Indeed, a selective caspase-3 inhibitor prevented the effects of DFO. During the early induction period of apoptosis, GRP78 and HSP70 mRNA expression was not affected. In contrast, there was mainly increased mRNA expression of Growth Arrest and DNA Damage-inducible gene 153 (GADD153), which seemed to be at the level of transcription rather than mRNA stability. Furthermore, fortifying cells with antioxidants did not prevent the increased GADD153 mRNA expression, and no evidence of single-strand breaks in DNA was found, suggesting that neither reactive oxygen species nor DNA damage was involved in triggering GADD153 gene activation. DFO also caused GADD153 protein to be expressed. Because GADD153 is recognized as a pro-apoptotic gene, these findings generate the notion that GADD153 might help mediate apoptosis in iron-deficient cells.

Taurine prevents the decrease in expression and secretion of extracellular superoxide dismutase induced by homocysteine: amelioration of homocysteine-induced endoplasmic reticulum stress by taurine.

Hyperhomocysteinemia is an independent risk factor for atherosclerosis. Homocysteine has been shown to induce endoplasmic reticulum (ER) stress in vascular endothelial cells. ER stress is a condition in which glycoprotein trafficking is disrupted and unfolded proteins accumulate in the ER. ER molecular chaperons, such as GRP78, are induced and an ER resident kinase, PERK, is activated when cells are subjected to ER stress. Conversely, taurine is reported to have antiatherogenic effects by unknown mechanisms. To elucidate the mechanisms by which homocysteine induces atherosclerosis and taurine prevents it, we examined whether homocysteine and taurine affect the expression and secretion of extracellular superoxide dismutase (EC-SOD), a glycoprotein secreted from vascular smooth muscle cells (VSMCs) that protects the vascular wall from oxidative stress. We assessed the expression of EC-SOD and GRP78 mRNA in cultured rat VSMCs by Northern blot analysis. The EC-SOD protein secreted into the culture medium was examined by Western blot analysis. Homocysteine (5 mmol/L) and other ER stress inducers, including A23187, were found to decrease EC-SOD mRNA expression and protein secretion. Furthermore, they upregulated GRP78 mRNA expression and activated PERK. Taurine (0.5 to 10 mmol/L), conversely, prevented these actions induced by homocysteine. Homocysteine induces ER stress and reduces the secretion and expression of EC-SOD in VSMCs, leading to increased oxidative stress in the vascular wall. Taurine restores the secretion and expression of EC-SOD by ameliorating ER stress induced by homocysteine.

TNF-alpha and heat-shock protein gene expression in ischemic-injured liver from fasted and non-fasted rats. Role of donor fasting in the prevention of reperfusion injury following liver transplantation.

We have previously shown that livers from long-term-fasted rats acquire tolerance to warm ischemic injury following transplantation, despite the fact that fasting depletes glycogen and ATP from the liver. The precise mechanism of the protective effect induced by donor fasting, however, is still a matter of controversy. In this experiment we determined heat-shock protein (GRP78) mRNA expression in livers during long-term fasting and TNF-alpha mRNA expression in transplanted livers exposed to warm ischemia. We also measured the concentration of TNF-alpha by ELISA in the ascitic fluid of fed and fasted rats injected intraperitoneally with zymosan to investigate why livers from fasted rats tolerate ischemic injury better. There seemed to be a positive correlation between GRP78 mRNA expression and survival. TNF-alpha secretion into the ascitic fluid of fasted rats was markedly suppressed, and fasting donor animals induced cytoprotective substances, such as GRP78, in the liver. These factors may contribute to the tolerance to ischemic injury produced by donor fasting.

TNF-α and heat-shock protein gene expression in ischemic-injured liver from fasted and non-fasted rats. Role of donor fasting in the prevention of reperfusion injury following liver transplantation

Abstract We have previously shown that livers from long-term-fasted rats acquire tolerance to warm ischemic injury following transplantation, despite the fact that fasting depletes glycogen and ATP from the liver. The precise mechanism of the protective effect induced by donor fasting, however, is still a matter of controversy. In this experiment we determined heat-shock protein (GRP78) mRNA expression in livers during long-term fasting and TNF-α mRNA expression in transplanted livers exposed to warm ischemia. We also measured the concentration of TNF-α by ELISA in the ascitic fluid of fed and fasted rats injected intraperitoneally with zymosan to investigate why livers from fasted rats tolerate ischemic injury better. There seemed to be a positive correlation between GRP78 mRNA expression and survival. TNF-α secretion into the ascitic fluid of fasted rats was markedly suppressed, and fasting donor animals induced cytoprotective substances, such as GRP78, in the liver. These factors may contribute to the tolerance to ischemic injury produced by donor fasting.