Glucose-regulated Protein 78 mRNA Research Articles

Nerve growth factor attenuates 2-deoxy-d-glucose-triggered endoplasmic reticulum stress-mediated apoptosis via enhanced expression of GRP78

The glucose analog 2-deoxy-d-glucose (2DG) depletes cells of glucose. Inhibition of glycosylation caused by glucose depletion induces endoplasmic reticulum (ER) stress with subsequent apoptosis. Glucose-regulated protein 78 (GRP78) is a molecular chaperone that acts within the ER. During ER stress, GRP78 expression is induced as part of the unfolded protein response (UPR). We found that nerve growth factor (NGF) prevented 2DG-triggered ER stress-mediated apoptosis, but not the induction of GRP78 expression, in PC12 cells. Surprisingly, GRP78 expression was further up-regulated when NGF was added to 2DG-treated PC12 cells. When a specific inhibitor of phosphatidylinositol 3-kinase (PI3-K), LY294002, was added to 2DG plus NGF-treated cells, both the effects of NGF on 2DG-induced apoptosis and GRP78 expression were significantly diminished. In addition, versipelostatin (VST), a specific inhibitor of GRP78 expression, and small interfering RNA (siRNA) against GRP78 mRNA also decreased both the effects of NGF on 2DG-induced apoptosis and GRP78 expression. RT-PCR and Western blot analyses revealed that enhanced production of nuclear p50 ATF6, but not spliced XBP1, mainly contributed to the NGF-induced enhancement of GRP78 expression in 2DG-treated cells. These results suggest that the NGF-activated PI3-K/Akt signaling pathway plays a protective role against ER stress-mediated apoptosis via enhanced expression of GRP78 in PC12 cells.

Characteristic Upregulation of Glucose-Regulated Protein 78 in an Early Lesion Negative for Hitherto Established Cytochemical Markers in Rat Hepatocarcinogenesis

Previously, we reported α2-macroglobulin (α2M) to be a novel marker characteristic of rat hepatocellular preneoplastic and neoplastic lesions negative for hitherto well-established markers. In the present study, we further examined other candidate markers with specificity for the same type of lesions. Glutathione S-transferase-placental form (GST-P)-negative hepatocellular altered foci (HAF) were generated using a two-stage (initiation and promotion) carcinogenesis protocol with N,N-diethylnitrosamine (DEN) and either Wy-14,643 or clofibrate, two peroxisome proliferators. Microarray analysis using total RNAs isolated from laser-microdissected GST-P-negative HAF (amphophilic cell foci) and adjacent normal tissues was conducted along with immunohistochemistry and real-time RT-PCR. Staining for glucose-regulated protein 78 (GRP78) was detected in GST-P-negative HAF and hepatocellular adenomas, and slightly increased GRP78 mRNA expression was observed in the lesions by real-time RT-PCR analysis. Thus, an early increase of GRP78 expression in hepatocarcinogenesis is likely a feature of the amphophilic subset of HAF.

GRP78 expression correlates with histologic differentiation and favorable prognosis in neuroblastic tumors

Glucose-regulated protein 78 (GRP78), an endoplasmic reticulum protein, is essential for the differentiation of neuroblastoma cells and is selectively induced when the cells are undergoing apoptosis. These findings suggest that GRP78 may affect the tumor behavior of neuroblastoma. Our study evaluates the association of clinicopathologic factors and patient survival with the expression of GRP78 in patients with neuroblastoma. GRP78 expression in 68 neuroblastic tumors was investigated semiquantitatively by immunohistochemistry. GRP78 mRNA and protein levels in 7 tumor tissues were also quantified by real-time PCR and Western blot respectively and correlated well with the immunohistochemical results. Forty (58.8%) of the 68 neuroblastic tumors showed positive GRP78 expression. The percentage of positive GRP78 immunostaining increased as the tumor histology became differentiated (p = 0.001). Furthermore, positive GRP78 expression strongly correlated with early clinical stages (P = 0.002) but inversely correlated with MYCN amplification (p = 0.001). Kaplan-Meier analysis showed that patients with positive GRP78 expression did have better survival than those with negative expression (5-year survival rate, 72.9% and 23.4% respectively, p < 0.001). Multivariate analysis further showed that GRP78 expression was an independent prognostic factor. Moreover, GRP78 expression predicted better survival in patients with either undifferentiated or differentiated histologies. GRP78 expression still had significant prognostic value when the analysis was restricted to tumors of advanced stages or without MYCN amplification. Thus, GRP78 can serve as a novel independent favorable prognostic factor for patients with neuroblastoma.

Hypothermic treatment restores glucose regulated protein 78 (GRP78) expression in ischemic brain

Mild hypothermia is a well-known method of reducing brain damage caused by traumatic, hypoxic, and ischemic injury. To elucidate the neuroprotective mechanism induced by hypothermic treatment, we compared gene expression profiles in the hippocampus of gerbils rendered ischemic for 15 min and then reperfused for 3 h under conditions of normothermia (37±0.5°C) or hypothermic treatment (34±0.5°C). Using the differential display method, we observed significantly reduced expression of the 78 kDa glucose regulated protein (GRP78), in ischemic gerbil hippocampus that underwent normothermic reperfusion, but normal GRP78 expression in animals that underwent hypothermic reperfusion. In situ hybridization and Northern blot analysis showed GRP78 mRNA expression was reduced in the CA1 region of the hippocampus under normothermic conditions, but was not reduced under hypothermic conditions. Western blot analysis also showed the levels of immunoreactive GRP78 protein decreased in neurons of the hippocampal CA-1 region under normothermia, but not under hypothermic treatments. Furthermore, adenovirus-mediated overexpression of GRP78 protects rat hippocampal neurons from cell death and inhibits the rise in intracellular calcium concentration normally induced by hydrogen peroxide. These results suggest that reduction in GRP78 expression contributes to cell damage in the ischemic brain and that hypothermia-mediated restoration of GRP78 expression is one mechanism that enhances neuronal survival.

Induction of glucose-regulated protein 78 in rat uterine glandular epithelium during uterine sensitization for the decidual cell reaction.

Endometrial receptivity for implantation and sensitization for decidualization in rodents is a transient state under the control of the ovarian steroids estrogen and progesterone. It is unclear, however, what molecular events mediate the onset of uterine receptivity. Messenger RNA differential display was performed on endometrial RNA from ovariectomized rats differentially sensitized for decidualization. Maximally sensitized uteri were at the equivalent of Day 5 of pseudopregnancy, and temporally nonsensitized uteri at Day 4 or 6; hormonally nonsensitized uteri were from animals on Day 5 treated with low or high doses of estradiol on Day 4. A cDNA with endometrial expression restricted to maximally sensitized uteri was isolated, cloned, and sequenced. The cDNA matched the sequence for glucose-regulated protein 78 (GRP78), a heat shock 70-related protein that resides in the lumen of the endoplasmic reticulum (ER) and has roles in several cellular processes including multimeric protein assembly, the degradation of proteins, and the storage and regulation of ER luminal calcium. Northern blot analysis indicated a dramatic increase in GRP78 mRNA levels restricted to the sensitized, Day 5 endometrium, suggesting a role in the onset of the sensitized phase. In situ hybridization and immunohistochemistry experiments localized the up-regulation of GRP78 within the receptive endometrium to the glandular epithelium.

Glucose regulation of GRP78 gene expression

The endoplasmic reticulum chaperone glucose-regulated protein 78 (GRP78) is essential for the proper glycosylation, folding and assembly of many membrane bound and secreted proteins. GRP78 mRNA is well known to be induced in cultured cells by lowering medium glucose concentrations from 4.5 to 0 mg/ml. Here we report a study designed to determine the effects of intermediate concentrations of glucose on GRP78 mRNA abundance. Progressive reduction in culture medium glucose from 4.5 to 1.0 mg/ml progressively reduced GRP78 mRNA to approximately 30% of the initial level. Induction of GRP78 mRNA by glucose starvation was observed in medium containing less than 1 mg/ml glucose. Determination of the amount of glucose consumed in these cultures showed that reduction of glucose concentrations led first to repression of GRP78 mRNA abundance, followed by induction of the mRNA only when glucose is nearly exhausted. Caloric restriction in mice both reduces fasting and mean 24 h glucose blood concentrations and GRP78 mRNA abundance in the liver. Thus, it is possible that negative regulation of GRP78 mRNA in the liver is due directly to reduced blood glucose concentrations.

Dietary Energy Restriction in Mice Negatively Regulates Hepatic Glucose-Regulated Protein 78 (GRP78) Expression at the Posttranscriptional Level

Dietary energy restriction delays age-related physiologic changes, increases maximum life span, and reduces cancer incidence. We showed previously that 50% energy restriction in mice reduces hepatic expression of glucose-regulated protein mRNA by 50 to 80%. Changes in glucose-regulated protein 78 (GRP78) levels can either decrease or increase the rate of secretion of specific proteins. Therefore, energy restriction probably produces a global change in the spectrum of proteins secreted by the liver. These studies were initiated to investigate the molecular basis for the negative regulation of the gene. By use of transfection and nuclear run-on techniques, the strong induction of GRP78 gene transcription in cultured cells subjected to acute, extreme glucose deprivation has been well characterized. However, negative regulation of GRP78 gene expression in vivo by energy restriction is not as well understood. In our studies, a reduction in GRP78 protein levels determined using Western blotting closely paralleled a reduction in hepatic GRP78 mRNA measured by Northern and dot blotting. In each case the changes were statistically significant. This close correspondence indicates that energy restriction does not influence the translation rate or the stability of GRP78 protein. No statistically significant difference in the rate of transcription of the gene was detected in energy-restricted mice by use of transcription run-on assays. These results strongly suggest that energy restriction results in destabilization of GRP78 mRNA, thereby repressing hepatic expression of the gene.