Induction Of GRP94 Research Articles

Protective Effects of Glucose-Related Protein 78 and 94 on Cisplatin-Mediated Ototoxicity.

Cisplatin is a widely used chemotherapeutic drug for treating various solid tumors. Ototoxicity is a major dose-limiting side effect of cisplatin, which causes progressive and irreversible sensorineural hearing loss. Here, we examined the protective effects of glucose-related protein (GRP) 78 and 94, also identified as endoplasmic reticulum (ER) chaperone proteins, on cisplatin-induced ototoxicity. Treating murine auditory cells (HEI-OC1) with 25 μM cisplatin for 24 h increased cell death resulting from excessive intracellular reactive oxygen species (ROS) accumulation and caspase-involved apoptotic signaling pathway activation with subsequent DNA fragmentation. GRP78 and GRP94 expression was increased in cells treated with 3 nM thapsigargin or 0.1 μg/mL tunicamycin for 24 h, referred to as mild ER stress condition. This condition, prior to cisplatin exposure, attenuated cisplatin-induced ototoxicity. The involvement of GRP78 and GRP94 induction was demonstrated by the knockdown of GRP78 or GRP94 expression using small interfering RNAs, which abolished the protective effect of mild ER stress condition on cisplatin-induced cytotoxicity. These results indicated that GRP78 and GRP94 induction plays a protective role in remediating cisplatin-ototoxicity.

OASIS/CREB3L1 Is Induced by Endoplasmic Reticulum Stress in Human Glioma Cell Lines and Contributes to the Unfolded Protein Response, Extracellular Matrix Production and Cell Migration

OASIS is a transcription factor similar to ATF6 that is activated by endoplasmic reticulum stress. In this study we investigated the expression of OASIS in human glioma cell lines and the effect of OASIS knock-down on the ER stress response and cell migration. OASIS mRNA was detected in three distinct glioma cell lines (U373, A172 and U87) and expression levels were increased upon treatment with ER stress-inducing compounds in the U373 and U87 lines. OASIS protein, which is glycosylated on Asn-513, was detected in the U373 and U87 glioma lines at low levels in control cells and protein expression was induced by ER stress. Knock-down of OASIS in human glioma cell lines resulted in an attenuated unfolded protein response to ER stress (reduced GRP78/BiP and GRP94 induction) and decreased expression of chondroitin sulfate proteoglycan extracellular matrix proteins, but induction of the collagen gene Col1a1 was unaffected. Cells in which OASIS was knocked-down exhibited altered cell morphology and reduced cell migration. These results suggest that OASIS is important for the ER stress response and maintenance of some extracellular matrix proteins in human glioma cells.

Heightened Induction of Proapoptotic Signals in Response to Endoplasmic Reticulum Stress in Primary Fibroblasts from a Mouse Model of Longevity

Previous work from our laboratory has shown that primary fibroblasts from long-lived Snell dwarf mice display a higher sensitivity to the lethal effects of endoplasmic reticulum (ER) stressors, such as thapsigargin, than cells from normal mice. Here we show that thapsigargin induces higher expression of CHOP, enhanced cleavage of caspase-12, higher caspase-3 activity, and increased phosphorylation of c-JUN, all indicators of enhanced apoptosis, in dwarf fibroblasts. Dwarf and normal fibroblasts show no genotypic difference in up-regulating BiP, GRP94, and ERp72 proteins after exposure to thapsigargin. However, dwarf fibroblasts express lower basal levels of a number of putative XBP1 target genes including Armet, Edem1, Erdj3, p58(IPK) and Sec61a1, as well as Ire1α itself. Furthermore, when exposed to thapsigargin, dwarf fibroblasts display attenuated splicing of Xbp1, but similar phosphorylation of eIF2α, in comparison to normal fibroblasts. These data support the notion that IRE1/XBP1 signaling is set at a lower level in dwarf fibroblasts. Diminished Xbp1 splicing in dwarf-derived fibroblasts may tilt the balance between prosurvival and proapoptotic signals in favor of apoptosis, thereby leading to higher induction of proapoptotic signals in these cells and ultimately their increased sensitivity to ER stressors. These results, together with recent findings in Caenorhabditis elegans daf-2 mutants, point to a potential interplay between insulin/IGF-1 signals and unfolded protein response signaling.

Cytosolic phospholipase A 2-α enhances induction of endoplasmic reticulum stress

Induction of endoplasmic reticulum (ER) stress by the complement membrane attack complex is enhanced by activation of cytosolic phospholipase A 2-α (cPLA 2). To address mechanisms by which cPLA 2 may modulate ER stress, we produced a mutant cPLA 2, containing an ER targeting domain (cPLA 2-ERmut). After transfection and fractionation of COS-1 cells, cPLA 2-ERmut was present mainly in the membrane fraction, whereas wild type (wt) cPLA 2 was principally in the cytosol. By fluorescence microscopy, cPLA 2-ERmut was enriched in a perinuclear distribution under basal conditions, colocalizing with the ER protein, calnexin, while cPLA 2-wt was mainly cytosolic. Both forms of cPLA 2 transiently expressed in COS cells showed basal phosphorylation at serine 505, which correlates with catalytic activity. Expression of cPLA 2-wt was ∼ 5-fold greater, compared with cPLA 2-ERmut, but both enzymes produced comparable increases in free arachidonic acid, implying that cPLA 2-ERmut effectively hydrolyzed ER membrane phospholipids. Although transfection of cPLA 2-ERmut or wt did not induce ER stress independently, cPLA 2-ERmut and wt enhanced the induction of ER stress by tunicamycin, dithiothreitol and ionomycin (monitored by induction of grp94 and C/EBP homologous protein-10), and the effect was dependent on the catalytic activity. cPLA 2-ERmut enhanced production of superoxide. Induction of ER stress in tunicamycin-treated cells expressing cPLA 2-ERmut was attenuated in the presence of the antioxidant, N-acetyl cysteine, and reduced glutathione, and was exacerbated by dl-buthionine-(S,R)-sulfoximine (which depletes glutathione). Expression of cPLA 2-ERmut exacerbated tunicamycin-induced apoptosis. Thus, induction of ER stress is facilitated by the activation of cPLA 2 at the ER. The mechanism involves ER membrane phospholipid hydrolysis, and accumulation of reactive oxygen species.

Endoplasmic reticulum stress (ER-stress) by 2-deoxy-D-glucose (2DG) reduces cyclooxygenase-2 (COX-2) expression and N-glycosylation and induces a loss of COX-2 activity via a Src kinase-dependent pathway in rabbit articular chondrocytes

Endoplasmic reticulum (ER) stress regulates a wide range of cellular responses including apoptosis, proliferation, inflammation, and differentiation in mammalian cells. In this study, we observed the role of 2-deoxy-D-glucose (2DG) on inflammation of chondrocytes. 2DG is well known as an inducer of ER stress, via inhibition of glycolysis and glycosylation. Treatment of 2DG in chondrocytes considerably induced ER stress in a dose- and time-dependent manner, which was demonstrated by a reduction of glucose regulated protein of 94 kDa (grp94), an ER stress-inducible protein, as determined by a Western blot analysis. In addition, induction of ER stress by 2DG led to the expression of COX-2 protein with an apparent molecular mass of 66-70kDa as compared with the normally expressed 72-74 kDa protein. The suppression of ER stress with salubrinal (Salub), a selective inhibitor of eif2-alpha dephosphorylation, successfully prevented grp94 induction and efficiently recovered 2DG- modified COX-2 molecular mass and COX-2 activity might be associated with COX-2 N-glycosylation. Also, treatment of 2DG increased phosphorylation of Src in chondrocytes. The inhibition of the Src signaling pathway with PP2 (Src tyrosine kinase inhibitor) suppressed grp94 expression and restored COX-2 expression, N-glycosylation, and PGE2 production, as determined by a Western blot analysis and PGE2 assay. Taken together, our results indicate that the ER stress induced by 2DG results in a decrease of the transcription level, the molecular mass, and the activity of COX-2 in rabbit articular chondrocytes via a Src kinase-dependent pathway.

Essential role of the unfolded protein response regulator GRP78/BiP in protection from neuronal apoptosis

Neurodegenerative diseases are often associated with dysfunction in protein quality control. The endoplasmic reticulum (ER), a key site for protein synthesis, senses stressful conditions by activating the unfolded protein response (UPR). Here we report the creation of a novel mouse model where GRP78/BiP, a major ER chaperone and master regulator of UPR, is specifically eliminated in the Purkinje cells (PCs). GRP78 depleted PCs activate UPR including induction of GRP94, PDI, CHOP and GADD34, feedback suppression of eIF2α phosphorylation and apoptotic cell death. In contrast to current models of protein misfolding where abnormal accumulation of ubiquitinated protein is prominent, cytosolic ubiquitin staining is dramatically reduced in GRP78 null PCs. Ultrastructural evaluation reveals that the ER shows prominent dilatation with focal accumulation of electron-dense material within the ER. The mice show retarded growth and severe motor coordination defect by week 5 and cerebellar atrophy by week 13. Our studies uncover a novel link between GRP78 depletion and reduction in cytosolic ubiquitination and establish a novel mouse model of accelerated cerebellar degeneration with basic and clinical applications.

Aberrant endoplasmic reticulum stress response in lymphoblastoid cells from patients with bipolar disorder

Impaired endoplasmic reticulum (ER) stress response has been suggested as a possible pathophysiological mechanism of bipolar disorder (BD). The expression of ER stress-related genes, spliced form or unspliced form of XBP1, GRP78 (HSPA5), GRP94 (HSP90B1), CHOP (DDIT3), and calreticulin (CALR), were examined in lymphoblastoid cells derived from 59 patients with BD and 59 age- and sex-matched control subjects. Basal mRNA levels and induction by 4 h or 12 h of treatment with two ER stressors, thapsigargin or tunicamycin, were examined using real-time quantitative reverse transcription-polymerase chain reaction. Induction of the spliced form of XBP1 as well as total XBP1 by thapsigargin was significantly attenuated in patients with BD. Induction of GRP94 by thapsigargin was also decreased in the BD group. A haplotype of GRP94, protective against BD, exhibited significantly higher GRP94 expression upon ER stress. This report confirms and extends earlier observations of impaired ER stress response in larger samples of lymphoblastoid cell lines derived from BD patients. Altered ER stress response may play a role in the pathophysiology of BD by altering neural development and plasticity.

Phosphorylated PKR contributes the induction of GRP94 under ER stress

Phosphorylated double-stranded RNA-dependent protein kinase (PKR) is thought to play an important role during ER stress induced cell death, but its molecular mechanism of action has not yet been clarified completely. To resolve this issue, we employed a PKR inhibitor together with ER stress inducers (tunicamycin, thapsigargin, and 2-deoxyglucose) and found that this treatment applied to SK-N-SH and HepG2 cells suppressed the expressional induction of 94 kDa glucose regulated protein (GRP94) but not GRP78 proteins at both protein and mRNA levels. Although GRP94 mRNA increased, no significant difference was observed in the mRNA level of spliced X box binding protein 1 (XBP1) and reporter gene assay using GRP78 and GRP94 promoter with an ER stress response element (ERSE) showed that PKR inhibitor did not affect their activity. These results suggest that a novel mechanism other than ERSE-dependent mRNA transcription is required for the induction of GRP94 and phosphorylation of PKR contributes to the induction of GRP94 under ER stress.

Calpain Inhibition but not Reticulum Endoplasmic Stress Preconditioning Protects Rat Kidneys from p-Aminophenol Toxicity

p-Aminophenol (pAP, 225 mg/kg) administration to rats induced renal failure and has been associated with markers of endoplasmic reticulum (ER) stress, as well as calpain and caspase-12 activation in kidneys. To determine the importance of ER stress and calpain during pAP-induced nephrotoxicity, rats were pretreated with low, nontoxic, doses of ER stress inducers or with the selective calpain inhibitor PD150606 (3 mg/kg). Prior ER stress induced by tunicamycin and oxidized dithiothreitol did not result in protection against renal failure, but PD150606 administration was protective and decreased significantly the rise in creatinine and blood urea nitrogen observed after 24-h post-pAP administration. pAP-induced XBP1 upregulation was not modified by calpain inhibition, but a trend to lower GRP94 induction was determined, suggesting that pAP-induced ER stress was mostly calpain independent. In contrast, pAP-induced caspase-12 cleavage products were significantly decreased with PD150606 pretreatment, demonstrating that caspase-12 activation was calpain dependent. This study reveals the importance of calpain in pAP-induced renal failure. Further research with other nephrotoxicants needs to be performed to determine if calpain activation is a common feature of drug-induced renal failure.

Calpain-Induced Endoplasmic Reticulum Stress and Cell Death following Cytotoxic Damage to Renal Cells

Calpains and endoplasmic reticulum (ER) stress have both been implicated in renal cell death following exposure to reactive chemical toxicants (RCTs). Therefore, we explored the link between ER stress, calpain, and cell death in renal cell injury due to model RCTs (iodoacetamide, menadione, tert-butyl hydroperoxide) and ER stress inducers (tunicamycin [TUN], thapsigargin [THAPS]). The calpain inhibitor, PD150606, significantly reduced the RCT and TUN-induced cell death in the renal cell line LLC-PK1, but not death induced by THAPS. ER stress was confirmed by the significant induction of GRP78 following exposure to RCTs and ER stress inducers. While GRP94 induction was observed following RCTs and TUN, it was not statistically significant because of variability. THAPS at 5 microM significantly induced GRP94, while 20 mmicroM caused a calpain-dependent cleavage of GRP94. Caspase-12 and m-calpain were variably induced and/or cleaved following exposure to all toxicants, supporting activation of these signaling pathways. Inhibition of calpain blocked the induction of GRP78 following exposure to RCTs suggesting that calpain was contributing to the observed ER stress following RCTs. In contrast, calpain inhibition did not block ER stress protein induction following exposure to nontoxic concentrations of TUN or THAPS, indicating that calpain inhibition did not block the ER stress protein induction pathways directly. These studies demonstrate a previously unappreciated link between calpain activation and ER stress-associated cell death in renal cells. While further studies are required to clarify the molecular events involved, these results confirm that calpain activation and the ER are important related players in chemically induced renal cell damage.

De-regulation of GRP stress protein expression in human breast cancer cell lines.

The stress-inducible glucose regulated proteins (GRPs), a class of calcium-binding molecular chaperones localized in the endoplasmic reticulum, have been implicated in the development of tumorigenicity, drug resistance, and cytotoxic immunology. This study investigates the expression pattern of GRP94 and GRP78 in a panel of breast carcinoma cell lines, as compared to two independently derived normal human breast epithelial cell lines. Here we report that a 3- to 5-fold increase in the basal level of the GRP94 protein was observed in all five breast carcinoma cell lines examined. The increase was independent of either the p53 or estrogen receptor status of the breast carcinomas. In carcinoma cells deprived of glucose, mimicking the conditions in poorly vascularized solid tumors, up to 9-fold induction of GRP94 was observed relative to the basal level expressed in a normal breast epithelial cell line. Interestingly, while the majority of the breast cancer cell lines can respond to tunicamycin- and thapsigargin-induced stress by increasing the steady state levels of grp94 and grp78 transcripts, the induction at the GRP protein level is variable and does not always correspond with the transcript level. Further, we discovered that one of the human breast carcinoma cell lines, MCF-7, has specifically lost its ability to respond to tunicamycin stress.

Induction of glucose-regulated protein (glucose-regulated protein 78/B:P and glucose-regulated protein 94) and heat shock protein 70 transcripts in the immature rat brain following status epilepticus

Prior to 21 days of age, the immature rat brain is relatively resistant to excitotoxicity caused by the glutamate analogue, kainate. As stress-inducible proteins (GRP78, GRP94 and HSP70) have been proposed to possess molecular chaperone activity and protect cells from the deleterious effects of damaged proteins, we examined the pattern of expression of their respective messenger RNAs following systemic kainate at different postnatal ages. In untreated rats between seven and 21 days old, there was a higher basal level of grp78 and grp94 expression compared to hsp70. Unlike hsp70, which was inducible only in 21-day-old rats, kainate-mediated grp94 induction occurred in several regions of the brain as early as postnatal day 7. Grp78 messenger RNA expression was also increased by kainate treatment in 14-day-old rats, and the induction was most pronounced in the kainate-resistant dentate gyrus. With increasing age, longer lasting expression of both grp78 and grp94 messenger RNAs was observed in kainate-vulnerable regions, similar to observations in the adult rat brain. These results demonstrate non-overlapping expression patterns of glucose-regulated proteins and HSP70 in the immature central nervous system, suggesting that they serve different functions. While hsp70 induction could be a marker for potential cell injury and death, increased expression of grp78 and grp94 could play a neuroprotective role in the developing rat brain.

Conformation-defective herpes simplex virus 1 glycoprotein B activates the promoter of the grp94 gene that codes for the 94-kD stress protein in the endoplasmic reticulum.

GRP94 is a major glycoprotein in the endoplasmic reticulum with calcium-binding properties. Recently, GRP94 has been shown to bind to unassembled forms of multimeric proteins and peptides. We report here that GRP94 forms a stable association with the mutated form of the herpes simplex type virus 1 (HSV-1) glycoprotein B, but not with the fully processed viral protein. Both the glycosylated and unglycosylated forms of GRP94 are capable of complexing with the mutated, conformation-defective viral glycoprotein. Cotransfection of expression vectors for gB and grp94 promoter fusion genes revealed that the grp94 promoter is strongly activated by the mutant form of gB. Analysis of the grp94 promoter mutants showed that two regions in the promoter, a highly conserved element referred to as grp core and the CCAAT element most proximal to the TATA element (C1), mediate the induction of grp94 by malfolded protein. We further determined that the grp94 core and C1 element bind to common as well distinct nuclear factors from grp78, a commonly coregulated gene. Through UV cross-linking, site competition, and immunocross-reactivity, we identified that the heteromeric CCAAT-binding protein (CBF) is one component of the grp94 C1 complex.

Generation of a Mammalian Cell Line Deficient in Glucose-regulated Protein Stress Induction through Targeted Ribozyme Driven by a Stress-inducible Promoter

GRP94 is an endoplasmic reticulum (ER) localized glycoprotein with Ca2+ binding and protein chaperoning properties. Using a ribozyme driven by a stress-inducible promoter and targeted against grp94 mRNA, we have generated a cell line deficient in its ability to induce GRP94. The effect of the ribozyme is mediated by the cleavage of the grp94 message just downstream of the initiation codon, and not by an antisense effect, as determined by the level of intact grp94 mRNA. Unexpectedly, this cell line's ability to induce GRP78 is also impaired. Transient overexpression of recombinant human lysosomal hydrolase alpha-L-iduronidase in the ribozyme expressing cells indicates that the secretion ratio of this enzyme is reduced by about 6-fold. Additionally, the ribozyme expressing cells showed increased sensitivity to Ca2+ depletion from ER caused by either A23187 or thapsigargin, an ER-Ca(2+)-ATPase inhibitor, but not to tunicamycin. These combined results show that the induction of GRP94 may play important roles in ER to nuclear signaling, protein sorting and secretion, and specific protection against Ca2+ depletion stress.