Role Of GRP94 Research Articles

Grp94 Inhibitor HCP1 Suppressed the Replication of SVA in BHK-21 Cells and PK-15 Cells.

Glucoregulatory protein 94 (Grp94) is necessary for the post-viral life cycle and plays a quality control role in viral proteins, but the role of Grp94 in regulating viral replication in host cells is not well known. Therefore, finding a compound that can regulate Grp94 will help us to study the mechanism of viral replication. Previously, we synthesized a coumarin pyrazoline derivative HCP1 that is an effective inhibitor of Grp94. We suppose that HCP1 may inhibit viral replication. This study aimed to investigate the effect of HCP1 on the replication ability of Senecavirus A (SVA), so as to provide a target and a leading compound for revealing the pathogenic mechanism of the virus and developing antiviral drugs. Rat cell lines BHK-21 and porcine cell lines PK-15 were infected with SVA, and the infected cells were treated with different concentrations of HCP1. The cell viability (CCK-8), virus titer (TCID50), autophagy level, and Grp94 expression were measured. The results showed that a low concentration of HCP1 decreased viral titer and viral load in BHK-21 and PK-15 cells, and 5μM HCP1 significantly decreased the expression of SVA VP2 protein. In addition, SVA infection can lead to an increased level of autophagy, and HCP1 can inhibit host cell autophagy caused by SVA infection, thereby inhibiting viral replication and infection. These findings reveal that Grp94 is a key factor in controlling SVA replication, and its inhibitor HCP1 suppresses SVA replication by inhibiting the increase of Grp94 protein level and autophagy induced by SVA. This study will contribute to the development of a new class of small-molecule antiviral drugs.

The HSP GRP94 interacts with macrophage intracellular complement C3 and impacts M2 profile during ER stress

The role of GRP94, an endoplasmic reticulum (ER) stress protein with both pro- and anti-inflammatory functions, has not been investigated in macrophages during ER stress, whereas ER stress has been reported in many diseases involving macrophages. In this work, we studied GRP94 in M1/LPS + IFNγ and M2/IL-4 primary macrophages derived from human monocytes (isolated from buffy coats), in basal and ER stress conditions induced by thapsigargin (Tg), an inducer of ER calcium depletion and tunicamycin (Tm), an inhibitor of N-glycosylation. We found that GRP94 was expressed on the membrane of M2 but not M1 macrophages. In M2, Tg, but not Tm, while decreased GRP94 content in the membrane, it induced its secretion. This correlated with the induction of a pro-inflammatory profile, which was dependent on the UPR IRE1α arm activation and on a functional GRP94. As we previously reported that GRP94 associated with complement C3 at the extracellular level, we analyzed C3 and confirmed GRP94-C3 interaction in our experimental model. Further, Tg increased this interaction and, in these conditions, C3b and cathepsin L were detected in the extracellular medium where GRP94 co-immunoprecipitated with C3 and C3b. Finally, we showed that the C3b inactivated fragment, iC3b, only present on non-stressed M2, depended on functional GRP94, making both GRP94 and iC3b potential markers of M2 cells. In conclusion, our results show that GRP94 is co-secreted with C3 under ER stress conditions which may facilitate its cleavage by cathepsin L, thus contributing to the pro-inflammatory profile observed in stressed M2 macrophages.

High expression of endoplasmic reticulum chaperone grp94 is a novel molecular hallmark of malignant plasma cells in multiple myeloma.

BackgroundMultiple myeloma (MM) is a hematologic malignancy that is characterized by the proliferation of abnormal bone marrow plasma cells (BMPC) and overproduction of immunoglobulin or light chains with evidence of end-organ damage such as bone damage, anemia, hypercalcemia, and renal dysfunction. The pathogenesis of MM is closely linked to dysregulated unfolded protein response (UPR) in the endoplasmic reticulum (ER). Constitutive activation of UPR in mice, as demonstrated by transgenic expression of a master UPR transcription factor XBP1s (a UPR-specific splice variant of X-box binding protein 1), causes myeloma. grp94 (gp96) is a key downstream chaperone in the ER that mediates the UPR as a part of the protein quality control mechanism in the secretory pathway. Our recent study has shown that the persistence of plasma cells as well as the development of myeloma in XBP1s-transgenic mice is critically dependent on grp94. However, the role of grp94 in the initiation and progression of human MM is still unknown.MethodsThe expression level of grp94 in BMPCs was measured by flow cytometry, real-time RT-PCR, and Western blot analysis. We compared the expression levels of grp94 in BMPCs in a spectrum of patients including MM, monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), as well as non-plasma cell disorders (NPC).ResultsWe found that grp94 was highly expressed in malignant plasma cells in patients with MM, but not in BMPCs in patients with MGUS/SMM and NPC. The expression level of grp94 correlated significantly with CD138 expression level. We also found that the grp94 expression level in BMPCs from International Staging System (ISS) stage III MM patients is higher than those in ISS stage I/II MM patients.Conclusionsgrp94 is highly expressed in BMPCs in MM, which correlates with the advanced stage of this disease. Our data demonstrated that grp94 is a novel diagnostic and prognostic biomarker. It also positioned grp94 as a promising therapeutic target for MM.

Inhibition of immunoglobulin secretion from peripheral blood mononuclear cells by glucose-regulated protein94 (Grp94) in allergic subjects

Glucose-regulated protein94 (Grp94) is the most represented endoplasmic reticulum-resident HSP with the unique property to modulate the immune response. This has opened the way to the use of Grp94 as effective therapeutic agent in both depressed and exaggerated activity of the immune system. We investigated the effect of native Grp94 on peripheral blood mononuclear cells (PBMCs) isolated from blood of two subjects with a different history of bronchial allergic asthma. Whereas in subject 1 an elevated basal level of Ig and altered morphological aspects of PBMCs suggested an intense antigen-driven stimulation of the immune system, subject 2 had an apparently normal basal humoral response. However, Grp94 reduced in a concentration-dependent manner the Ig secretion from PBMCs of both subjects, inhibition being maximal at 100 ng/ml Grp94 after 15 days. The effect was apparently related to inhibition of intra-cellular content of both IgG and IgE, and in subject 1 was still observed a year after the first examination. Dot-blot experiments revealed the presence of anti-Grp94 antibodies in Ig secreted from PBMCs and in plasma of both subjects, confirming the role of Grp94 as antigen responsible for activation of the immune system, even in the absence of clinical signs of asthma. Anti-Grp94 antibodies significantly decreased after PBMC treatment with Grp94 at 100 ng/ml. Results demonstrate that inhibition of the humoral response by Grp94 crucially depends on being Grp94, the antigen challenging the immune system in these allergic subjects, thus supporting the role of Grp94 as immuno-modulatory agent in pathologies with exaggerated immune response.

Overexpression of glucose-regulated protein 94 after spinal cord injury in rats

Glucose-regulated protein (GRP) 94 is a member of the stress protein family, which is localized in the endoplasmic reticulum (ER). Spinal cord injury (SCI) induced ER stress that results in apoptosis. However, the role of GRP94 in injury of the central nervous system remains unknown. In this study, we performed SCI in adult rats and investigated acutely the protein expression and cellular localization of GRP94 in the spinal cord. Western blot analysis revealed that GRP94 was low in normal spinal cord. It rose at 6 h after SCI, peaked at 1 day, remained for another 3 days, then declined to basal levels at 5 days after injury. Immunohistochemistry further confirmed that GRP94 immunoactivity was expressed at low levels in gray matter and white matter in normal condition and increased after SCI. Double immunofluorescence staining showed that GRP94 was co-expressed with NeuN (neuronal marker), and GFAP (astroglial marker). In addition, caspase-12, caspase-3 and phospho-c-Jun NH2-kinase (p-JNK) levels increased at 6 h, peaked at 1 day, and then gradually reduced to normal levels for 2 weeks after SCI by western blot analysis. Co-localization of GRP94/caspase-12 and GRP94/p-JNK was detected in neurons and glial cells. Taken together, these data suggest GRP94 involvement in the injury response of the adult spinal cord of the rats.

Silencing of GRP94 expression promotes apoptosis in pancreatic cancer cells

As a molecular chaperone, GRP94 is the most abundant glycoprotein in the endoplasmic reticulum, playing an important role in maintaining cellular homeostasis. Here, we investigated the expression and the role of GRP94 in regulating cell growth and apoptosis in pancreatic cancer cells. GRP94 mRNA levels were analyzed by QRT-PCR. Immunohistochemistry was performed to localize GRP94 in tissues of the normal pancreas (n=20), chronic pancreatitis (n=20) and pancreatic ductal adenocarcinoma (n=44). Silencing of GRP94 expression was carried out by transfection with specific siRNA oligonucleotides. Apoptosis was induced by treatment with actinomycin D. Compared to normal pancreatic tissues, median mRNA levels of GRP94 were 1.5- and 3.7-fold (p<0.05) lower in chronic pancreatitis and pancreatic cancer tissues, respectively. GRP94 protein was strongly expressed in normal acinar cells and moderately expressed in normal ductal cells. GRP94 expression was lost in 48% of the cancer cases. Moderate or strong staining in cancer cells was observed in 32 and 20% of pancreatic cancer tissues, respectively. Silencing GRP94 by siRNA increased apoptosis of pancreatic cancer cells in vitro. Patients with higher than the median expression have a tendency for a worsened survival. When the small number of patients with the highest expression (n=3) were compared with the rest of the group (n=41), the survival difference was significantly worse (5 vs. 18 months, respectively, p=0.006). Down-regulation of GRP94 decreases apoptosis resistance in pancreatic cancer cells. Clinically, patients with high GRP94 expression show a tendency for a worsened survival.

Geldanamycin, an hsp90/GRP94-binding drug, induces increased transcription of endoplasmic reticulum (ER) chaperones via the ER stress pathway.

Geldanamycin, a benzoquinone ansamycin, binds specifically to hsp90 and GRP94 in vitro and in vivo. Treatment of cells with geldanamycin alters the molecular chaperone function of hsp90, and as a result, blocks certain cytosolic proteins from reaching their mature form, inhibits their activity, and/or affects their stability. In contrast, little is known about either the effects of geldanamycin on GRP94, the endoplasmic reticulum (ER) homologue of hsp90, or the role of GRP94 in protein folding. In this study, we demonstrate in a variety of cell lines that geldanamycin is a potent inducer of the cellular response to stress in the ER, resulting in the transcriptional up-regulation of ER chaperones and expression of the gadd153/CHOP transcription factor. Their induction occurs through the unfolded protein response pathway originating in the ER and is not due to effects of the drug on hsp90. Geldanamycin increases the association of nascent proteins with BiP, which indicates that their folding and/or assembly has been altered. These data suggest that GRP94 may play an essential role in the maturation of a number of secretory pathway proteins.