内质网分子伴侣GRP78在小鼠脑发育过程中的时空表达

多细胞生物体发育的实质是基因的选择性表达,表达蛋白的成熟有赖于分子伴侣的协助,但迄今分子伴侣特别是内质网分子伴侣在脑发育过程中的作用尚不清楚。本文应用RT—PCR、蛋白质免疫印迹和免疫组织化学的方法研究了小鼠发育不同阶段的脑组织中GRP78的表达和定位分布随时间变化的情况。结果发现GRP78在小鼠脑发育过程中呈时空性表达：胚胎早期表达较高,胚胎发育末期逐渐下降,出生后逐渐升高,至生后一周时达到成年鼠的水平;在胚胎期GRP78蛋白在脑组织的表达呈现从端脑到后脑逐渐降低的趋势。研究结果还表明GRP78蛋白的免疫染色阳性产物在神经细胞中出现的时间早于神经胶质细胞。这些首次观察到的结果提示GRP78与脑的早期发生和形态建成有一定的关系。     

Enhanced accumulation of mRNA for 78-kilodalton glucose-regulated protein (GRP78) in tissues of nonobese diabetic mice

Using 78-kildalton glucose-regulated protein cDNA as a probe of Northern blots, we have examined the distribution and inducibility of mRNA encoding the 78-kilodalton glucose-regulated protein in three tissues of nonobese diabetic mice. The gene was constitutively expressed in normal, unstressed cells of liver, brain, and spleen. Developing diabetes correlated with elevated expression in only liver and brain of diabetic mice. This induction of gene expression was associated with the transition from the prediabetic stage to the onset of hyperglycemia and coincided with falling levels of plasma insulin and rising hyperglycemia. The activation of 78-kilodalton glucose-regulated protein gene expression appeared to be transient. We suggest that the temporally differential, tissue-specific expression of this gene in adult nonobese diabetic mice offers an opportunity to study a physiologically relevant regulation of this stress-induced gene.     

Overexpression of the 78-kDa glucose-regulated protein/immunoglobulin-binding protein (GRP78/BiP) inhibits tissue factor procoagulant activity

Previous studies have demonstrated that overexpression of GRP78/BiP, an endoplasmic reticulum (ER)-resident molecular chaperone, in mammalian cells inhibits the secretion of specific coagulation factors. However, the effects of GRP78/BiP on activation of the coagulation cascade leading to thrombin generation are not known. In this study, we examined whether GRP78/BiP overexpression mediates cell surface thrombin generation in a human bladder cancer cell line T24/83 having prothrombotic characteristics. We report here that cells overexpressing GRP78/BiP exhibited significant decreases in cell surface-mediated thrombin generation, prothrombin consumption and the formation of thrombin-inhibitor complexes, compared with wild-type or vector-transfected cells. This effect was attributed to the ability of GRP78/BiP to inhibit cell surface tissue factor (TF) procoagulant activity (PCA) because conversion of factor X to Xa and factor VII to VIIa were significantly lower on the surface of GRP78/BiP-overexpressing cells. The additional findings that (i) cell surface factor Xa generation was inhibited in the absence of factor VIIa and (ii) TF PCA was inhibited by a neutralizing antibody to human TF suggests that thrombin generation is mediated exclusively by TF. GRP78/BiP overexpression did not decrease cell surface levels of TF, suggesting that the inhibition in TF PCA does not result from retention of TF in the ER by GRP78/BiP. The additional observations that both adenovirus-mediated and stable GRP78/BiP overexpression attenuated TF PCA stimulated by ionomycin or hydrogen peroxide suggest that GRP78/BiP indirectly alters TF PCA through a mechanism involving cellular Ca(2+) and/or oxidative stress. Similar results were also observed in human aortic smooth muscle cells transfected with the GRP78/BiP adenovirus. Taken together, these findings demonstrate that overexpression of GRP78/BiP decreases thrombin generation by inhibiting cell surface TF PCA, thereby suppressing the prothrombotic potential of cells.     

ER-stress-induced secretion of circulating glucose-regulated protein 78kDa (GRP78) ameliorates pulmonary artery smooth muscle cell remodelling

Pulmonary arterial hypertension (PAH) is driven by vascular remodelling due to inflammation and cellular stress, including endoplasmic reticulum stress (ER stress). The main ER-stress chaperone, glucose-regulated protein 78 kDa (GRP78), is known to have protective effects in inflammatory diseases through extracellular signalling. The aim of this study is to investigate its significance in PAH. Human pulmonary arterial smooth muscle cells (PASMC) were stimulated with compounds that induce ER stress, after which the secretion of GRP78 into the cell medium was analysed by western blot. We found that when ER stress was induced in PASMC, there was also a time-dependent secretion of GRP78. Next, naïve PASMC were treated with conditioned medium (CM) from the ER-stressed donor PASMC. Incubation with CM from ER-stressed PASMC reduced the viability, oxidative stress, and expression of inflammatory and ER-stress markers in target cells. These effects were abrogated when the donor cells were co-treated with Brefeldin A to inhibit active secretion of GRP78. Direct treatment of PASMC with recombinant GRP78 modulated the expression of key inflammatory markers. Additionally, we measured GRP78 plasma levels in 19 PAH patients (Nice Group I) and correlated the levels to risk stratification according to ESC guidelines. Here, elevated plasma levels of GRP78 were associated with a favourable risk stratification. In conclusion, GRP78 is secreted by PASMC under ER stress and exhibits protective effects from the hallmarks of PAH in vitro. Circulating GRP78 may serve as biomarker for risk adjudication of patients with PAH.Graphical abstractProposed mechanism of ER-stress-induced GRP78 secretion by PASMC. Extracellular GRP78 can be measured as a circulating biomarker and is correlated with favourable clinical characteristics. Conditioned medium from ER-stressed PASMC reduces extensive viability, ROS formation, inflammation, and ER stress in target cells. These effects can be abolished by blocking protein secretion in donor cells by using Brefeldin A. Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-022-01292-y.     

The nucleotide sequence encoding the hamster 78-kDa glucose-regulated protein (GRP78) and its conservation between hamster and rat

The complete nucleotide (nt) sequence encoding the hamster 78-kDa glucose-regulated protein has been determined using a cDNA plasmid p3C5. Comparison of the nucleotide sequences from rat and hamster showed a strong conservation in the coding region as well as 5'- and 3'-untranslated regions (UTRs). The relatively long (206 nt for rat) 5'UTR shares 72% sequence homology between rat and hamster in the 142 nt upstream from the ATG start codon. This conserved region contained an imperfect inverted-repeat sequence. The long 5'UTR region is capable of forming stable dyad structures. The homology within the rat and hamster protein-coding region is 93.7%, with most of the differences resulting in silent site mutations. Out of the 654 amino acids, only four changes are detected, two of which are located in the signal peptide. While the sizes of the 3'UTR are different between the two species compared, strong sequence homologies (95%) were observed throughout the entire UTRs. Also, the 3'UTR was not rich in A + T residues as found in other eukaryotic mRNAs.     

The rat 78,000 dalton glucose-regulated protein (GRP78) as a precursor for the rat steroidogenesis-activator polypeptide (SAP): the SAP coding sequence is homologous with the terminal end of GRP78

Based on the striking sequence identity between the amino acid sequence of rat steroidogenesis-activator polypeptide (SAP) and the carboxyl terminus of the 78,000 dalton glucose-regulated protein (GRP78), the precursor-product relationship between GRP78 and SAP was investigated in Leydig cells. Immunoblot analysis with peptide antibodies specific for GRP78 and SAP showed that the putative SAP precursor is also immunoreactive with the anti-GRP78 antibody. Genomic blot hybridizations further revealed that GRP78 is neither rearranged nor amplified in the H-540 Leydig cell tumor, the original source for SAP. Further, there appears to be a single copy of the SAP coding sequence within the rat genome. This sequence resides within the last exon of GRP78. Our observations support the hypothesis that, in steroidogenic cells, SAP is likely to be derived from posttranslational processing of a very minor fraction of GRP78.     

Coordinated induction of two unrelated glucose-regulated protein genes by a calcium ionophore: human BiP/GRP78 and GAPDH

The induction of human BiP/GRP78 and GAPDH protein genes by the calcium ionophore A23187 was determined. Steady-state levels of mRNA for both the glucose starvation-responsive BiP/GRP78 gene and the glucose-responsive GAPDH gene were dramatically induced in a variety of human cells. There is a homologous palindromatic sequence GCCGTTAACGGC in the active promoter region of the two genes that is known to be required for the induction of mammalian BiP/GRP78 by A23187. The evidence confirms in general the function of this element in the regulation of calcium-associated gene activity.     

Analysis of the expression of a glucose-regulated protein (GRP78) promoter/CAT fusion gene during early Xenopus laevis development

Developmental regulation of the expression of a glucose-regulated gene encoding a 78 kd protein, GRP78, has been characterized by microinjection of a rat GRP78/CAT chimeric gene into early Xenopus embryos. Tunicamycin-induced expression of the chimeric gene during Xenopus development was similar to the pattern of endogenous GRP78 protein synthesis, with expression first being detected at gastrula and increasing at least until the tailbud stage. Deletion analysis of the rat GRP78 promoter revealed that sequences between -154 and -130 were necessary for full tunicamycin-inducible and constitutive expression of the fusion gene. These results suggest that there is conservation of regulatory elements of the GRP78 promoter between rat and Xenopus.     

Expression of glucose-regulated proteins (GRP78 and GRP94) in hearts and fore-limb buds of mouse embryos exposed to hypoglycemia in vitro

Hypoglycemia, the classic inducer of glucose-related protein (GRP) synthesis, is dysmorphogenic in rodent embryos and detrimentally affects the heart. This study compares GRP induction in a target vs non-target tissue by evaluating GRP expression in hearts and fore-limb buds of mouse embryos following exposure to hypoglycemia in vitro. Gestational day 9.5 embryos were exposed to 2, 6, and 24 h of either mild (80 mg/dl glucose) or severe (40 mg/dl glucose) hypoglycemia using the method of whole-embryo culture. GRP78 increased in a dose- and time-dependent fashion in embryonic hearts exposed to either 40 mg/dl or 80 mg/dl glucose, whereas GRP94 levels increased in hearts only after 24 h of hypoglycemia. In contrast to the heart, GRP induction in fore-limb buds occurred only with GRP78 following the most severe level and duration of hypoglycemia. RT-PCR analysis demonstrated an elevation in GRP78 and GRP94 message levels in embryonic hearts following severe hypoglycemia. However, mRNA levels did not increase in response to mild hypoglycemia. Overall, these data demonstrate the preferential induction of GRPs in the heart as compared to fore-limb buds in mouse embryos exposed to hypoglycemia. Increases in GRP protein levels may be a more reliable biomarker of stress than message levels. However, both tissues and methods should be examined for enhanced biomarker sensitivity.     

The 78 kDa glucose-regulated protein (GRP78/BIP) is expressed on the cell membrane,is released into cell culture medium and is also present in human peripheral circulation

In human rabdomiosarcoma cells (TE671/RD) chronic exposure to 500 nM thapsigargin (a powerful inhibitor of the endoplasmic reticulum Ca²⁺-ATPases) resulted in the induction of the stress protein GRP78/BIP. Making use of the surface biotinylation method, followed by the isolation of the GRP78 using ATP-agarose affinity chromatography, it was found that a fraction of the thapsigargin-induced GRP78 is expressed on the cell surface. The presence of GRP78 on the membrane of thapsigargin-treated cells was confirmed by fractionation of cell lysates into a soluble and a membrane fraction, followed by Western blot analysis with an anti-GRP78 antibody. It was also found that conspicuous amounts of GRP78 are present in the culture medium collected from thapsigargin-treated cultures. This extracellular GRP78 originates mostly by an active release from intact cells and does not result solely from the leakage of proteins from dead cells. Moreover, small amounts of circulating, free GRP78 and naturally-occurring anti-GRP78 autoantibodies were detected in the peripheral circulation of healthy human individuals.     

GRP78 induction by cyclosporin A in human HeLa cells

Immunosuppressive drugs such as cyclosporin A (CsA) and FK506 are known to have pleiotropic effects on cells. Here we demonstrate that treatment of HeLa cells with low concentrations of CsA (but not of FK506) induces the synthesis of a stress protein, GRP78, located inside the endoplasmic reticulum. High concentrations of CsA lead to a general decrease in protein synthesis. When cells are stressed (heat-shocked) during the CsA treatment, the synthesis of heat shock proteins is reinforced. FK506 has no detectable effects at any concentration. The mechanism of induction of GRP78 by CsA remains presently unknown. Whatever the mechanism involved, GRP78 overexpression might be responsible for some of the physiological effects of CsA.     

The interaction between ubiquitin C-terminal hydrolase 37 and glucose-regulated protein 78 in hepatocellular carcinoma

The ubiquitin C-terminal hydrolase (UCH) is a subfamily of deubiquitinating enzymes, which consists of four members: UCH-L1, UCH-L3, UCH37, and BRCA1-associated protein-1. Although there is growing evidence that UCH enzymes and human malignancies are closely correlated, there have been few studies on UCH37, especially on its interactions with other proteins. In the current study, a functional proteomic analysis was performed to screen UCH37-interacting proteins in hepatocellular carcinoma (HCC), and glucose-regulated protein 78 was identified as one interacting with UCH37, which was confirmed by co-immunoprecipitation and confocal laser scanning microscopy analysis, suggesting that their interaction could provide a new insight into the mechanism of HCC.     

Novel mechanism of anti-apoptotic function of 78-kDa glucose-regulated protein (GRP78): endocrine resistance factor in breast cancer, through release of B-cell lymphoma 2 (BCL-2) from BCL-2-interacting killer (BIK)

Activation of the intrinsic apoptotic pathway represents a major mechanism for breast cancer regression resulting from anti-estrogen therapy. The BH3-only protein BIK is inducible by estrogen-starvation and anti-estrogen treatment and plays an important role in anti-estrogen induced apoptosis of breast cancer cells. BIK is predominantly localized to the endoplasmic reticulum where it regulates BAX/BAK-dependent release of Ca(2+) from the endoplasmic reticulum stores and cooperates with other BH3-only proteins such as NOXA to cause rapid release of cytochrome c from mitochondria and activate apoptosis. BIK is also known to inactivate BCL-2 through complex formation. Previously, we demonstrated that apoptosis triggered by BIK in estrogen-starved human breast cancer cells is suppressed by GRP78, a major endoplasmic reticulum chaperone. Here we described the isolation of a novel clonal human breast cancer cell line (MCF-7/BUS-10) resistant to long-term estrogen deprivation. These cells exhibit elevated level of GRP78, which protects them from estrogen starvation-induced apoptosis. Our studies revealed that overexpression of GRP78 suppresses apoptosis induced by BIK and NOXA, either alone or in combination. Surprisingly, the interaction of GRP78 with BIK does not require its BH3 domain, which has been implicated in all previous BIK protein interactions. We further showed GRP78 and BCL-2 form independent complex with BIK and that increased expression of GRP78 decreases BIK binding to BCL-2. Our findings provide the first evidence that GRP78 can decrease BCL-2 sequestration by BIK at the endoplasmic reticulum, thus uncovering a potential new mechanism whereby GRP78 confers endocrine resistance in breast cancer.     

Involvement of androgen receptor and glucose-regulated protein 78 kDa in human hepatocarcinogenesis

Previous studies demonstrated that androgen receptor (AR) is expressed in human hepatocellular carcinoma (HCC), one of the male-dominant diseases. Glucose-regulated protein 78 kDa (GRP78/Bip), which has a role in cancer development, is one of the androgen response genes in prostate cell lines. The aim of this study was to investigate the impact of AR on endoplasmic reticulum (ER)-stress signaling in human hepatoma. AR and GRP78 expressions were examined in human liver tissue panels. Human hepatoma cells stably expressing short hairpin RNA targeting AR and cells over-expressing AR were generated. The expressions of ER-stress molecules and AR were measured by real-time RT-PCR and Western blotting. The effect of AR on ER-stress responsive gene expression was examined by reporter assay. Strong positive correlation between AR mRNA and GRP78 mRNA was observed in stage I/II-HCCs. AR enhanced ER-stress responsive element activities and GRP78 expression, and regulated ER-stress response in hepatocytes. Sorafenib strongly induced significant apoptosis in HepG2 cells by the inhibition of AR and inhibition of the downstream GRP78. AR seems a co-regulator of GRP78 especially in earlier-stage HCC. AR plays a critical role in controlling ER-stress, providing new therapeutic options against HCC.     

Binding to membrane proteins within the endoplasmic reticulum cannot explain the retention of the glucose-regulated protein GRP78 in Xenopus oocytes.

We have studied the compartmentation and movement of the rat 78-kd glucose-regulated protein (GRP78) and other secretory and membrane proteins in Xenopus oocytes. Full length GRP78, normally found in the lumen of rat endoplasmic reticulum (ER), is localized to a membraneous compartment in oocytes and is not secreted. A truncated GRP78 lacking the C-terminal (KDEL) ER retention signal is secreted, although at a slow rate. When the synthesis of radioactive GRP78 is confined to a polar (animal or vegetal) region of the oocyte and the subsequent movement across the oocyte monitored, we find that both full-length and truncated GRP78 move at similar rates and only slightly slower than a secretory protein, chick ovalbumin. In contrast, a plasma membrane protein (influenza haemagglutinin) and two ER membrane proteins (rotavirus VP10 and a mutant haemagglutinin) remained confined to their site of synthesis. We conclude that the retention of GRP78 in the ER is not due to its tight binding to a membrane-bound receptor.