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。     

Down-regulation of glucose-regulated protein (GRP) 78 potentiates cytotoxic effect of celecoxib in human urothelial carcinoma cells

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor that has been reported to elicit anti-proliferative response in various tumors. In this study, we aim to investigate the antitumor effect of celecoxib on urothelial carcinoma (UC) cells and the role endoplasmic reticulum (ER) stress plays in celecoxib-induced cytotoxicity. The cytotoxic effects were measured by MTT assay and flow cytometry. The cell cycle progression and ER stress-associated molecules were examined by Western blot and flow cytometry. Moreover, the cytotoxic effects of celecoxib combined with glucose-regulated protein (GRP) 78 knockdown (siRNA), (-)-epigallocatechin gallate (EGCG) or MG132 were assessed. We demonstrated that celecoxib markedly reduces the cell viability and causes apoptosis in human UC cells through cell cycle G1 arrest. Celecoxib possessed the ability to activate ER stress-related chaperones (IRE-1α and GRP78), caspase-4, and CCAAT/enhancer binding protein homologous protein (CHOP), which were involved in UC cell apoptosis. Down-regulation of GRP78 by siRNA, co-treatment with EGCG (a GRP78 inhibitor) or with MG132 (a proteasome inhibitor) could enhance celecoxib-induced apoptosis. We concluded that celecoxib induces cell cycle G1 arrest, ER stress, and eventually apoptosis in human UC cells. The down-regulation of ER chaperone GRP78 by siRNA, EGCG, or proteosome inhibitor potentiated the cytotoxicity of celecoxib in UC cells. These findings provide a new treatment strategy against UC.     

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.     

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.     

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 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.     

Endothelin-1 stimulates c-fos mRNA expression and acts as a modulator on cell proliferation of rat FRTL5 thyroid cells.

In FRTL5 thyroid cells, endothelin (ET)-1 alone had no effect on DNA synthesis but caused a transient increase in c-fos mRNA levels and stimulated IGF-I induced DNA synthesis and cell proliferation. By contrast, ET-1 inhibited the stimulatory effects of TSH actions on DNA synthesis, cell proliferation and c-AMP production. 8-Bromo-cAMP-induced DNA synthesis was also inhibited by ET-1, suggesting that ET-1 exerts its inhibitory effects at step(s) involving cAMP production and post cAMP pathway. ET-1-induced suppression of TSH actions were reversed by a C-kinase inhibitor, H-7. These results suggest that the effect of ET on functions of FRTL5 cells is, at least, in part mediated by C-kinase dependent pathway.     

Studies on the promoter region of the c-Ha-ras gene in FRTL5 rat thyroid cells

The functional activity of the promoter region of the rat c-Ha-ras gene was examined in FRTL5 rat thyroid cells, the cell type from which this promoter was cloned. A plasmid (p035-ras-CAT) was constructed containing the untranslated-1 exon as well as 172 base pairs (bp)5' to this exon inserted upstream of the chloramphenicol acetyl transferase (CAT) reporter gene. These 172 bp of 5'-flanking region contain two 10 bp GC box consensus sites and two CAAT boxes. Very weak promoter activity was observed in experiments involving transient transfection of FRTL5 cells with this plasmid, as well as with another plasmid (p5kb-ras-CAT) containing a much more extensive (3.5 kb) 5'-flanking region of the gene. In contrast, strong promoter activity was observed when the same plasmids were transfected into mouse 3T3 fibroblasts. When other promoters (pfos, RSV, and MMTV) were used to drive CAT activity, CAT activity in FRTL5 cells was about 10-fold less than in NIH-3T3 cells and rat embryo fibroblasts. However c-Ha-ras promoter activity was reduced out of proportion in FRTL5 thyroid cells relative to the other cell types (approximately 50-fold less). DNA gel-shift assays performed using crude extracts of FRTL5 and 3T3 nuclear proteins revealed quantitatively similar binding to the same promoter region in the c-Ha-ras 5'-flanking sequence. These data demonstrate that promoter activity of the rat c-Ha-ras gene is contained within the 172 bp 5'-flanking region of the gene. This promoter activity is expressed at a much lower level in slow-growing FRTL5 cells relative to other more rapidly growing cell types.     

Thyrotropin receptor gene expression in oncogene-transfected rat thyroid cells: correlation between transformation, loss of thyrotropin-dependent growth, and loss of thyrotropin receptor gene expression.

Rat FRTL-5 and PC-Cl-3 thyroid cells are continuously cultured, clonal lines which require thyrotropin to grow and function. Both can be efficiently transformed when infected with RNA or DNA viruses carrying oncogenes or when directly transfected with activated oncogenes. Transformation, assayed by the appearance of cell growth in agar and by tumorigenicity in syngeneic rats or nude mice, is associated with the loss of thyrotropin-dependent cell division and thyrotropin-regulated functions such as thyroglobulin synthesis. In 16 clones of FRTL-5 or PC-Cl-3 cells transformed with different oncogenes, we show that loss of thyrotropin-dependent growth and function correlates with the loss of thyrotropin receptor gene expression, measured with a rat thyrotropin receptor cDNA probe.     

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.