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The vertebrate neuromuscular junction (NMJ) is considered as a “tripartite synapse” consisting of a motor axon terminal, a muscle endplate, and terminal Schwann cells that envelope the motor axon terminal. The neuregulin 1 (NRG1)-ErbB2 signaling pathway plays an important role in the development of the NMJ. We previously showed that Grb2-associated binder 1 (Gab1), a scaffolding mediator of receptor tyrosine kinase signaling, is required for NRG1-induced peripheral nerve myelination. Here, we determined the role of Gab1 in the development of the NMJ using muscle-specific conditional Gab1 knockout mice. The mutant mice showed delayed postnatal maturation of the NMJ. Furthermore, the selective loss of the gab1 gene in terminal Schwann cells produced delayed synaptic elimination with abnormal morphology of the motor endplate, suggesting that Gab1 in both muscles and terminal Schwann cells is required for proper NMJ development. Gab1 in terminal Schwann cells appeared to regulate the number and process elongation of terminal Schwann cells during synaptic elimination. However, Gab2 knockout mice did not show any defects in the development of the NMJ. Considering the role of Gab1 in postnatal peripheral nerve myelination, our findings suggest that Gab1 is a pleiotropic and important component of NRG1 signals during postnatal development of the peripheral neuromuscular system.
Subject(s)
Animals , Mice , Mice, Knockout , Motor Endplate , Muscle, Skeletal , Muscles , Myelin Sheath , Neuregulin-1 , Neuromuscular Junction , Peripheral Nerves , Presynaptic Terminals , Protein-Tyrosine Kinases , Schwann Cells , Synapses , VertebratesABSTRACT
Background and purpose:More and more evidence has showed that Grb2 binding protein-2 (Gab2) is associated with tumor invasion and metastasis. However, the relationship between Gab2 and epithelial-mesenchymal transition (EMT) in breast cancer is not clear. The aim of this study is to investigate the effect of Gab2 on EMT markers and the mechanism of Gab2 on breast cancer invasion and metastasis.Methods:Immunohistochemical methods were used to detect the expressions of Gab2, E-cadherin and vimentin in 80 cases of breast cancer tissues, and the correlations between them were analyzed. Western blot was used to detect the expression of Gab2 in breast tissues. After MDA-MB-231 cells were transfected with siRNA plasmid, wound healing assay was used to detect the invasive ability of transfected cells induced by epithelial growth factor (EGF) in vitro. Then Western blot was used to analyze the protein expressions of E-cadherin, vimentin, phosphorylated GSK-3β (p-GSK-3β) and nuclear Snail.Results:Gab2 was negatively correlated with the expression of E-cadherin and positively correlated with the expression of vimentin in breast cancer tissues (P<0.05). The expression of Gab2 in breast cancer tissues was higher than that in normal breast tissues adjacent to breast cancer. In vitro, Gab2 expression was significantly knocked down in MDA-MB-231 cells transfected with Gab2 siRNA plasmid (SiGab2/MDA-MB-231cells). Meanwhile, the invasive ability of SiGab2/MDA-MB-231cells was decreased with EGF stimulation. The expression of E-cadherin was increased in SiGab2/MDA-MB-231cells. However, the expressions of vimentin, p-GSK-3β and nuclear Snail were decreased in SiGab2/MDA-MB-231cells.Conclusion:Gab2 can promote the invasion and metastasis of breast cancer by EMT through GSK-3β/Snail signaling pathway.
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AIM:To investigate the expression of Grb2-associated binding protein 2 (Gab2) in human osteo-sarcoma cells and its relationship with the invasion and metastases of human osteosarcoma cells.METHODS: The tech-nique of small RNA interference was used to transfect human osteosarcoma U2-OS cell lines.Western blotting and RT-PCR were used to detect the protein and mRNA expression of Gab2 in transfected U2-OS cells.After transfection, through chem-otaxis and invasion assays in vitro, the cell migration and invasion abilities were detected.RESULTS:After transfection, the expression of Gab2 at mRNA and protein levels in Gab2 siRNA transfected cells ( SiGab2/U2-OS) was lower than that in scrambled siRNA transfected cells ( Scr/U2-OS ) and U2-OS cells.After stimulation with epidermal growth factor ( EGF) at concentration of 10 μg/L, the migration SiGab2/U2-OS cells was significantly less than Scr/U2-OS cells and U2-OS cells ( P<0.01 ) .The number of invasion cells of SiGab2/U2-OS group was significantly lower than the other 2 control groups ( P<0.01) .CONCLUSION:Inhibition of Gab2 expression obviously attenuates the migration and invasion abilities of human osteosarcoma U2-OS cell line.
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ObjectiveTo investigate the expression of growth factor receptor-bound protein-2 (Grb2), matrix metalloproteinase-3 (MMP-3), and MMP-9 in cholangiocarcinoma and its significance. MethodsThe expression of Grb2, MMP-3, and MMP-9 in cholangiocarcinoma tissues of 47 cases and normal tissues was measured using immunohistochemistry, and the correlations of Grb2 expression with clinical pathology and MMP-3 and MMP-9 expression were analyzed. Comparison of continuous data was made using t test, and the correlation of Grb2 expression with MMP-3 and MMP-9 expression was analyzed using the multivariate linear regression model. ResultsThe expression of Grb2 in cholangiocarcinoma tissues was significantly higher than that in normal bile duct tissues (t=5935, P＜0.001); the expression of Grb2 in cholangiocarcinoma tissues and normal bile duct tissues showed no significant correlation with age, sex, and differentiation level; the expression of Grb2 in cholangiocarcinoma tissues with lymph node or distant metastasis was significantly higher than that in cholangiocarcinoma tissues without metastasis (t=3.882, P=0.003). The expression of Grb2 was positively correlated with the expression of MMP-3 and MMP-9 (r2=0.3667, P=0.018; r2=0.5133, P=0.007). ConclusionThe expression of Grb2 in cholangiocarcinoma tissues is higher than that in normal bile duct tissues, and it is closely related to the invasion and metastasis of carcinoma. Further study shows that the expression of Grb2 is positively correlated with the expression of MMP-3 and MMP-9.
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Objective: To testify the location of recombinant adenovirus Ad5/F35-HF2S in chronic myeloid leukemia K562 cells and its interaction with the fusion protein Bcr-Abl. Methods: The recombinant adenovirus Ad5/F35-HF2S was infected into K562 cells, then the expression of fusion protein HF2S in K562 cells was assessed by RT-PCR (reverse transcription-PCR) and Western blotting. The location of HF2S protein in K562 cells was demonstrated by immunofluorescence and Western blotting assay. The immunofluorescence was performed to verify the co-localization of HF2S and Bcr-Abl. The co-immunoprecipitation assay was employed to detect the interaction between HF2S and Bcr-Abl. Results: The leukemia cell line K562 was effectively infected by the adenovirus Ad5/F35-HF2S. The fusion protein HF2S and Bcr-Abl were co-located and interacted with each other in the cytoplasm. Conclusion: HF2S can be successfully expressed and interacted with Bcr-Abl in the cytoplasm of K562 cells. These results provide the basement of the targeted therapy for chronic myeloid leukemia. Copyright © 2013 by TUMOR.
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Objective To explore the role of growth factor receptor-bound 2 (GRB2) in homo sapiens eukaryotic translation elongation factor 1 alpha 2 (EEF1A2) promoting the carcinogenesis and development of pancreatic cancer.Methods The human pancreatic cancer cell line SW1990 with low expression of EEF1A2,was transfected by Ad5/F35-EEF1A2 plasmid.The expression of EEF1A2 in human pancreatic cancer cell lines BxPC-3 cells with high expression of EEF1A2,was down-regulated by small interfering RNA (siRNA) interference.The expressions of GRB2 in SW1990 and BxPC-3 cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot before and after interference.The pancreatic cancer BxPC-3 cells were interfered by specific and efficient chemical synthesized siRNA fragment.The changes of BxPC-3 cell proliferation and migration were observed by methyl thiazoly tetrazolium (MTT) and Transwell assay before and after interference.The data were analyzed by one-way analysis of variance.Results After human pancreatic cancer cell line SW1990 was transfected with Ad5/F35-EEF1A2 plasmid,the expression of GRB2 at mRNA and protein level both increased significantly (F=5.67,6.39,both P＜0.05).After the expression of EEF1A2 was inhibited by EEF1A2-siRNA,the expression of GRB2 at mRNA and protein level both decreased significantly (F=6.59,4.69,both P＜0.05).After BxPC-3 cells was transfected with siRNA-GRB2 for 72 hours,the results of MTT assay indicated that the absorbance value was 1.22±0.14,compared with negative control group (1.42±0.15) and blank control group (1.39 ± 0.15) the difference was statistically significant (F =6.63,P＜ 0.05).The results of transwell assay showed that the migration ability of cells in siRNA-GRB2 group decreased.After 24 hours,the number of migrated cells was 24.10±4.25,compared with negative control group (54.72±5.24) and blank control group (51.26 ± 6.23) the difference was statistically significant (F=55.00,P＜ 0.05).Conclusion GRB2 is key molecule in EEF1A2 promoting the progenesis and development of pancreatic cancer.
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Objective To analyze the influence of peptidimer-c on the gene expression profiling of K562 cells and investigate the mechanism of peptidimer-c inducing the apoptosis and inhibiting proliferation of K562cells.Methods Trypan blue staining technique was performed for counting the number of living K562 cells treated with peptidimer-c.The ultrastructure changes of K562 cells treated with peptidimer-c was observed under transmission electron microscope.The Human U133 Plus 3.0 gene chips were used to detect the differentially expressed genes of K562 cells treated with peptidimer-c.Reverse transcription PCR was conducted to confirm some genes identified by gene chips.Results Peptidimer-c could induce the apoptosis and inhibit the proliferation of K562 cells.Peptidimer-c caused widely changes of the gene expression profiles of K562 cells.The chip data suggested that there were 529 differentially expressed genes,of which 455 genes were up-regulated and 74 genes were down-regulated.The relevant apoptotic genes were down-regulated markedly,including JUN,AXUD1,TNFRSF10B,etc.Fifteen of the differentially expressed genes were detected by RT-PCR,which was consistent with the chip data.Conclusion Peptidimer-c may induce aooptosis of K562 cells by activating the TNF/TNFR family and the JUN family.
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Objective: To observe the expression of phosphorylated mammalian target of rapamycin(p-mTOR), epidermal growth factor receptor(EGFR), growth factor receptor-bound protein 2(Grb2) and vascular endothelial growth factor(VEGF) in human colorectal cancer tissues and to explore their roles in the carcinogenesis of colorectal cancer. Methods: Tissue microarray containing 185 colorectal cancer tissues was constructed and the expression of EGFR, Grb2, p-mTOR and VEGF in the colorectal cancer tissues and the corresponding adjacent tissues was examined by immunohistochemistry methods. The relationship between their expression with the clinicopathological characteristics such as age, sex, invasion depth, lymphatic metastasis, clinical stage and differentiation degree was analyzed. Results: EGFR, Grb2, p-mTOR and VEGF were scarcely expressed or absent in the corresponding adjacent tissues; their positive rates in the colorectal caner tissues were 21.1%, 44.9%, 42.2% and 54.1%, respectively, which were significantly higher than those in the corresponding adjacent tissues (P<0.05). The expression of EGFR, Grb2, p-mTOR and VEGF was not correlated with the patients' sex, age and differentiation degree of cancer. Overexpression of EGFR was found significantly associated with the invasion depth and clinical stage of cancer(P<0.05); and overexpression of p-mTOR and VEGF was significantly associated with lymphatic metastasis, invasion depth and clinical stage(P< 0.05). There was a correlation between every two of the four proteins (r=0.245-0.567, P<0.05). Conclusion: Over-expression of EGFR, Grb2, p-mTOR and VEGF is closely associated with the development and progresssion of colorectal cancer, and they may be worth further studying as new targets for the molecular target therapy of colorectal cancer.
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Objective To analyze the effects of an inhibitor of the SH3 (Src homology) domains of Grb2 on the growth and proliferation of K562 cells. Methods The peptidimer [(VPPPVPPRRR)2-K], penetratin (RQIKIWFQNRRMKWKK) and peptidimer-c [poptidimer linked to penetratin: (VPPPVPPRRR)2-K-Aha-RQIKIWFQNRRMKWKK] were synthesized by solid-phase synthesis using Fmoc chemistry, and purified by high performance liquid chromatography (HPLC) on a C18 column. Purity was evaluated by HPLC, and the identity of the peptides was checked by electrospray mass spectroscopy (MS). A pull-down assay was used to observe the specific binding of peptidimer-c to the Grb2 of K562 cell lysates. The inhibition of peptidimer-c on K562 cell proliferation was evaluated by trypan blue exclusion assay, the cytostatic effect was tested by clonogenic assay, and the cytotoxicity was examined by WST-1 method. A further experiment was performed with clonogenic assay to analyze the co-effect of peptidimer-c respectively combined with Gleevec, Hydroxyurea and Cytarabine by Jing's method. Results The HPLC analysis showed only a simple peak, which means that the peptide is in high purity. MS analysis showed the peptides were coincided with the design. The molecular weight of peptidimer-c was of 4794.0 and that of the penetratin 2246.7. Pull-down assay demonstrated that the peptidimer-c, not the penetratin, could bind to Grb2 specifically. The trypan blue assay showed that the peptidimer-c could inhibit the proliferation of K562 significantly in a dose-dependent manner, even 3～6 h after the cells were exposed to the drug, and penetratin alone did not influence the cell proliferation. Gleevec inhibited the growth of K562 not only in a dose-dependent manner, but also in a time-dependent manner. WST-1 test showed the cytotoxieity of peptidimer-c or Gleevec on K562 cells, the IC50 of peptidimer-c was (17±2) μmol/L and the IC50 of Gleevec was (0.25±0.05) μmol/L. In the methylcellulose semi-solid medium system, the colony formation of K562 was greatly decreased by peptidimer-c as compared to the penetratin, and the colony number decreased as the dose of peptidimer-c increased. The IC50 value ofpeptidimer-c on K562 colony formation was (3.9±0.9) μmol/L, IC50 of Gleevec was (0.03±0.02) μmol/L, IC50 of Hydroxyurea was (15±7) μmol/L, and that of cytarabine was (0.014±0.012) μmol/L. There were synergistic effects of peptidimer-c with Gleevec, Hydroxyurea or Cytarabine on K562 by colonogenic assay. Combination of 1.5 μmol/L peptidimer-c and 0.05 μmol/L Gleevec showed synergistic effect on K562, as well as the combination of 1.5 μmol/L peptidimer-c and 0.006 μmol/L or 0.01 μmol/L Cytarabine. Conclusion These results suggested that peptidimer-c had an inhibitory effect on K562 cells and combination of peptidimer-c with other drugs would increase the anti-cancer effects.
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The growth factor receptor -bound protein-2 (Grb2) is an adapter protein in cells. Over expression of Grb2 is found in many kinds of tumor and plays an important role in tumor proliferation and progression. So it may be a molecular target for anti-tumor therapy.
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Chronic rheumatoid arthritis (RA) is characterized by the hyperplasia of synovial tissue, which results from the combined influence of the proliferation and antiapoptosis of the synovial cells. In this study, to identify candidate factors involved in the regulation of synovial hyperplasia, the expression profile of 205 apoptosis-related genes in a rheumatoid synovium was analyzed in comparison with that in an osteoarthritis (OA) synovium using a cDNA microarray. Upregulated genes in the RA synovium include TNFR2, GRB2, RBL2, CDC25B, MAPK p38, CDK-like kinase 2, and FLICE2, whereas 5 genes including SARP1 were down-regulated relative to OA. Among them, importantly, the expression levels of GRB2 and FLICE2 genes were remarkably enhanced in RA but not OA synoviocytes in response to TNF -alpha treatment. Therefore, TNF-alpha inducibility to GRB2 and FLICE2 genes abnormally enhanced in RA synoviocytes might represent the increased transcripts of these two genes in rheumatoid sunovial tissues. Moreover, these results suggest that RA-specific signals by TNF-alpha, including GRB2 and FLICE2, are involved in the pathogenic processes of synovial hyperplasia.