Objective To study the function and mechanism of G protein coupled receptor kinase interacting protein 1(GIT1) RNA hairpin (GIT1-RNAh) in osteoblast migration. Methods The sixth passage osteoblasts were divided into 2 groups and were infected by GIT1-RNAh (experimental group) and green fluoresence protein RNA hairpin (GFP-RNAh) (control group) adenovirus for 12 hours respectively. Each group was further classfied into two groups according to with or without platelet-drived growth factor (PDGF) stimulation. The GIT1 expression and Paxillindistribution was analyzed by immunofluorescence staining. Paxillin phosphorylation was detected by Western Blot. The localization of Paxillin was determined by co-immunofluorescence staining after transfection with cyanine fluorescence protein tagged GIT1RNAh (CFP-GIT1-RNAh)(experimental group) and GFP-RNAh (CFP-GFP-RNAh)(control group). The role of GIT1-RNAh (experimental group) and GFP-RNAh (control group) adenovirus in osteoblasts migration was determined by wound healing assay. Results Immunofluorescence staining results showed that the GIT1-RNAh significantly inhibited endogenous GIT1 expression, interfered Paxillin distribution.Western Blot results showed that Paxillin phosporylation was obviously inhibited in osteoblasts infected with GIT1-RNAh adenovirus (P＜0.05). The wound healing assay results howed that GIT1-RNAh adenovirus significantly inhibited osteoblast migration induced by PDGF. Conclusion GIT1-RNAh inhibits osteoblasts migration by interfering paxillin distribution and decrease Paxillin phosphorylation.
ObjectiveTo investigate the mechanism of G protein coupled receptor kinase interacting protein 1 (GIT1) affecting angiogenesis by comparing the differentiation of bone marrow mesenchymal stem cells (BMSCs) differentiated into endothelial cells between GIT1 wild type mice and GIT1 gene knockout mice.MethodsMale and female GIT1 heterozygous mice were paired breeding, and the genotypic identification of newborn mice were detected by PCR. The 2nd generation BMSCs isolated from GIT1 wild type mice or GIT1 gene knockout mice were divided into 4 groups, including wild type control group (group A), wild type experimental group (group A1), GIT1 knockout control group (group B), and GIT1 knockout experimental group (group B1). The cells of groups A1 and B1 were cultured with the endothelial induction medium and the cells of groups A and B with normal cluture medium. The expressions of vascular endothelial growth factor receptor 2 (VEGFR-2), VEGFR-3, and phospho-VEGFR-2 (pVEGFR-2), and pVEGFR-3 proteins were detected by Western blot. The endothelial cell markers [von Willebrand factor (vWF), platelet-endothelial cell adhesion molecule 1 (PECAM-1), and vascular endothelial cadherin (VE-Cadherin)] were detected by flow cytometry. The 2nd generation BMSCs of GIT1 wild type mice were divided into 4 groups according to the different culture media: group Ⅰ, primary cell culture medium; group Ⅱ, cell culture medium containing SAR131675 (VEGFR-3 blocker); group Ⅲ, endothelial induction medium; group Ⅳ, endothelial induction medium containing SAR131675. The endothelial cell markers (vWF, PECAM-1, and VE-Cadherin) in 4 groups were also detected by flow cytometry.ResultsWestern blot results showed that there was no obviously difference in protein expressions of VEGFR-2 and pVEGFR-2 between groups; and the expressions of VEGFR-3 and pVEGFR-3 proteins in group A1 were obviously higher than those in groups A, B, and B1. The flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group A1 than in groups A, B, and B1 (P<0.05), and in group B1 than in groups A and B (P<0.05); but no significant difference was found between groups A and B (P>0.05). In the VEGFR-3 blocked experiment, the flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group Ⅲ than in groupsⅠ, Ⅱ, and Ⅳ, and in group Ⅳ than in groups Ⅰ and Ⅱ (P<0.05); but no significant difference was found between groups Ⅰ and Ⅱ (P>0.05).ConclusionGIT1 mediates BMSCs of mice differentiation into endothelial cells via VEGFR-3, thereby affecting the angiogenesis.