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Role Of C-kit-Gαi/Gab1 Signaling Axis In Promoting The Bone Mesenchymal Stem Cells Osteogenic Differentiation

Posted on:2017-02-22Degree:MasterType:Thesis
Country:ChinaCandidate:S J FangFull Text:PDF
GTID:2284330488961828Subject:Surgery
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Background and objectiveFracture healing is a complex cell and histology repair processes, including cell proliferation, differentiation, migration and regulation of multiple cytokines. Stem cell factor(SCF) interacted with its receptor c-Kit specifically and induced the dimerization or oligomerization of c-Kit, which then activated multiple downstream signaling pathways including PI3K-Akt-mTORC pathway and ERK-MAPK pathway and played an important role in bone formation and metabolism. However, the effects of SCF/c-Kit on bone mesenchymal stem cells(BMSCs) osteogenic differentiation and signaling from SCF/c-kit to its downstream of PI3K-Akt-mTORC pathway and ERK-MAPK pathway was still not entirely clear. In recent years, studies have confirmed Gαi and Gab1 protein coupling with tyrosine kinase receptors( RTKs) induced activation of downstream signaling pathways,such as: EGFR, FGFR, PDFGR.c-Kit receptor is a type Ⅲ RTKs may also mediated downstream signaling pathways through Gαi-Gab1 complex. So this study was designed to investigate the effects of SCF on BMSCs and the signaling from SCF /c-kit to its downstream pathways.Method(1)MTT assay and colony formation of MG63,U-2OS and BMSCs were detected after different concentration of SCF to assess the proliferation at 48 hor 14day.(2) To investigate anti-apoptotic effect of SCF, MG63, U-2OS and BMSCs were incubated with different concentrations of SCF and/or dexamethasone and then flow cytometry was used to analysis the apoptosis at 24 h.(3) After Gαi-shRNA lentivirus interfered the expression Gαi in BMSCs, BMSCs were plated in osteogenic differentiation medium(10-8 mol/liter dexamethasone, 10mmol/literβ-glycerophosphate and 5μg/ml ascorbic acid 2-phosphate),ALP staining and ALP activity assay were implemented to test osteogenic differentiation of BMSCs at 7 day.(4) To explore roles for Gαi and Gab1 proteins in the mechanism of SCF-mediated activation of the Akt-mTORC1 pathway and ERK-MAPK pathway, we used mouse embryo fibroblasts(MEFs) derived from wild-type(WT) mice or from mouse embryos deficient in Gai1, Gai2, Gai3 or Gab1, as well as those from embryos doubly deficient in Gαi1 and Gαi3. shRNA-lentivirus was used to interfere the expression of Gαi protein in both wild-type MEFs and BMSCs. After treated with SCF at different concentrations and time points, Western blot was used to analysis the expression and phosphorylation of Akt(Thr308 and Ser473)、mTORC1、S6、GSK、Gab1、c-Kit、S6K、ERK in cells mentioned above.Co-Immunoprecipitation(Co-IP) was used to test the coupling of Gαi1-Gab1 and c-Kit both in wild-type MEFs and BMSCs.Result(1)SCF promoted the proliferation and colony formation of MG63, U-2OS and BMSCs in a dose-dependent manner at 48 h or 14day(p<0.05).(2) SCF inhibition of dexamethasone-induced BMSCs apoptosis at 24h(p <0.05).(3) ALP staining and Activity assay showed that SCF increased the number of ALP positive cells and the ALP activity compared with control, however that decreased after Gαi-shRNA lentivirus interfered the expression ofGαi protein in BMSCs.(4)c-Kit receptor was detected in MG63, U-2OS and BMSCs. SCF induced the activation of Akt-mTORC1 and ERK- MAPK signaling pathway with evidence that the phosphorylation of Akt(Thr308 and Ser473), mTORC1, S6, GSK, Gab1, S6 K, ERK and c-Kit were increased at different time points and concentrations in BMSCs and MEFs. However, Loss of Gαi1, Gαi3 and Gab1 severely impaired the activation of Akt(Thr308 and Ser473), mTORC1, S6, GSK, Gab1, S6 K, ERK and c-Kit in response to SCF in MEFs. Signaling was restored with the supplement of Gαi1 and Gαi3 protein. Co-IP results that Gαi1 coupled with Gα1-Gab1 complex.Conclusion: SCF promoted BMSCs survival and osteogenic differentiation, inhibited apoptosis induced by dexamethasone. Gαi-Gab1 complex coupling with c-Kit receptor mediated SCF activated Akt-mTORC and ERK-MAPK signaling pathway.
Keywords/Search Tags:SCF/c-Kit, Gαi-Gab1 complex, BMSCs, Akt-m TORC1, ERK-MAPK, Osteogenic differentiation
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