EphB4 Signaling Pathway Regulates Proliferation,Differentiation And Apoptosis Of Human Embryonic Neural Stem Cells In Vitro

Objective Ischemic stroke is a devastating neurological disorder and one of the leading causes of morbidity and mortality.Neural regeneration and restoration after ischemic brain damage is a critical procedure for the treatment of ischemic stroke.Stroke could induce endogenous neural stem cells(NSCs)proliferation,differentiation and movement into peri-infarct regions.Multiple signaling pathways involved in regulating the behavior of NSCs.Endogenous NSCs proliferation and differentiation could be enhanced by adjusting the specific molecular mechanism after brain damage.Now,classic signaling pathways that are in involved in regulating NSCs proliferation and differentiation include Wnt signaling pathway,Notch signaling pathway?Shh(Sonic Hedgehog,Shh)signaling pathway and BMP(bone morphogenetic protein,BMP)signaling pathway.In our early research of Wnt signaling pathway,we found that ?-catenin is one of downstream proteins of Eph as well as the key regulator of the Wnt signaling pathway.Thus,we focus on the Eph/ephrin signaling pathway.The erythropoietin-producing hepatocellular(Eph)receptors form the largest family of receptor tyrosine kinases.Upon interaction of the Eph receptors with their ligands the ephrins,bidirectional signaling cascades are initiated downstream of receptor and ligand.In recent years,it is reported that Eph receptors and ephrin ligands are involved in regulating the proliferation,differentiation,survival and migration of neural stem cells(NSCs).However,all studies on EphB4 are focused on tumor and little is known about its role in NSCs.Considering that many tumors are sustained by a minor population of tumor-initiating cells that share many properties with stem cells,cancers may arise by transformation of normal tissue stem cells.These suggest that EphB4 might also play important roles in the proliferation and differentiation of NSCs.This study aimed to examine whether EphB4 is involved in proliferation and differentiation and apoptosis of human embryonic neural stem cells(hNSCs)in vitro and to explore its downstream signaling pathway for treatment of ischemic stroke.Methods(1)We identified hNSCs through immunofluorescence staining with Nestin after culturing 3 days,immunofluorescence staining with ? ?-tubulin and GFAP to label neurons and astrocytes respectively after differentiation 10 days,and immunofluorescence staining with Galc to label oligodendrocytes after differentiation 16 days.(2)hNSCs were cultured and transfected with over-expression and sh RNA knockdown lentivirus to up-and down-regulate EphB4 m RNA level and protein level.Then,the hNSCs that had been transduced with lentivirus were divided into scrambled-sh RNA group,EphB4-sh RNA group,EphB4-control group and EphB4-overexpression group.The suppression and up-regulation of m RNA expression were analyzed by real-time reverse transcription-polymerase chain reaction(RT-PCR),and EphB4 protein levels were detected using western blot.We examined the effect of EphB4 on cell proliferation through clone analysis and immunofluorescent labeling of Brd U.We examined the effect of EphB4 on cell differentiation through immunofluorescent labeling and RT-PCR analysis of Dcx,? ?-tubulin,GFAP and NG2 and Western blot analysis of Mash1.To determine the effect of EphB4 on apoptosis,hNSCs were immunostained for cleaved-caspase 3 and caspase 8,western blot analysis of cleaved-caspase 8 and flow cytometric analysis of apoptosis.We performed cell cycle analysis and used Cyclin D1/CDK4 inhibitor FAS,Abl inhibitor Gleevec and Abl activator DPH to inhibit and activate the activity of Cyclin D1,CDK4 and Abl,then further explored the downstream signaling pathway of EphB4.Results(1)Results showed that the proportion of Nestin~+ cells is 77.37%.Besides,these cells could differentiate into neurons(? ?-tubulin~+),astrocytes(GFAP~+)and oligodendrocytes(Galc~+).(2)Compared with the control group,EphB4 m RNA levels and proteinexpression levels were down-regulated after being transduced with EphB4-sh RNA lentivirus and up-regulated after being transduced with EphB4-OE lentivirus,respectively.The EphB4 knockdown and over-expression model in hNSCs was established successfully.(3)Results of clone analysis and immunofluorescent staining analysis showed that EphB4-deficient cells showed a distinct reduction in the clone diameter,neurosphere frequency and Brd U~+ cells,compared with the scrambled-sh RNA group.hNSCs in the EphB4-OE group formed more and larger neurospheres than control hNSCs.Immunostaining for Brd U and quantification of the immunopositive cells confirmed that EphB4 overexpression significantly increased the number of proliferating cells compared to the control group.(4)Results of immunofluorescent staining analysis RT-PCR analysis and western blot analysis showed that the number of Dcx~+ cells and ? ?-tubulin~+ cells,the m RNA levels of Dcx and ? ?-tubulin,the protein level of Mash1 decreased after infection with EphB4-sh RNA lentivirus.Moreover,EphB4 deficient lead to increased GFAP~+ cells and NG2~+ cells and increased m RNA levels of GFAP and NG2 compared to scrambled-sh RNA group.Conversely,over-expression of EphB4 increased the number of Dcx~+ cells and ? ?-tubulin~+ cells,the m RNA levels of Dcx and ? ?-tubulin,the protein level of Mash1,but decreased the number of GFAP~+ cells and NG2~+ cells,m RNA levels of GFAP and NG2 compared to the control group.(5)Results of immunofluorescent staining analysis,western blot analysis and flow cytometric analysis showed that the number of cleaved-caspase3~+ cells,caspase8~+ cells,expression of cleaved-caspase8 and flow cytometric analysis of apoptosis showed no differences after knockdown or overexpression of EphB4,compared to respective control group.(6)Result of flow cytometric analysis showed that,compared with the scrambled-sh RNA group,EphB4 knockdown in hNSCs significantly enhanced the proportion of cells in G0/G1,and this was associated with reduced proportions of cells in S phase and G2/M.Conversely,EphB4 over-expression in hNSCs led to a reduction in the proportion of cells in G0/G1,which was associated with an increased proportion of cells in S phase and G2/M.Knockdown and over-expression of EphB4 affected the protein levels of Cyclin D1 and CDK4.Moreover,effects of up-regulated EphB4 on cell self-renewal and proliferation can be abolished by FAS.Thus,the effects of EphB4 on cell self-renewal and proliferation are mediated by Cyclin D1/CDK4.(7)Western blot analysis showed that knockdown of EphB4 resulted in significantly reduced P-Abl levels in hNSCs.In contrast,P-Abl levels were increased in EphB4-overexpressing cultures.Abl inhibitor Gleevec and Abl activator DPH could impact the expression of Cyclin D1 and CDK4.Moreover,the effects of EphB4 over-expression on cell proliferation can be abolished by Gleevec.Thus,EphB4-mediated signaling to induce cell proliferation goes through Abl to reach Cyclin D1/CDK4.(8)Treatment with Abl inhibitor Gleevec and Cyclin D1/CDK4 inhibitor FAS did not influence the effects of EphB4 overexpression on neuronal differentiation and glial differentiation.Thus,Abl-Cyclin D1/CDK4 pathway is not involved in EphB4 mediated cell differentiation.Conclusion Knockdown of EphB4 inhibits proliferation and neuronal differentiation,promotes glial differentiation and does not affect apoptosis of hNSCs.Over-expression of EphB4 promotes proliferation and neuronal differentiation,inhibits glial differentiation and does not affect apoptosis of hNSCs.The effect of EphB4 on cell proliferation is mediated by the Abl-Cyclin D1/CDK4 pathway.However,Abl-Cyclin D1/CDK4 pathway is not involved in the effect of EphB4 on differentiation.In conclusion,EphB4 receptor participates in regulating proliferation of hNSCs and EphB4 is crucial for the balance between neuronal differentiation and glial differentiation from hNSCs.