The Study For The Roles Of Akt2 In Glioma Cells Invasion And Macrophages Chemotaxis

ObjectivesTo investigate the role of Akt2 in glioma cell invasion and illustrate the mechanismsof Akt2 signaling pathway in glioma cell invasion.To investigate the role of Akt2 in macrophage chemotaxis and illustrate themechanisms of Akt2 signaling pathway in macrophage chemotaxis.MethodsGlioma cells were transfected with small RNA interference plasmids to disrupt Akt2expression. Akt2 protein levels and mRNA levels were detected using Westernblotting and RT-PCR. Scratch assay and chemotaxis assay were examined to detectthe migration and chemotaxis ability of gliloma cells. The in vitro invasion ability ofgliloma cells was examined using matrigel invasion assay. F-actin content of glilomacells was analyzed with F-actin polymirazation assay. Western Blotting was used tocheck the level of phosphorylated cofilin, integrinβ1 and Girdin. WesternBlotting was used to check the expression level of MMP-2 and MMP-9. The rat brainglioma invasion model was set up to examine the in vivo invasion ability of glilomacells.THP-1 cells were transfected with a small RNA interference plasmid to disrupt Akt2expression. The Akt2 protein levels and mRNA levels were detected with Westernblotting and RT-PCR. The CSF-1 induced chemotaxis ability of THP-1 cells wasdetected using chemotaxis assay. F-actin content of THP-1 cells was analyzed withF-actin polymirazation assay. Western Blotting was used to check the level ofphosphorylated LIMK/cofilin and PKCζ. Mouse peritoneal macrophages weretransfected with small interfering RNA duplex oligoribonucleotide to disrupt Akt2expression.ResultsWestern blotting results showed significant decrease in Akt2 protein levels, consistentwith a decrease in mRNA levels. The Akt2-reduced glioma cells showed decreasedchemotaxis ability compared with the control cells (P＜0.01). When a scratch wascreated in the fluent monolayer cells, it took the Akt2-reduced glioma cells a longertime to fill the gap (P＜0.01). Adhesion assay showed the number of the adheringcells was decreased for reduction of Akt2 within 5 and 15 minutes (P＜0.01). Theinvasion assay showed prominent differences between the Akt2-reduced glioma cells and the control cells (P＜0.01). In the Akt2-reduced glioma cells, the actinpolymerization in response to the EGF stimulation was significantly reduced.Western blotting results showed that the level of phosphorylated cofilin,phosphorylated integrinβ1 and phosphorylated Girdin was inhibited for the Akt2reduction. The MMP-9 exspression in Akt2-reduced glioma cells was lower thancontrol cells. The rat brain glioma invasion assay showed that the number ofsatellite tumors has been decreased in the Akt2-reduced glioma group (P＜0.05).Western blotting results showed significant decrease in Akt2 protein levels, consistentwith a decrease in mRNA levels. The Akt2-reduced THP-1 cells showed decreasedchemotaxis ability compared with the control cells (P＜0.01). In the Akt2-reducedTHP-1 cells, the actin polymerization in response to the CSF-1 stimulation wassignificantly reduced. Western blotting results showed that the level ofphosphorylated LIMK/cofilin and phosphorylated PKCζwas inhibited for the Akt2reduction. The Akt2-reduced mouse peritoneal macrophages showed decreasedchemotaxis ability compared with the control cells (P＜0.01).Conclusions(1) Akt2 is required in glioma cells migration and EGF-induced chemotaxis byregulating F-actin polymerization and phosphorylation of Girdin.(2) Akt2 regulates the EGF-induced activation of cofilin, which regulated the actinpolymerization.(3) Akt2 regulates cell adhesion signal pathway directly through phosphorylation ofintegrinβ1.(4) Akt2 regulates the expression of MMP-9.(5) Akt2 plays an important role in glioma cells invasion in rat brain in vivo.(6) Akt2 is required in macrophages CSF-1 and MCP-1 induced chemotaxis byregulating F-actin polymerization.(7) Akt2 regulates the CSF-1-induced activation of LIMK/cofilin, which regulated theactin polymerization. Akt2 regulates cell chemotaxis signal pathway throughphosphorylation of PKCζ.(8) The two chemotactic signaling pathways, mediated by receptor tyrosine kinasesand G protein coupled receptor respectively, shares the same downstream signalingcomponents. Akt2 is required for both CSF-1 and MCP-1 induced macrophagechemotaxis. Akt2 is an ideal target to inhibit macrophage migration and chemotaxis.