The Functional Study Of Protein Kinase Cξ In The Chemotaxis Of Glioma Cells And Monocytes/Macrophages

Background and ObjectiveBrain glioma is the highest morbidity tumors in nervous system.We aimed toexplain the relationship between PKCζand cell migration,adhesion and actinpolymerization.In our study,we aimed to explain the influence of PKCζdown-regulation onmonocytes/macrophages chemotaxis and the related molecular mechanism.Methods1)We studied the role of atypical PKC chemokine-mediated chemotaxis of gliomaand monocytes/macrophages by using various PKC specific inhibitors.2)Chemotaxis assay and chemokinesis assay demonstrated the influence of PKCζon the chemotaxis ability of glioma cells and monocytes/macrophages.3)F-actin polymerization assay explained the changes of actin polymerizationstimulated by chemokines in different times.Western blotting assay detected theexpression ofphospho-LIMK and cofilin in siPKCζcells and control cells.4)Treated the cells by the specific myristolated PKCζpseudosubstrate to furtherdemonstrate the influence of PKCζon the cell migration.5)Using a small interference RNA strategy to attenuate PKCζexpression in mouseperitoneal macrophages and demonstrated the influence of PKCζon macrophage.6)Adhesion assay detected the influence of down-regulated PKCζon the adhesionof glioma cells.Western blotting assay detected the expression ofphospho-integrinβ1 siPKCζglioma cells and control glioma cells.7)We used the subcutaneous mouse xenograft model and invasion in vitro assay tovalidate the role of PKCζin glioblastoma invasion in vivo.Results1)Using specific inhibitors of PKC isotypes demonstrated that atypical PKC playsan important role in glioma cells and monocytes/macrophages chemotaxis.2)After transfected glioma and monocytes/macrophages by small interference RNA, the exression of PKCζsignificantly decreased.3)PKCζ-reduced glioma cells and monocytes/macrophages showed decreasedchemotaxis ability compared with control cells (P＜0.05).4)PKCζ-reduced glioma cells and monocytes/macrophages showed decreased actinpolymerization ability compared with the control cells (P＜0.05).The expressionofphospho-LIMK/cofilin was down regulated.5)The adherent cell of PKCζ-reduced glioma cells is significantly reducedcompared with control cells (P＜0.05).The expression of phospho-integrinβ1 wasdown regulated.6)Both in vivo and in vitro assay demonstrated that the invasion ability ofPKCζ-reduced glioma cells is significantly reduced compared with controlcells(P＜0.05).The expression of MMP-9 was also down regulated in siPKCζcells.7)When knock-down of PKCζby Stealth^TMRNA in mouse peritoneal macrophages,it took the PKCζ-reduced macrophages a longer time to fill the gap (P＜0.05).8)The chemotaxis ability of PKCζ-reduced mouse peritoneal macrophages issignificantly decreased compared with control cells induced by CSF-1 andMCP-1 (P＜0.05).Conclusions1)PKCζparticipated in EGF-induced glioma cells migration and invasion.PKCζaccommodated the activation of LIMK/cofilin and integrinβ1 to controlcytoskeletal rearrangement and adhesion.2)PKCζparticipated in CSF-1 induced monocytes/macrophages chemotaxis.PKCζaccommodate the activation of LIMK/cofilin to control cytoskeletalrearrangement.3)PKCζmay be required for both GPCR and RTK mediated chemotaxis signalingpathways.