Effects Of 0.4 MT PFMF On Focal Adhesion Signaling Pathways In FL Cells

In the previous researches,we have found that the 0.4 mT power frequency magnetic fields(PFMF)promoted the cytoskeleton reconstruction and motility of human amniotic epithelial(FL)cells by activating the epidermal growth factor receptor(EGFR)signaling pathway.However,it is not clear whether this reaction also involves the activation of another cytoskeleton component-the focal adhesion signaling pathway that is also closely involved in the cell migration.This paper starts from EGFR signaling pathway which is related with focal adhesion.The distributions,expression and activities of significant regulatory proteins in focal adhesion signaling pathway such as FAK,Paxillin were detected by using immunofluorescence and western blotting methods.Cell migration was evaluated to verify how PFMF affects the reaction of cell motility through activating FAK.The results showed that,compared to the sham group,PFMF exposure induced 1)the total amount of focal adhesion spots increased,the significant signaling protein FAK and Paxillin were more concentrated at the edges of cells and had symbiotic relationship with flake pseudopodia;2)the total content of Paxillin increased;the FAK protein level had no significant change,but its Y397 phosphorylation level increased significantly.3)These changes caused by PFMF were similar to that of EGFR ligand epidermal growth factor(EGF)did.4)inhibitions by either PD153035(PD)to inhibit the activity of EGFR or FAK Inhibitor 14(FI)to inhibit the phosphorylation of Y397-FAK,all results mentioned above induced by either PFMF or EGF disappeared;5)Cell scratch experimental results showed that similar to the function of EGF,PFMF also promoted cell migration,but the promoting effects of PFMF on cell migration was almost entirely disappeared when Y397-FAK was inhibited by FI.Then this paper makes a preliminary study of effects of PFMF on another focal adhesion signaling pathway-Integrin.The distributions,contents of significant regulatory proteins in Integrin signaling pathway such as Integrin?1?Talin?Vinculin were detected.Cell migration assay was performed to verify how PFMF affects the reaction of cell motility through Integrin.The results show that,compared to the sham group,PFMF exposure induced 1)signaling proteins in integrin pathway such as Integrin?1,Talin and Vinculin were also found concentrated at the cell edges and increased.2)the phenomena of focal adhesion proteins distributing at cell edges and increasing contents which were induced by PFMF disappeared when using GLPG0187(G)to inhibite Integrin;3)Cell scratch experimental results showed that the promoting effects of PFMF on cell migration were entirely disappeared when Integrin was inhibited by G.The above results suggested that 0.4 mT PFMF regulates the formation and distribution of FAK,Paxillin focal adhesion proteins by activating EGFR and probably through Integrin simultaneously to regulate the distributions and contents of Integrin?1,Talin and Vinculin to activate Integrin focal adhesion signaling pathway,thus promoting cell adhesion and migration,so as to participate in the stress reaction mechanism of PFMF–induced cell motility along with the reactions of the actin cytoskeleton.