Focal Adhesions Regulate The VEGF Induced Migration Of MSCs In Neural Differentiation

Mesenchymal stem cells (MSCs) which are capable of differentiating intoectodermal origin neurons, have a tropism for brain tumors or migrate directionallytoward chemotactic agents and cytokines expressed in injured brain or gliomas.However, the chemotactic signals mediating the migration of MSCs in various neuraldifferentiating states remain poorly understood. The assembly and distribution of focaladhesions (FAs) and the arrangement of cytoskeleton are involved in the process of cellmigration. In the present study, we investigated the mechanism of MSCs migratingtoward vascular endothelial growth factor (VEGF) focusing on cell adhesion signaling.The formation of FAs and F-actin cytoskeletons during the cell spreading were observed.Cell adhesions formed small focal complexes to mature big FAs with plating timeextended. F-actin assembled as circular bundles at the early stage and then formed stressfibres that made the cells posses polarity. The tyrosine phosphorylation activation ofY397-FAK, Y31-paxillin and Y118-paxillin which regulated the assembly of FAs andthe arrangement of F-actin cytoskeletons were changed during the spreading. In thisresearch, bFGF, BHA and DMSO were used to induce the neural differentiation ofMSCs in vitro. Undifferentiated MSCs were treated with10ng/ml basic fibroblasticgrowth factor (bFGF) for24h to perform pre-induction, followed by treated with200μM butylated hydroxyanisole (BHA) plus2%dimethylsulfoxide (DMSO) for5h.Finally, H-DMEM plus1%N2was used to maintain the differentiation for48h. Wefound that MSCs in various differentiation states showed different responses to VEGF:First, the activation of Y397-FAK, Y31-paxillin and Y118-paxillin of undifferentiatedMSCs were affected by different concentrations of VEGF. The quantity and distributionof FAs had the similar tendencies along with the changes of tyrosine kinases. Under the stimulation with the same concentration of VEGF, the activated phosphorylation of FAKand paxillin, the formation of FAs and the peripheral/intermediate ratio of FAs werechanged along with the handling time extented. Second, the features of FAs and F-actincytoskeletons were various among the neural differentiation states. Under the treatmentof VEGF, the abilities of differentiating cells to form lamellipodia were different andthe pre-induced MSCs possesed the most motility to protrude large board lamellipodiaand formed cell polarity at the begining of directional treatment of VEGF. FA assemblyrate of VEGF-induced migrating cells was faster than in untreated cells. Collectively,these results demonstrated that the states of neural differentiation and cell adhesionsignaling including the formation and distribution of FAs, the activation of focaladhesion proteins and the rearrangement of F-actin cytoskeletons, coordinately regulatethe chemotactic responses of neural differentiating MSCs toward VEGF.