FAK Regulates The VEGF-induced Migration Of NSCs Under Various Differentiation States Through PI3K/Akt, MAPKs And Rac1 Pathways

Precise migration of neural stem/progenitor cells(NSCs) is crucially important for neurogenesis and repair in the nervous system. Many neurodegenerative diseases are closely related to the deficient migration of NSCs. NSCs, either endogenous or transplanted, are highly motile and display a unique tropism for areas of pathology in the adult brain. Our previous studies showed that the different chemotactic responses of NSCs to vascular endothelial growth factor(VEGF) are depended on the cell differentiation status, dynamics of focal adhesions(FAs) and reorganization of F-actin. Focal adhesion kinase(FAK) is a cytoplasmic tyrosine kinase that plays a fundamental role in growth factor mediated signalling and is an important player in cell migration. However, the role of FAK in FA dynamics during chemotactic migration of NSCs at certain differentiation states is unknown. In the present study, we focus on the role of tyrosine phosphorylation of FAK in the remodeling of FA distribution and turnover during VEGF-induced NSC migration. We also verify the effects of FAK, PI3K/Akt and MAPKs pathway on VEGF-induced migration of NSCs and examined the PI3K/Akt and MAPKs signalling pathways and Rac1 in relation to FAK in VEGF-induced FA dynamics of NSCs under various differentiation states.To assess the function of FAK in VEGF-induced migration of NSCs, we examined the directed migration of C17.2 cells(a neural stem cell line) under varying neural differentiation states. Using a microchemotaxis Boyden chamber, we tested the transfilter migration of these cells toward VEGF in the presence of FAK inhibitor PF-228. A noticeable decrease in NSC migration under varying differentiation was observed in the presence of PF-228. To assess further the mechanisms of FAK that regulate the VEGF-induced migration of NSCs, a Dunn chamber was used to observe directly the migratory behavior of NSCs in the presence of PF-228. We found that inactivation of FAK by PF-228 led to a deficit of chemotaxis of both undifferentiated and 1 day differentiating NSCs, with a decreased migration speed and FMI, although no effects were observed on cells of 3 days differentiation. Using primary neurospheres isolated from the subventricular zone(SVZ) of newborn rat brains or the cerebral cortex of fetal mice, we verified the effects of FAK on VEGF-induced NSC migration. Neurospheres treated with VEGF displayed a marked spreading, whereas cells showed modest migration from the neurospheres and lacked the cellular organization after incubation with PF-228.Cell migration requires the coordinated formation and disassembly of FAs, a phenomenon known as FA turnover. It has been shown that FAK regulates the migration of NSCs, then we examined whether FAK is involved in the formation of FAs and reorganization of F-actin in cells under varying differentiation states. VEGF-treated NSCs exhibited a characteristic polarized morphology, exhibiting a leading edge with extension of lamellipodia and an increase of small dot-like FAs mainly concentrated at cell periphery. Cells treated with PF-228 exhibited elongated fibrillar or large rounded-to-ellipsoid adhesions. The number of total FAs is significantly decreased in both undifferentiated and differentiating cells, and the ratio of elongated FAs is increased in NSCs of undifferentiation and 1d differentiation. We also found that the phosphorylation of Y397-FAK and Y31/Y118-paxillin in NSCs of undifferentiation and differentiating states was significantly increased in the presence of VEGF, while decreased when incubated with PF-228.Directed cell migration requires front-rear cell polarization. By using a wound model in which a wound area was produced by scratching a monolayer of NSCs in varying differentiation states and cells migrated into the wound area, we found that more FAs were formed at the protruding lamellipodia with an increased ratio of FAs in the front to those at cell rear under VEGF induction. Whereas FAs localized to a more stable and narrow region near the peripheral membrane after inhibiting FAK by PF-228. In order to visualize the effect of FAK on the dynamics of FAs, especially the role of its phosphorylation at Y397, we directly observed the assembly of FAs and lamellipodia protrusion during VEGF-induced chemotactic migration. VEGF promoted the formation of focal complexes at the leading edge. At the trailing edge, we also observed the fast disassembly of FAs. The FA assembly was defective and the motility of cell protrusion was reduced when the FAK phosphorylation at Y397 was inhibited by PF-228. We further observed the assembly of FAs when NSCs were transfected with FAK-Y397 F mutant. Y397-FAK cells displayed predominantly larger and unusually shaped FA that localized to the periphery of cells, the length of FAs was significantly increased compared with those in wild-type FAK cells. Under VEGF stimulation, the number of FAs in wild-type FAK cells was increased notably and the length of those FAs was shortened, while no effects were observed in cells expressing FAK-Y397 F.FAK mediates cell adhesion and migration via its downstream signalings, such as PI3K/Akt and MAPKs pathways. When inhibition of FAK, the phosphorylation of all these downstream signalling effectors was significantly attenuated, suggesting that a functionally active FAK may affect the activation of PI3K/Akt and MAPKs pathways during VEGF-induced NSC migration. We found that VEGF-induced migration of neurospheres was critically abolished by the ERK and JNK inhibitors for 72 h. However, the inhibition of VEGF-induced migration of neurospheres by inhibitors of PI3K/Akt and p38 MAPK was only sustained up to 24 h. Directed inhibition of PI3K/Akt and MAPKs pathways also impared FA formation in undifferentiated and differentiating cells and significantly decreased distribution ratio of FAs in the front during cell migration. The phosphorylation of FAK and paxillin in VEGF-induced NSCs was also decreased when inhibition of PI3K/Akt and MAPKs, indicate that FAK and PI3K/Akt and MAPKs pathways may affect each other in VEGF-induced FA formaiton.Rac1, which is activated at lamellipodia, predominantly localizes to FAs in the membrane ruffles and involved in the regulation of FAs. Based on these findings, the functional roles of Rac1 in VEGF-induced migration of NSCs were evaluated. Overexpression of the constitutive activated mutants of Rac1(Rac1-Q61 L and Rac1-G12V) improved the migration efficiency of NSCs with a high FMI by forming a single dominant lamellipodia in the direction of movement. Rac1-Q61 L and Rac1-G12 V expressing cells possess an increased number of FAs in cells of undifferentiation and 1 day differentiation states. Reversely, the number of FAs was decreased in cells overexpressing the dominant negative Rac1-T17 N, confirming the role of Rac1 in FA organization. Under VEGF stimulation, the number of FAs was not further increased in Rac1-Q61 L and Rac1-G12 V cells, while FAs were increased in p IRES2-EGFP or wild type Rac1 cells of 1 day differentiation. However, there was no difference in the number of FAs in Rac1-T17 N cells with or without VEGF treatment, suggesting that the activation of Rac1 is required for the VEGF-stimulated FA formation. When inhibition of FAK by PF-228, the number of FAs was noticeable decreased in cells expressing wild type and constitutive activated mutants of Rac1. These data indicate that activation state of Rac1 regulates its role for FA formation and lamellipodia protrusion in a FAK-dependent manner during VEGF-induced migration.Taken together, these findings demonstrate that a robust role of FAK in regualting dynamics of FAs and VEGF-induced NSC migration, via the activations of PI3K/Akt, MAPKs and Rac1 signalling, correlates closely with their differentiation states.