PRKD1 Regulates Neuronal Differentiation Of Neural Stem Cells Induced By Fibronectin

ObjectiveNeural stem cells(NSCs)are characterized with the abilities of self-renewal and multilineage differentiation towards neural cells.It has been proposed that microenviroment is an essential issue for the proliferation and differentiation of NSCs.In this study,we aimed to investigate the effect of protein kinase PRKD1 on fibronectin induced differentiation of NSCs and the underlying mechanism which might BE involved in the interaction with PAX6.Methods1.The cortical neural stem cells of rat were purified and the differentiation of NSCs was induced by fibronection coating and withdrawing of growth factors EGF and FGF in the medium.Differentiation of NSCs were identified by immunofluorescence assay.2.The proliferation and differentiation of NSCs was evaluated by mophorlogical observation,immunofluorescence assay and Ed U incoporation assay.3.The spatial and temporal expression profile of FAK,PRKD1,PAX6 during differentiation of NSCs were determined by Western blot,nuclear separation assay and cellular immunofluorescence analysis.4.The FAK inhibitor14 was used to inhibit activity of FAK,and CRT 0066101 and si RNA were used to inhibit the activity of PRKD1.5.The PRKD1 interacted molecules such as PAX6 and FAK were identified using Co-IP,GST-pull down analysis.Results1.Rat cortical NSCs were successfully isolated and cultured in vitro.2.Growth factor removal and Fibronectin coating induce neural stem cells to differentiate into TUBB3 positive cells with the ratio of75%.3.Fibronectin induced up-regulation of p-FAK during the differentiation of NSCs,and the treatment with FAK inhibitor FAK Inhibitor14 decreased the level of p-FAK and inhibited the differentiation of NSCs.4.During the differentiation of NSCs induced by Fibronectin,both of the expression level of total PRKD1 and phosphorylated PRKD1 was significantly increased,and the nuclear separation experiments showed the PRKD1 was resendent in cytoplasm and nucleus.5.Co-IP experiments in the differentiation of NSCs induced by Fibronectin demonstrated that PRKD1 interacts with PAX6 and FAK,and GST-Pull down experiments propsoed that PRKD1 might interact directly with PAX6.6.The treatment of PRKD1 inhibitor CRT 0066101 or PRKD1 si RNA inhibited the differentiation of NSCs,increased the proliferation and upregulated the expression of PAX6.Conclusion The expression of PRKD1 is increased during the differentiation induced by Fibronectin.The inhibition of PRKD1 activity and the interference of PRKD1 expression affect the differentiation and proliferation of neural stem cells.PRKD1 promoted FN-induced differentiation;PRKD1 interacted with transcription factor PAX6 and focal adhesion kinase FAK,and had direct interaction with PAX6;PRKD1 was initially identified as a downstream molecule of FAK,which is an upstream molecule of PAX6 during the differentiation,a signalpathway of FAK?PRKD1?PAX6 regulates the differentiation of NSCs.It is speculated that PRKD1 may phosphorylate PAX6 and subsequently modulate its degradation.