NGF regulated the expression of Tyro3 receptor in transcription or protein level

【摘要】：正Objective:A newly identified family of receptor tyrosine kinases,called TAM family,has been shown to provide growth trophic support for a variety of cells.This family is comprised of three members,Tyro3,Ax1 and MerTK.Tyro3 is preferentially expressed in the neocortex and CA1,CA3 of hippocampus,and mainly associated with neurons.To date,however,there is no evidence showing that the Tyro3 receptor under the regulation of NGF and function in coordination with this neurotrophic factor in support of the neuron differentiation.The study is to investigate the Tyro3 expression induced by NGF and the role in promoting differentiation of Tyro3 receptor on PC12 cells.Methods:We cultured PC12 pheochromocytoma cells to perform these experiments.The PC12 cells were transfected by CMV-full length-tyro3 or tyro3 promoter-CFP plasmid,respectively,and then induced to differentiate by treatment with NGF at a final concentration of 10 ng/mL in RPMI medium 1640 serum-free medium for 24 h.By the end of induction,the PC12 cells were subsequently used to perform the experiments of western blotting and observation of living cells with confocal microscope.Results:We observed significantly increased fluorescent intensity and immunoreactivity of CFP in PC 12 cells transfected with CMV-full-length-Tyro- 3-CFP chimera after administration of NGF compared with that of cultures non-treated with NGF.The same phenomenon was observed in Tyro-3-promotor-GFP plasmid transfection experiments but not in CMV-GFP vector transfection experiments.We also found that NGF affects intracellular localization of full-length-Tyro-3-CFP in PC12 cells by confocal microscopy.Moreover,we detected that Tyro3-overexpressing PC12 cells exhibit longer neurite outgrowth than that of control PC 12 cells.It is suggestive that the expression of Tyro3 receptor triggered by NGF has the effect on neuron differentiation.Conclusion In summary,our studies for the first time demonstrate that Tyro3 receptor is involved potently in neuronal differentiation and under regulations of NGF both in transcription and in protein level.