神经生长因子对PC12细胞内源性FoxO3a亚细胞分布的影响及其作用机制

目的研究神经生长因子(nerve growth factor,NGF)对内源性FoxO3a转录因子亚细胞定位的影响及其作用机制。方法 PC12细胞分为血清剥夺组、K252a处理组、NGF处理组及K252a+NGF处理组,通过免疫细胞化学(immunocytochemistry)研究给予NGF及K252a处理后,FoxO3a的亚细胞定位情况。应用表达FoxO3a-GFP融合蛋白的质粒,转染PC12细胞,经NGF及K252a处理,观察FoxO3a-GFP的亚细胞定位。通过蛋白免疫印迹(Western blot)研究NGF及K252a处理对TrkA磷酸化水平的影响;应用PI3K、Akt及ERK信号通路抑制剂研究NGF及K252a对TrkA下游Akt、ERK及FoxO3a信号蛋白磷酸化的影响。结果 PC12细胞剥夺血清及剥夺血清加K252a后,内源性FoxO3a主要分布于细胞核内;给予NGF处理后,内源性FoxO3a则外排至细胞核外;NGF+K252a处理后,K252a则可以阻断NGF的作用,FoxO3a位于细胞核内;过表达外源性GFP-FoxO3a质粒的结果与此一致,Western blot结果显示给予NGF处理后,NGF受体TrkA及TrkA下游Akt、ERK及FoxO3a的磷酸化水平显著上升,K252a则阻断了这一作用。同时,PI3K的抑制剂LY294002及Akt的抑制剂Akt inhibitorⅧ也阻断了NGF诱导的FoxO3a的磷酸化,但MAPK信号通路的抑制剂PD98059则没有这一作用。结论 NGF可促进内源性FoxO3a磷酸化并转位出核,NGF的作用与激活TrkA受体及其下游激酶有关。

Effect and mechanisms of nerve growth factor on the subcellular location of FoxO3a transcription factor in PC12 cells

Objective To investigate the role of nerve growth factor(NGF) on the nuclear/plasma shuttling of FoxO3a.Methods PC12 cells were divided into serum-free group,serum free plus TrkA specific inhibitor K252a group,NGF-treated group,and NGF plus K252a group.Immunocytochemistry(ICC) with anti-FoxO3a antibody was applied to observe the subcellular location of endogenous FoxO3a.Overexpression of FoxO3a-green fluorescent protein was performed to confirm the ICC results.Western blot was used to analyze the phosphorylation of TrkA,Akt,MAPK and FoxO3a before or after the treatment of NGF and K252a.The PI3K inhibitor LY294002,Akt inhibitor Ⅷ and ERK inhibitor were applied to study the underlying mechanisms.Results Endogenous FoxO3a was mainly located in the nucleus in serum-free condition and K252a-treated groups.FoxO3a was translocated to cytoplasma when PC12 cells were treated with NGF,which was blocked with K252a.Compared with serum-free group,NGF significantly induced phosphorylation of TrkA,Akt and FoxO3a,which was also blocked by K252a.Moreover,the phosphorylation of FoxO3a was blocked by PI3K inhibitor LY294002 and Akt inhibitor Ⅷ,though the MAPK pathway inhibitor PD98059 showed no effect.Conclusion NGF promotes endogenous FoxO3a translocation to cytoplasma,and this effect may be related with the phosphorylation of TrkA and its downstream molecular induced by NGF.