FoxO3a转录因子对神经肽W表达的调控及其在慢性应激中的意义

Neuropeptide W (NPW) is a peptide hormone which plays an important role in regulation of feeding behavior and in the stress response. Recent studies indicated the involvement of NPW in the regulation of HPA axis function in stress response. FoxO3a is a member of FoxO family of transcription factors which is involved in cellular proliferation, diferentation and apoptosis. However, the role of FoxO3a in stress response and the function of the HPA axis is not known. Our preliminary data has shown that: (1) glucocorticoids increased FOXO3a expression while decreased NPW expression in PC12 cells and cultured hypothalamic cells. (2) FOXO3a overexpression suppressed NPW expression while FOXO3a knock-down blocked the inhibitory effect of glucocorticoids on NPW expression indicating an inhibitory role of FOXO3a on NPW expression. Moreover, the promoter region of the NPW gene has FOXO binding sites and FOXO3a depressed NPW promoter activity in PC12 cells. Thus our working hypothesis is that NPW is a new direct target of FOXO3a and by regulating NPW, FOXO3a may be involved in the regulation of the HPA axis and stress response. Using multiple approaches such as microarray, RT-PCR, Western blot, transfection, siRNA, reporter gene, EMSA and Chip assay, we will examine the possibility that NPW is a direct FOXO3a target and investigate the role of FOXO3a in the inhibition of glucocorticoids on the expression of NPW and its implication in the function of the HPA axis in stress response in vitro and in vivo.

FoxO3a参与老化及肿瘤等的调控，是当今的研究热点, 但它和神经肽W (NPW)的关糸以及它是否参与慢性应激及HPA轴的调控还不清楚。 NPW在下丘脑广泛表达，参与应激反应及HPA轴的调控。 我们发现糖皮质激素(GC)显著增强FoxO3a活性并抑制NPW的表达。NPW的启动子区有多个FoxO3a的结合位点, 过表达FoxO3a抑制NPW的表达而敲除它则增加NPW的表达并拮抗GC对NPW的作用。这些结果高度提示NPW为FoxO3a的直接下游靶基因, FoxO3a可能通过调控NPW而参与应激反应。本项目拟采用电转染、印迹, PCR、siRNA、ChIP和行为学分析等方法在细胞及动物证明NPW是FoxO3a的直接新靶标, FoxO3a参与GC抑制NPW表达的作用，从而证实FoxO3a参与应激反应及HPA轴的调控。本项目将为FoxO作为应激相关疾病如抑郁症等提供新的发病机制及药物新靶点.

FoxO3a转录因子广泛参与细胞的代谢,增殖,分化及凋亡等的调控。是当今生物科学的研究热点,但FoxO3a转录因子是否参与慢性应激反应及下丘脑-垂体-肾上腺(HPA)轴的调控还不清楚。神经肽W(NPW)在下丘脑广泛表达，参与应激情绪反应及HPA轴的调控。本项目采用电转染、PCR、siRNA、荧光报告基因、ChIP分析及行为学实验等方法在PC12细胞,原代培养下丘脑细胞及体内实验中证明NPW是FoxO3a的直接新靶标,糖皮质激素通过诱导FoxO3a的表达抑制NPW表达,为FoxO3a参与应激反应及HPA轴的调控提供更可靠的依据。结果显示:1.NPW是神经细胞中FoxO3a转录因子的新下游靶标;Foxo3a可结合到NPW启动子区域中FoxO3a的结合位点(FHRE),抑制NPW基因启动子活性而抑制NPW转录和表达;2.地塞米松(DEX)抑制细胞NPW的表达,其作用是通过激活细胞糖皮质激素受体,降低PI3K/Akt/FoxO3a磷酸化而促进FoxO3a的核转位和功能,从而抑制NPW的表达。3.皮质类固醇的长期给药可显著增加体内FoxO3a表达,使NPW的mRNA和蛋白水平降低,并降低大鼠体重,蔗糖摄入量和强制游泳时间(诱发抑郁)。4.NGF/BDNF能通过Trk/PI3K/Akt/FoxO3a诱导NPW的表达,FoxO3a抑制NGF对NPW表达的刺激作用。这些发现说明FoxO3a可通过调节下丘脑NPW表达参与下丘脑-垂体-肾上腺(HPA)轴的调控及慢性应激反应(诱发抑郁)。另外, 我们也发现 1.NGF通过TrkA/PI3K/Akt/FoxO3a诱导GAP-43的表达。2.常春藤皂苷元通过PI3K/Akt/FoxO3a保护PC12细胞免受皮质酮诱导的损伤。3.青蒿素通过PI3K/Akt抑制FoxO3a的核转位,降低FoxO3a的活性,促进了NPW表达。青蒿素预处理抑制了皮质酮诱发的抑郁。 .发表学术论文25篇,平均影响因子>6,通讯作者文章24篇。会议论文>30篇。获邀参编Encyclopedia of Gerontology and Population Aging百科全书。申请发明专利二个。毕业博士研究生3名(澳门优秀博士毕业生一名),博士后3名。