PTEN 调节 ERK5 信号影响神经细胞分化的机制研究

Neural cell aberrant regeneration, differentiation or degeneration causes diverse neural disorders. PTEN and MAPKs signalling play Yin and Yang roles respectively in regulating cell growth and differentiation, keeping a balance between cell survival and apoptosis, proliferation and differentiation. Emerging evidence shows that PTEN deletion or inactivation leads the cell undue growth and premature differentiation, but in which mechanism remains unknown. Our previous studies shows the forced PTEN knocked-down in PC12 cells caused neurite growth out in regular growth medium and the phosphorylation level of ERK5 significantly increased with NGF stimulation in detection of western blotting. Based on our previous studies and literature, we hypothesize that PTEN regulates ERK5 signalling activity, ERK5 capacity of entering nucleus, and/or the down-stream gene transcription of ERK5, finally influences process of PC12 differentiation. In the proposed project, we will employ the major methods in molecular biology and cell biology, clarify how PTEN regulates activity of MEKK2/3-MEK5-ERK5 signaling pathway, and how PTEN alters the ERK5 capacity of entering the nucleus where the differentiation related genes are properly transcribed in PC12 cells or in new born neurons. Meanwhile, new molecule(s) may involved in the modulation of ERK5 activity will be identified in the project. It is expected that mechanism of PTEN regulating MEKK2/3-MEK5-ERK5 on PC12 differentiation will be clarified through this project. The results not only provide insight into understanding new mechanism of the neural cell differentiation, also provide us a new avenue for artificially controlling neural cell differentiation, leading to develop new strategy for therapy of neural degeneration disorders.

神经细胞再生与分化障碍或退化涉及到许多疾病。PTEN和MAPKs信号在细胞生长与分化过程中起着类似阴阳调节作用，决定着细胞的生存与调亡、生长与分化间的平衡。而PTEN缺失或失活可促进神经细胞增殖并且过早分化，但详细机制不明。我们以往的研究显示，在PC12细胞内抑制PTEN表达后，ERK5的磷酸化水平明显增高，且细胞突起生长显著。这些促使我们推测PTEN可能调节ERK5信号通路，经影响ERK5的活性、入核能力以及对下游分化相关基因的转录，进而影响神经细胞的分化。本项目将利用现有的细胞生物学和分子生物学手段，探索PTEN调节ERK5所在信号通路活性、ERK5入核能力及其下游基因表达调控的分子机制，同时寻找可能参与调节ERK5活性的协同分子，综合分析PTEN调节ERK5的活性对PC12细胞以及新生神经元的分化影响。结果既为我们深入了解神经细胞分化的过程，又为潜在的应用研究提供理论基础。

PTEN是一种磷酸酶，既可以去磷酸磷脂，又可去磷酸化蛋白质底物，在细胞内信号转导中下调PI3K-Akt信号活性。利用稳定转染的PTEN knock-down PC12细胞系，我们观察到PTEN表达缺失有利于PC12细胞分化，同时检测到ERK5（一种MAPKs）的磷酸化水平较其他MAPKs明显升高，且有利于PC12细胞的神经突起生长，促进分化。在系统分析ERK5信号途径的活性中，我们发现，PTEN可直接降低ERK5的磷酸化水平，对其上游分子Cot和MEK5的磷酸化水平没有明显影响。随后我们深入研究了PTEN调节ERK5活性的分子机制，以及这种调节对PC12细胞分化的影响。进一步的研究表明，PTEN去磷酸化ERK5抑制PC12细胞分化，并在ERK5的过表达和表达抑制实验中得到进一步证实。ERK5的过表达能明显促进PC12分化，同时抑制细胞增殖。利用分子生物学手段，鉴定了几个与分化相关的ERK5下游分子，并进行了初步功能分析。同时，我们利用CRISPR/Cas9技术在基因组水平对PTEN基因进行编辑，使得PTEN-long的表达水平明显升高，分泌的PTEN-long具有远程抑制细胞增殖和迁移的能力。到结题时间止，标注该基金的已发表的SCI论文8篇，其中的核心内容尚在投稿中。