蛋白磷酸酶PP2A催化亚基β（PPP2CB）调节红细胞发育与分化的作用及机制研究

Erythropoiesis is tightly regulated at various levels. PPP2CB is beta catalytic subunit of serine/threonine protein phosphatase 2A (PP2A) which plays key roles in cell proliferation, apoptosis and tumorigenesis through de-phosphorylation of given proteins. However, the role of PPP2CB in erythropoiesis remains largely unknown. Previously our group suggest that during erythroid differentiation of human cord blood CD34+ cells, PPP2CB is upregulated significantly. Overexpression of PPP2CB in K562 cells induces spontaneous erythroid differentiation. Furthermore, PPP2CB interacts with the erythroid transcription factor GATA1 and increases the transactivation activity of GATA1. These data indicate that PPP2CB might be a critical regulator of erythroid differentiation. In the present study, we would use human primary cord blood CD34+ cells and xenograft transplantation mouse model to investigate the effect of PPP2CB on erythroid differentiation of human HSC cells in vitro and in vivo. Using vav-cre transgenic mice and PPP2Cbflox/flox mice to generate conditional knockout mice in which PPP2CB is knockout in hematopoietic cells, we evaluate the role of PPP2CB in erythroid development and differentiation globally. Furthermore, we will detect the regulatory mechanism of PPP2CB on GATA1 activity in detail, then illustrating the novel mechanism of protein phosphatase in regulating erythroid differentiation. Finally, we will examine the expression level in MDS patient with anemia and confirm the physiological significance of PPP2CB in erythroid differentiation and development.

红细胞生成过程受到精确的调控。PPP2CB是丝/苏氨酸磷酸酶PP2A的催化亚基，可参与细胞增殖、凋亡及肿瘤发生等过程，但在红细胞生成中的作用尚无报道。本课题组前期发现PPP2CB在CD34+细胞向红系分化过程中表达上调，在K562细胞中过表达PPP2CB可促进红系分化，还可与红系转录因子GATA1相互作用并增强GATA1转录活性，提示PPP2CB是调节红细胞分化的重要因子。本研究拟利用人脐带血CD34+细胞及异种造血干细胞移植模型，明确PPP2CB对人HSC体外、体内向红系分化的影响；制备血细胞特异敲除PPP2CB的小鼠模型，从整体水平评价PPP2CB对红细胞发育分化的影响；深入研究PPP2CB调节GATA1活性的分子机制，阐明磷酸酶介导的去磷酸化调节红细胞分化的新机制；最后在红系分化障碍病人中检测PPP2CB的表达及作用，进一步揭示PPP2CB调节红细胞分化成熟的生理学意义。

T细胞活化的精准调节对于机体全身免疫动态平衡至关重要，其异常活化与众多免疫系统疾病密切相关。蛋白磷酸酶2A（PP2A）是一类丝苏氨酸磷酸酶，在机体内广泛表达。PP2A全酶是包含结构亚基（A亚基）、调节亚基（B亚基）以及催化亚基（C亚基）的异源三聚体复合物。其中，催化亚基包括α亚型（PPP2CA）和β亚型（PPP2CB）。目前已知PPP2CA调控T细胞发育和分化过程，但PPP2CB的生理功能尚不清楚。本研究中我们构建了T细胞特异敲除PPP2CB的Lck-Cre;Ppp2cbfl/fl（cKO）小鼠，发现PPP2CB条件敲除小鼠T细胞发育基本正常。我们进一步利用T细胞体外激活模型，检测PPP2CB对T细胞活化的影响。结果显示PPP2CB缺失不影响CD3/CD28抗体与ConA等刺激的TCR介导的T细胞活化，以及IL-15及IFNα等刺激的细胞因子受体介导的T细胞的增殖。进一步采用PMA/离子霉素刺激T细胞活化模型，检测PPP2CB对细胞膜受体依赖的T细胞活化作用，发现PPP2CB敲除的T细胞增殖更加明显，CD25、CD69表达水平和IL2、IFNγ、TNF分泌量显著升高；进入细胞周期的细胞明显增多，表明PPP2CB敲除会特异促进PMA/离子霉素刺激的T细胞活化。. 为探究PPP2CB敲除促进PMA/离子霉素引发的T细胞活化机制，我们检测了胞内信号通路，发现PPP2CB敲除会引起Akt、mTOR以及ERK1/2等的磷酸化异常增强。PI3K特异抑制剂wortmannin能够完全逆转PPP2CB敲除T细胞的过度活化。以上结果表明，敲除PPP2CB通过增强PI3K/Akt通路，促进PMA/离子霉素引发的T细胞过度活化。最后，我们利用基于质谱的非标记磷酸化蛋白质组技术，发现PMA/离子霉素刺激下，PPP2CB敲除的T细胞中有47种蛋白质的磷酸化上调，其中13种蛋白质已报道与PI3K/Akt信号密切相关，并且多数会激活PI3K/Akt信号通路。. 综上所述，我们利用PPP2CB条件敲除小鼠模型，明确了PPP2CB不影响T细胞发育，揭示了PPP2CB负调控PI3K/Akt通路，进而抑制PMA/离子霉素刺激的T细胞活化。本研究为进一步认识PPP2CB分子功能提供了重要线索。