PIM1激酶调控哺乳动物红细胞脱核的分子机制研究

Erythropoiesis is a highly regulated multistage process by which hematopoietic stem cells proliferate, differentiate and eventually form mature erythrocytes. Among this step-by-step process, erythroblast enucleation is the most unique biological event in mammals. Although this distinctive feature has been discovered for more than a century, the molecular mechanisms remain largely unclear. This bottleneck also restricts the generation of functional erythrocytes using embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Our RNA-seq analysis of erythroid cells at each developmental stage revealed that serine/threonine kinase PIM1 was significantly up-regulated in both human and mouse orthochromatic erythroblasts and was the highest expressed protein kinase in this stage. To explore the role of PIM1 in human erythroblast enucleation, we knocked down PIM1 by shRNA in human CD34+ cells and found that PIM1 knockdown specifically resulted in remarkable decrease in enucleation with no effects on cell growth, differentiation and apoptosis. Moreover, comparison of PIM1 sequence revealed that specific amino acid sites existed between mammals and non-mammals. Base on these findings, we hypothesize that PIM1 plays important roles in mammalian erythroblast enucleation. In this application, we will explore the molecular mechanisms by which PIM1 regulate enucleation using state-of-the art approaches including flow cytometry, cell sorting, cell imaging, phosphoproteomics in conjunction with animal models. Successful accomplishment of proposed studies will not only uncover novel mechanisms of mammalian erythroblast enucleation, but may also help in developing strategy for generation of functional erythrocytes ex vivo.

红系发育是造血干细胞增殖分化最终形成成熟红细胞的精细调控过程。其中红细胞脱核是哺乳动物特有的生物学现象。虽然该现象早已被发现，但其机制尚不明确。这也是制约我们利用胚胎干或诱导多能干细胞体外生成功能性红细胞的一个瓶颈。本课题组红系发育各阶段转录组数据显示：PIM1表达水平在晚幼红细胞中显著上调，且是晚幼红细胞表达最高的蛋白激酶。前期我们对PIM1在红系发育中的作用进行了初步研究，发现敲低人CD34+细胞的PIM1不影响红细胞增殖、分化和凋亡，只特异性地影响脱核。此外比对哺乳和非哺乳动物PIM1蛋白序列，我们发现PIM1存在特异变化的氨基酸位点。由此我们推测其在哺乳动物红细胞脱核过程中发挥了重要的调控作用。本项目拟利用流式、细胞成像、磷酸化组学、动物模型等多手段深入探讨PIM1在脱核中的分子机制。项目的完成不仅有助于揭示红细胞脱核的分子调控机制，也有望为体外生成功能性红细胞提供了新的理论策略。

红系发育是造血干细胞增殖分化最终形成成熟红细胞的精细调控过程。其中红细胞脱核是哺乳动物特有的生物学现象。虽然该现象早已被发现，但其机制尚不明确。在此研究中，我们发现丝氨酸/苏氨酸蛋白激酶PIM1在晚幼红细胞中显著上调，且是晚幼红细胞表达最高的蛋白激酶；当在人CD34+细胞中敲低PIM1的表达时，我们发现PIM1的敲低并不影响红细胞的增殖，也未诱导红系细胞的凋亡，并且对红系祖细胞的分化以及随后的红系终末分化也没有显著影响，但是特异性地影响红细胞的正常脱核；进一步利用ImageStream和免疫荧光技术探索PIM1不足对脱核过程的影响时发现：PIM1敲低不影响红细胞核的极化，但显著影响极化后F-actin依赖的肌动蛋白收缩环的形成和内吞囊泡的产生与运输。随后，我们利用EpoR-tdTomato-Cre工具鼠获得了PIM1红系条件性敲除小鼠（Pim1fl/fl EpoRcre）。血常规结果显示：Pim1fl/fl EpoRcre小鼠呈现出一定程度的小细胞低色素贫血（红细胞体积下降、血红蛋白含量下降）；但流式细胞技术检测发现Pim1fl/fl EpoRcre小鼠骨髓中造血干祖细胞稳态、红系祖细胞生成、有核红细胞分化、红系造血岛巨噬细胞发育均未发生显著变化；然而我们发现Pim1fl/fl EpoRcre小鼠的原始红细胞、早幼红细胞、中幼红细胞和晚幼红细胞的细胞大小均与对照小鼠一致，而其脱核后的细胞（网织红细胞和成熟红细胞）的体积却明显小于对照小鼠；此外，Pim1fl/fl EpoRcre小鼠红细胞中F-actin的组装和囊泡运输也均出现明显紊乱。最后，我们分选PIM1敲低组和对照组的人晚幼红细胞并进行了磷酸化蛋白质组学测定，发现PIM1缺乏导致与F-actin组装和膜泡运输相关的调控小GTPase活性的大量蛋白磷酸化修饰发生了明显变化。综上，本项目通过体内外实验首次证实PIM1在红细胞脱核中的重要功能，并阐释了PIM1可能通过调节小GTPase蛋白磷酸化活性从而调控脱核过程中F-actin的组装及内吞囊泡的产生和运输。项目的完成拓展了PIM1在正常发育进程中的作用，丰富了对哺乳动物红细胞脱核的认识，并有望为体外功能性红细胞的生成提供新的理论策略。