Pim激酶调控切应力诱导内皮祖细胞迁移和分化的FRET研究

Endothelial progenitor cells (EPCs) have been extensively studied in cardiovascular diseases and accumulating evidence highlights their importance in vascular repair and tissue remodelling. Thus, understanding the molecular mechanisms involved in the differentiation and migration of EPCs is essential to design effective therapeutic strategies. Shear stress, generated by blood flow or tissue fluid flow, can accelerate the proliferation, differentiation, and capillary-like tube formation of EPCs. Pim proteins consisting of three isoforms (Pim-1, Pim-2, and Pim-3) are a family of serine/threonine kinases that regulate fundamental cellular responses such as cell growth, differentiation, and apoptosis. Our data suggest that Pim and Akt display cooperativity in the regulation of cell growth and migration, indicating that cross-talk exists between the Pim and Akt kinases. We also found that Pim kinases are sensitive to mechanical forces, and shear stress can strongly up-regulate the expression of Pim kinases in EPCs. Thus, based on fluorescence resonance energy transfer (FRET), we will focus on the activation Pim kinases by shear stress, the cross-talk between Pim and Akt kinases, and biological impact of their interaction during EPC migration and differentiation. The final goal of these studies is to explore the mechanical biological mechanisms of stem cell differentiation, and to improve the efficacy of EPC transplantation for vascular repair.

内皮祖细胞（EPCs）的迁移和分化是血管内膜损伤修复中的关键事件。切应力可提高EPCs功能活性并诱导其向内皮细胞的定向分化。Pim是一类丝/苏氨酸蛋白激酶家族，课题组前期在对Pim激酶家族的系列研究中，观察到Pim和蛋白激酶B（Akt）之间在功能上有交叉重叠，提示Pim 和Akt激酶可能存在交互对话（cross-talk）。预实验又发现Pim对应力敏感，切应力能早期显著上调EPCs中Pim表达量。本项目将以Pim蛋白和Akt蛋白之间的“cross-talk”为切入点，利用荧光共振能量转移技术（FRET），主要研究：①受切应力刺激的EPCs，Pim的激活与Akt之间是否存在交互对话？②Pim和Akt “cross-talk”是否是切应力条件下EPCs迁移和分化的关键信号节点？本项目对以上问题的探讨不仅为干细胞分化中的力学生物学机制提供新思路，也为临床开展应力介导的干细胞治疗提供基础实验依据。

PIM是一类丝/苏氨酸蛋白激酶家族。围绕Pim1以及Akt/eNOS信号，我们首先在原代培养的脐静脉内皮细胞（HUVECs）中，Lip3000转染Pim1过表达质粒后，免疫印迹结果显示，Pim1蛋白高表达，同时eNOS Ser1177和eNOS Ser633位点磷酸化表达均升高。用RNA干扰实验沉默Pim1或抑制剂SMI-4a抑制Pim1，免疫印迹结果显示，Pim1蛋白呈低表达，同时eNOS Ser1177和eNOS Ser633位点磷酸化表达均降低。上述结果表明Pim1可能参与了Akt下游的eNOS Ser1177和eNOS Ser633两个位点的磷酸化调节。随后，将原代培养的血管内皮祖细胞（EPCs）加载层流切应力（1.5Pa），发现生理大小层流切应力可通过Pim1调节EPCs中eNOS Ser1177和eNOS Ser633位点磷酸化，诱导EPCs血管形成能力改变。将HUVECs加载层流切应力（1.5Pa），发现切应力可以通过Pim1/eNOS途径调节HUVECs增殖、NO的分泌，这可能是切应力调控内皮细胞NO合成和释放的新途径，为临床心血管疾病的发病机制研究提供了新的力学生物学思路。另外，利用荧光共振能量转移（FRET）技术原理，我们成功构建了ECFP-PIM-Linker-Hsp90-Ypet探针，并通过定点突变PCR设计制备了PIM蛋白突变体探针EPHY_K67M、EPHY_P81SN82K和EPHY_D167A，为深入研究PIM蛋白活性调控及其参与细胞信号转导提供了有效的可视化工具。在此基础上，探索了阿霉素（Doxorubicin）对PIM蛋白活性的影响及其在阿霉素卵泡毒性中的作用与机制，阐明了Doxorubicin可能通过激活Src蛋白，引起细胞内钙库释放，进而引起卵泡细胞内PIM蛋白激活来调控卵泡的毒性，这为临床进行肿瘤药物治疗时减弱阿霉素的毒性作用提供了依据。