He Hua, Zhao Zhi-Hong, Han Fu-Sheng, Wang Xi-Fu, Zeng Yu-Jie
Department of Emergency Cardiology, Beijing Anzhen Hospital, Capital Medical University Beijing 100029, China.
Department of Cardiology, Pudong New Area District Zhoupu Hospital Shanghai 201318, China.
Int J Clin Exp Med. 2015 Jan 15;8(1):188-202. eCollection 2015.
to probe into the effects of PKCε on migration and paracrine functions of stem cells and potential molecular mechanisms.
Bone Marrow mesenchymal stem cells (BMMSCs) were obtained from rat femur and passaged. mRNA and protein levels of capital proteins in PKCε signaling, SDF-1/CXCR4 axis and PI3K/AKT pathway in the MSCs in different conditions treating with PKC agonist, specific PKCε inhibitor, CXCR4 antagonist or PI3K inhibitor for 24 hours were analyzed by real-time PCR and western blot, and migration abilities were observed by migration assay in vitro and the changes of paracrine factors in different treatments were analyzed by protein clips assay.
the levels of p-JNK, p-P38MAPK, SDF-1, CXCR4, PI3K and p-AKT increased significantly after treating with PKC agonist (P < 0.05) and decreased obviously after treating with specific PKCε inhibitor. Migration ability and paracrine function of MSCs were enhanced in PMA group and attenuated in PKCε inhibitor group, and inhibiting activity of CXCR4 or PI3K attenuated the effects of PKCε, but not abolished completely.
There was cross-talking between PKCε signaling and SDF-1/CXCR4 axis and PI3K/AKT pathway in signal transduction of MSCs. Activating PKCε could improve migration ability and paracrine function of MSCs partly at least independent of SDF-1/CXCR4 axis and PI3K/AKT pathway.
探讨蛋白激酶Cε(PKCε)对干细胞迁移和旁分泌功能的影响及其潜在分子机制。
从大鼠股骨获取骨髓间充质干细胞(BMMSCs)并传代培养。用PKC激动剂、特异性PKCε抑制剂、CXCR4拮抗剂或PI3K抑制剂处理骨髓间充质干细胞24小时后,采用实时荧光定量PCR和蛋白质印迹法分析不同处理条件下骨髓间充质干细胞中PKCε信号通路、SDF-1/CXCR4轴及PI3K/AKT信号通路中相关蛋白的mRNA和蛋白水平,通过体外迁移实验观察细胞迁移能力,采用蛋白质印迹法分析不同处理条件下旁分泌因子的变化。
PKC激动剂处理后,p-JNK、p-P38MAPK、SDF-1、CXCR4、PI3K及p-AKT水平显著升高(P<0.05),特异性PKCε抑制剂处理后明显降低。PMA组骨髓间充质干细胞迁移能力和旁分泌功能增强,PKCε抑制剂组减弱,抑制CXCR4或PI3K活性可减弱PKCε的作用,但不能完全消除。
骨髓间充质干细胞信号转导过程中PKCε信号通路与SDF-1/CXCR4轴及PI3K/AKT信号通路存在交互作用。激活PKCε至少部分可通过独立于SDF-1/CXCR4轴及PI3K/AKT信号通路的方式提高骨髓间充质干细胞的迁移能力和旁分泌功能。