Zhao Yin-Hua, Lv Xin, Liu Yan-Li, Zhao Ying, Li Qiang, Chen Yong-Jin, Zhang Min
State Key Laboratory of Military Stomatology, Department of General Dentistry and Emergency, School of Stomatology, Fourth Military Medical University, No. 145 West Changle Road, Xi'an 710032, China.
State Key Laboratory of Military Stomatology, Department of General Dentistry and Emergency, School of Stomatology, Fourth Military Medical University, No. 145 West Changle Road, Xi'an 710032, China.
Stem Cell Res. 2015 May;14(3):283-96. doi: 10.1016/j.scr.2015.02.006. Epub 2015 Feb 26.
Our previous studies have shown that hydrostatic pressure can serve as an active regulator for bone marrow mesenchymal stem cells (BMSCs). The current work further investigates the roles of cytoskeletal regulatory proteins Ras homolog gene family member A (RhoA) and Ras-related C3 botulinum toxin substrate 1 (Rac1) in hydrostatic pressure-related effects on BMSCs. Flow cytometry assays showed that the hydrostatic pressure promoted cell cycle initiation in a RhoA- and Rac1-dependent manner. Furthermore, fluorescence assays confirmed that RhoA played a positive and Rac1 displayed a negative role in the hydrostatic pressure-induced F-actin stress fiber assembly. Western blots suggested that RhoA and Rac1 play central roles in the pressure-inhibited ERK phosphorylation, and Rac1 but not RhoA was involved in the pressure-promoted JNK phosphorylation. Finally, real-time polymerase chain reaction (PCR) experiments showed that pressure promoted the expression of osteogenic marker genes in BMSCs at an early stage of osteogenic differentiation through the up-regulation of RhoA activity. Additionally, the PCR results showed that pressure enhanced the expression of chondrogenic marker genes in BMSCs during chondrogenic differentiation via the up-regulation of Rac1 activity. Collectively, our results suggested that RhoA and Rac1 are critical to the pressure-induced proliferation and differentiation, the stress fiber assembly, and MAPK activation in BMSCs.
我们之前的研究表明，流体静压可作为骨髓间充质干细胞（BMSCs）的一种活性调节因子。当前的工作进一步研究了细胞骨架调节蛋白Ras同源基因家族成员A（RhoA）和Ras相关C3肉毒杆菌毒素底物1（Rac1）在流体静压对BMSCs相关效应中的作用。流式细胞术分析表明，流体静压以RhoA和Rac1依赖的方式促进细胞周期启动。此外，荧光分析证实，在流体静压诱导的F-肌动蛋白应力纤维组装中，RhoA起积极作用，而Rac1起消极作用。蛋白质免疫印迹表明，RhoA和Rac1在压力抑制的细胞外信号调节激酶（ERK）磷酸化中起核心作用，并且Rac1而非RhoA参与压力促进的应激活化蛋白激酶（JNK）磷酸化。最后，实时聚合酶链反应（PCR）实验表明，在成骨分化早期，压力通过上调RhoA活性促进BMSCs中成骨标记基因的表达。此外，PCR结果显示，在软骨形成分化过程中，压力通过上调Rac1活性增强BMSCs中软骨形成标记基因的表达。总的来说，我们的结果表明，RhoA和Rac1对于压力诱导的BMSCs增殖和分化、应力纤维组装以及丝裂原活化蛋白激酶（MAPK）激活至关重要。
