Lee Fan-Yen, Zhen Yen-Yi, Yuen Chun-Man, Fan Raymond, Chen Yen-Ta, Sheu Jiunn-Jye, Chen Yi-Ling, Wang Ching-Jen, Sun Cheuk-Kwan, Yip Hon-Kan
Division of Thoracic and Cardiovascular Surgery, Department of Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of MedicineKaohsiung 88301, Taiwan.
Center for Shockwave Medicine and Tissue Engineering, Kaohsiung Chang Gung Memorial HospitalKaohsiung 83301, Taiwan.
Am J Transl Res. 2017 Apr 15;9(4):1603-1617. eCollection 2017.
Mechanotransduction (MTD) is an important physiopathological signalling pathway associated with cardiovascular disease such as hypertension. Phosphorylation of focal adhesion kinase (FAK) is a MTD-sensing protein. This study tested the hypothesis that mTOR-FAK MTD signaling axis was crucial for focal adhesion (FA) maturation and cell proliferation.
Shock-wave was adopted as a tool for MTD and mTOR-FAK signaling.
After demonstrating a failure in FAK phosphorylation after microfilament depolymerization, we attempted to identify the upstream regulator out of three kinases known to be activated in pressure-stimulated MTD [i.e., GSK-3β, Akt, and mTORC1 (mammalian target of rapamycin complex 1)]. Of the three specific inhibitors, only rapamycin, an inhibitor of mTORC1, was found to inhibit FAK phosphorylation, suggesting that mTORC1 is the upstream regulator in shock-wave-elicited FAK phosphorylation. Moreover, mTOR and its readout protein S6K were found to be activated by shock-wave stimulation. On the other hand, microscopic examination revealed not only MTD-induced increase in the number of actin stress fibers, but also alternative subcellular localization of mTORC1 as vesicle-like inclusions on microfilaments. Besides, rapamycin was found to destruct the granular pattern of mTORC1, while dissociation between F-actin and mTORC1 was noted after cytochalasin D administration. Since mTORC1 and FAK are essential for cell proliferation, we performed proliferation assay for mesenchymal stem cell (MSC) with and without shock-wave administration/rapamycin treatment/FAK depletion. The results demonstrated significant enhancement of cell proliferation after shock-wave stimulation but remarkable suppression after rapamycin and siFAK treatment.
Our findings suggest not only a co-ordinated regulation of FAK phosphorylation by mTORC1 and microfilaments, but also the participation of mTORC1-FAK signalling in MSC proliferation.
机械转导（MTD）是一种与心血管疾病（如高血压）相关的重要生理病理信号通路。粘着斑激酶（FAK）的磷酸化是一种MTD传感蛋白。本研究检验了mTOR-FAK MTD信号轴对粘着斑（FA）成熟和细胞增殖至关重要这一假设。
采用冲击波作为MTD和mTOR-FAK信号的工具。
在证明微丝解聚后FAK磷酸化失败后，我们试图从已知在压力刺激的MTD中被激活的三种激酶[即糖原合成酶激酶-3β（GSK-3β）、蛋白激酶B（Akt）和雷帕霉素靶蛋白复合体1（mTORC1）]中鉴定上游调节因子。在这三种特异性抑制剂中，仅发现雷帕霉素（一种mTORC1抑制剂）可抑制FAK磷酸化，这表明mTORC1是冲击波诱导的FAK磷酸化的上游调节因子。此外，发现mTOR及其读出蛋白核糖体蛋白S6激酶（S6K）可被冲击波刺激激活。另一方面，显微镜检查不仅显示MTD诱导肌动蛋白应力纤维数量增加，而且还显示mTORC1在微丝上呈囊泡样包涵体的替代性亚细胞定位。此外，发现雷帕霉素可破坏mTORC1的颗粒模式，而在给予细胞松弛素D后，观察到丝状肌动蛋白（F-肌动蛋白）与mTORC1之间的解离。由于mTORC1和FAK对细胞增殖至关重要，我们对给予或未给予冲击波/雷帕霉素处理/FAK缺失的间充质干细胞（MSC）进行了增殖测定。结果表明，冲击波刺激后细胞增殖显著增强，但雷帕霉素和小干扰RNA FAK（siFAK）处理后显著抑制。
我们的研究结果不仅表明mTORC1和微丝对FAK磷酸化有协同调节作用，而且表明mTORC1-FAK信号通路参与了MSC的增殖。
