Fan-Yen Lee1,2, Yen-Yi Zhen3, Chun-Man Yuen4,2, Raymond Fan5, Yen-Ta Chen6,2, Jiunn-Jye Sheu1, Yi-Ling Chen3, Ching-Jen Wang7,2, Cheuk-Kwan Sun8, Hon-Kan Yip3,9,2,10,11. 1. Division of Thoracic and Cardiovascular Surgery, Department of Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of MedicineKaohsiung 88301, Taiwan. 2. Center for Shockwave Medicine and Tissue Engineering, Kaohsiung Chang Gung Memorial HospitalKaohsiung 83301, Taiwan. 3. Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of MedicineKaohsiung 88301, Taiwan. 4. Division of Neurosurgery, Department of Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of MedicineKaohsiung 88301, Taiwan. 5. Department of Biochemistry, Hunter College, City University of New YorkNY 10065, U.S.A. 6. Divisions of Urology, Department of Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of MedicineKaohsiung 88301, Taiwan. 7. Department of Orthopedic Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of MedicineKaohsiung 88301, Taiwan. 8. Department of Emergency Medicine, E-Da Hospital, I-Shou University School of Medicine for International StudentsKaohsiung 82445, Taiwan. 9. Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial HospitalKaohsiung 83301, Taiwan. 10. Department of Medical Research, China Medical University Hospital, China Medical UniversityTaichung 40402, Taiwan. 11. Department of Nursing, Asia UniversityTaichung 41354, Taiwan, China.
Abstract
BACKGROUND: 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. METHODS: Shock-wave was adopted as a tool for MTD and mTOR-FAK signaling. RESULTS: 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. CONCLUSION: 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.
BACKGROUND: 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. METHODS: Shock-wave was adopted as a tool for MTD and mTOR-FAK signaling. RESULTS: 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. CONCLUSION: 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.
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