PDK1/AKT信号通路对肌腱组织受力刺激的保护机制研究

Tendons are mechanosensitive connective tissues that transfer force from muscle to bone. Although mechanical overload is one of the main causes of tendon injuries, physiological loading still plays an essential role in maintaining tendon integrity. In China, a huge health and economy burden is caused by tendon injuries. Therefore, better strategies are needed in tendon injury prevention, treatment development and rehabilitation. To achieve this goal, the fundamental understanding of how tendons maintain homeostasis subjected to mechanical stimulation is essential. In our previous studies, we identified that activation AKT signal cascade by the 6% uni-axial loading, plays an essential role in the tenogenesis and neo-tendon formation. Thus, we generated SCX-PDK1 mice strain with compromised AKT activation in tendon tissue, and demonstrated for the first time that even low-intensity treadmill training induced decreased tendon mass and rupture of Achilles tendon in SCX-PDK1 mice but not in WT mice. Based on our preliminary findings, we hypothesize that “PDK1/AKT signaling pathway is essential in maintaining homeostasis of tendon subjected to mechanical loading”. To address the hypothesis, we will execute the following four linked AIMs:.AIM 1: Investigate the role of PDK1/AKT activation in tendon development, maturation and in vitro neo-tendon formation ability using SCX-PDK1 mice. .AIM 2: Examine if transgenic inducible deletion of PDK1 impair tendon homeostasis subjected to mechanical overloading using tendon specific tamoxifen-inducible deletion PDK1 mice model (SCX-CreER-PDK1)..AIM 3: Investigate whether pharmacological manipulation of AKT activation improves tendon healing in tendon injury mice model..This project will provide a powerful tool for the further basic tendon study and possibly form the foundation on which to build future research into the development of novel strategies or refinement of existing pharmacological interventions for tendon health.

肌腱损伤是肌骨系统伤病中最常见的一种，其会引起疼痛，影响病人的日常生活,严重的话会导致残疾。在课题组前期研究中，我们首次发现了AKT通过将力学刺激信号转化成生物信号调节了肌腱组织的形成。于是我们针对AKT活化所需的上游激酶PDK1，构建了肌腱特异性PDK1基因敲除小鼠（SCX-PDK1）模型。在成年SCX-PDK1小鼠中，我们发现虽然其跟腱比野生型小鼠要小，但是并没有明显的行动障碍。值得注意的是，SCX-PDK1小鼠在仅仅接受低强度的跑步机训练后，就发生了跟腱变小和断裂的情况，而野生型小鼠却没有任何异常。基于前期研究基础，我们假设PDK1/AKT的激活是肌腱细胞转译力学信号的重要节点，其对肌腱的形成与力学保护其重要的作用。为了验证我们的假设，本项目拟结合体外基础研究和转基因小鼠体内模型，对肌腱受力保护机制进行深入的研究，为未来肌腱研究奠定基础和对肌腱损伤的治疗提供新的思路。

肌腱主要负责将肌肉收缩产生的拉力传导到骨骼系统上，从而驱动关节活动，由于长期受到反复的机械力刺激，肌腱损伤是肌骨系统伤病中最常见的一种，其会引起疼痛，影响病人的日常生活甚至导致残疾。我们发现，肌腱在受到机械力刺激后，肌腱细胞中AKT会被磷酸化，并且激活下游信号通路，最终调节肌腱组织的形成与修复。由于PDK1是AKT信号通路的上游分子，是激活AKT的核心条件。在肌腱特异性敲除SCX-PDK1小鼠中，我们发现.在胚胎阶段以及新生期，其肌腱与野生型小鼠无显著性区别，证明AKT的磷酸化激活并不影响小鼠跟腱的发育。在成年小鼠中，我们检查了8周，16周以及24周的小鼠，发现SCX-PDK1小鼠的跟腱比野生型小鼠要小18%。进一步地，我们对8周的小鼠进行16天，每天2小时的上坡跑步机训练，发现48%的SCX-PDK1小鼠经过锻炼后会发生跟腱断裂情况，而野生型小鼠跟腱结构完整，没有病变。我们在体外构建了3D单向力学刺激模型，从而模拟小鼠肌腱的受力拉伸情况分析细胞内信号通路的激活情况。我们发现正常情况下单向拉伸会引起细胞的内AKT蛋白活化从而激活下游负责蛋白合成的S6K最终使肌腱干细胞成肌腱细胞分化并生成一型胶原。但是在PDK1缺失型小鼠中我们发现即使AKT不能被活化，S6K的磷酸化被抑制从而导致一型胶原无法被合成，最终引起了跟腱无法修导致断裂。本项目通过体内外实验阐明了PDK1-AKT是肌腱感受机械压力的一个重要通路，为是肌腱损伤修复提供了一个潜在治疗靶点。