Mechanotransduction and the Regulation of Skeletal Muscle Mass

描述（申请人提供）：机械刺激在骨骼肌质量的调节中发挥着重要作用，肌肉质量的维持对疾病预防和生活质量做出了重大贡献。尽管几十年来人们已经认识到机械信号和肌肉质量调节之间的联系，但驱动这一重要过程的分子机制仍然未知。因此，我们研究的长期目标是定义机械刺激调节肌肉质量的分子事件。在这个项目中，我们的目标是确定机械刺激激活哺乳动物雷帕霉素靶点（mTOR）信号传导的机制。具体来说，现在已知 mTOR 可以发挥雷帕霉素敏感和雷帕霉素不敏感的信号传导事件，在本项目中，我们将重点关注雷帕霉素敏感的 mTOR (RSmTOR) 信号传导。我们关注 RSmTOR 信号传导，因为我们之前的工作表明：i) 机械刺激可以强有力地激活 RSmTOR 信号传导； ii) RSmTOR 信号传导对于机械诱导的肥大反应是必需的； iii) RSmTOR 信号传导的激活本身足以诱导肥大。由于机械刺激激活 RSmTOR 信号传导，因此必须存在机械转导途径，用于将机械信息转换为激活 RSmTOR 信号传导的生化事件。根据我们的初步数据，我们提出晚期内体/溶酶体系统（LEL）是该途径的核心组成部分。该项目的前三个目标将通过测试以下假设来解决这一概念：1）Raptor 对于将 mTOR 靶向 LEL 以及 RSmTOR 信号传导的机械激活是必要的； 2) RSmTOR 信号传导的机械激活部分归因于 LEL 处磷脂酸 (PA) 的二酰基甘油激酶 z (DGK z) 依赖性增加； 3) 机械刺激诱导马铃薯蛋白 (TSC2) 磷酸化增加，导致其与 LEL 解离，结果，LEL 处的 Rheb 被激活并刺激 RSmTOR 信号传导。除了测试这些假设之外，我们还将定义 Raptor、DGK z/PA 和 TSC2/Rheb 在多大程度上有助于机械诱导的蛋白质合成变化和诱导肥大。重要的是，通过使用先进技术，我们将能够在体内测试我们所有的假设（例如，用生物传感器进行体内转染、骨骼肌特异性诱导敲除小鼠、敲除小鼠的拯救实验等）。此外，在最后一个目标中，我们将使用最先进的质谱技术（NeuCode）来全局绘制机械调节的蛋白质组/磷酸化蛋白质组，并利用我们的通过这种方法，我们将能够确定哪些事件是下游介导的，哪些是上游/与 RSmTOR 信号激活平行的。因此，我们预计该项目的成果不仅将填补我们当前知识的关键空白，而且还将产生一个新的知识体系，指导未来研究的基本方向，旨在全面定义机械刺激如何调节骨骼肌质量。

DESCRIPTION (provided by applicant): Mechanical stimuli play a major role in the regulation of skeletal muscle mass, and the maintenance of muscle mass contributes significantly to disease prevention and quality of life. Although the link between mechanical signals and the regulation of muscle mass has been recognized for decades, the molecular mechanisms that drive this vital process are still not known. Hence, the long-term goal of our research is to defin the molecular events through which mechanical stimuli regulate muscle mass. In this project, we aim to identify the mechanisms via which mechanical stimuli activate signaling by the mammalian target of rapamycin (mTOR). Specifically, it is now known that mTOR can exert both rapamycin-sensitive and rapamycin-insensitive signaling events, and in this project we will focus on rapamycin-sensitive mTOR (RSmTOR) signaling. We are focusing on RSmTOR signaling because our previous work established that: i) mechanical stimuli can robustly activate RSmTOR signaling; ii) RSmTOR signaling is necessary for a mechanically-induced hypertrophic response; and iii) the activation of RSmTOR signaling, in and of itself, is sufficient to induce hypertrophy. Since mechanical stimuli activate RSmTOR signaling, it follows that a mechanotransduction pathway must exist for converting mechanical information into the biochemical events that activate RSmTOR signaling. Based on our preliminary data, we are proposing that the late endosomal / lysosomal system (LEL) is a central component of this pathway. The first three aims of this project will address this concept by testing the following hypotheses: 1) Raptor is necessary for the targeting of mTOR to the LEL and, in turn, the mechanical activation of RSmTOR signaling; 2) the mechanical activation of RSmTOR signaling is due, in part, to a diacylglycerol kinase ζ (DGKζ)-dependent increase in phosphatidic acid (PA) at the LEL; and 3) mechanical stimuli induce an increase in the phosphorylation of tuberin (TSC2), which causes it to dissociate from the LEL, and as a result, Rheb at the LEL becomes activated and stimulates RSmTOR signaling. In addition to testing these hypotheses, we will also define the extent to which Raptor, DGKζ/PA and TSC2/Rheb contribute to mechanically-induced changes in protein synthesis and the induction of hypertrophy. Importantly, through the use of advanced techniques, we will be able to test all of our hypotheses in-vivo (e.g., in-vivo transfection with biosensors, skeletal muscle specific inducible knockout mice, rescue experiments in knockout mice, etc.) Furthermore, in the last aim, we will use a state-of-the-art mass spectrometry technique (NeuCode) to globally map the mechanically-regulated proteome / phosphoproteome, and with our approach, we will be able to determine which events are mediated downstream versus upstream / parallel to the activation of RSmTOR signaling. Thus, we expect that the outcomes of this project will not only fill key gaps in our current knowledge, but they will also generate a new body of knowledge that will guide the fundamental direction of future studies that are aimed at fully defining how mechanical stimuli regulate skeletal muscle mass.