Mechanotransduction and the Regulation of Skeletal Muscle Mass

描述（由申请人提供）：机械刺激在骨骼肌质量的调节中起重要作用，肌肉质量的维持对疾病预防和生活质量有重要作用。虽然机械信号和肌肉质量调节之间的联系已经被认识了几十年，但驱动这一重要过程的分子机制仍然不为人知。因此，我们研究的长期目标是确定机械刺激调节肌肉质量的分子事件。在本项目中，我们旨在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事件，在本项目中，我们将重点研究雷帕霉素敏感的mTOR （RSmTOR）信号。我们专注于RSmTOR信号，因为我们之前的工作已经确定：1)机械刺激可以强烈激活RSmTOR信号；ii) RSmTOR信号对于机械诱导的肥厚反应是必要的；iii) RSmTOR信号的激活本身就足以诱导肥大。由于机械刺激激活RSmTOR信号，因此必须存在机械转导途径，将机械信息转化为激活RSmTOR信号的生化事件。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mTOR靶向到LEL是必要的，反过来，RSmTOR信号的机械激活；2) RSmTOR信号的机械激活部分是由于二酰基甘油激酶ζ (DGKζ)依赖于LEL处磷脂酸（PA）的增加；3)机械刺激诱导tuberin （TSC2）磷酸化增加，使其与LEL分离，结果，LEL上的Rheb被激活并刺激RSmTOR信号传导。除了测试这些假设，我们还将确定Raptor， DGKζ/PA和TSC2/Rheb在多大程度上促进了机械诱导的蛋白质合成变化和诱导肥厚。重要的是，通过使用先进的技术，我们将能够在体内测试我们所有的假设（例如，生物传感器的体内转染，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）来全球绘制机械调节的蛋白质组/磷蛋白质组，并通过我们的方法，我们将能够确定哪些事件是下游介导的，哪些是上游介导的/平行于RSmTOR信号的激活。因此，我们期望这个项目的结果不仅将填补我们目前知识的关键空白，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，旨在充分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.