Mechanotransduction and the Regulation of Skeletal Muscle Mass

描述(申请人提供)：机械刺激在骨骼肌群的调节中起主要作用，而肌群的维持对疾病预防和生活质量有重要贡献。虽然机械信号和肌肉质量调节之间的联系已经被认识到几十年了，但驱动这一重要过程的分子机制仍然不清楚。因此，我们研究的长期目标是明确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通过哺乳动物雷帕霉素靶标(MTOR)激活信号的机制。具体地说，目前已知mTOR可以同时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，我们将重点介绍雷帕霉素敏感的mTOR(RSmTOR)信号。我们之所以关注RSmTOR信号，是因为我们以前的工作证明：i)机械刺激可以强有力地激活RSmTOR信号；ii)RSmTOR信号是机械诱导的肥大反应所必需的；iii)RSmTOR信号本身的激活足以诱导肥厚。由于机械刺激激活RSmTOR信号，因此必须存在机械信号转导途径，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，我们认为晚期内体/溶酶体系统(LEL)是这一途径的中心组成部分。本项目的前三个目标将通过检验以下假设来解决这一概念：1)Raptor对于mTOR靶向LEL，进而机械激活RSmTOR信号是必需的；2)RSmTOR信号的机械激活，部分是由于依赖二酰甘油激酶ζ(DGKζ)的LEL上磷脂酸(PA)的增加；以及3)机械刺激诱导结节蛋白(TSC2)的磷酸化增加，这导致它与LEL解离，结果，LEL上的RHEB被激活并刺激RSmTOR信号。除了检验这些假说，我们还将确定Raptor、DGKζ/PA和TSC2/Rheb在机械诱导的蛋白质合成变化和诱导肥大中的作用程度。重要的是，通过使用先进技术，我们将能够在体内测试我们的所有假设(例如，生物传感器的体内转基因、骨骼肌特定诱导的基因敲除小鼠、基因敲除小鼠的救援实验等)。此外，在最后一个目标中，我们将使用最先进的质谱学技术(NeuCode)来绘制机械调节的蛋白质组/磷酸蛋白质组的全球图谱，通过我们的方法，我们将能够确定哪些事件是在RSmTOR信号激活的下游或上游/平行地介导的。因此，我们预计该项目的成果不仅将填补我们目前知识中的关键空白，而且还将产生一个新的知识体系，将指导未来旨在充分定义机械刺激如何调节骨骼肌质量的研究的基本方向。

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.