Role of AP-1 in Skeletal Myogenesis

AP-1 在骨骼肌生成中的作用

• 批准号: RGPIN-2018-05896
• 负责人: McDermott, John
• 金额: $ 2.62万
• 依托单位: York University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=659212
• 关键词: Role; AP; Skeletal; Myogenesis

Development, maintenance and regeneration of skeletal muscle is a primal feature of eumetazoans (all animals except sponges and placozoans). Muscles, as tissues specialized for contraction, are essential effectors of the nervous system underlying activities such as locomotion, escape, feeding, and defence. In previous work we implicated the AP-1 transcription complex in the control of skeletal muscle gene regulation during the fundamental process of myogenic differentiation. Recently, we reported that AP-1 is expressed not only in embryonic myoblasts but also in the resident stem cells (satellite cells) in adult skeletal muscle, We also documented that ERK signalling stabilizes the Fra2 subunit of the AP-1 complex. Satellite cells are crucial for the vital post-natal maintenance and repair of skeletal muscle in all mammals. In the proposed studies we plan to further assess the requirement for AP-1 in skeletal muscle development and regeneration. To do this we will address the following specific hypothesis driven objectives:******1) AP-1 is required for Notch signalling in myoblasts and satellite cells.***2) Conditional cjun/fosl2 deletion in mice will disrupt embryonic myogenesis and/or post-natal muscle regeneration.***3) Growth factor signaling controls Fra2 transcription complex composition and myogenesis.******Experimental Strategy:***Objective 1: Previous work identified that AP-1 transcriptionally targets a number of components of the Notch signaling pathway in muscle. The next step is to employ gain and loss of function analysis of AP1 to assess how this influences components and downstream targets of Notch signalling in primary myoblast and single muscle fiber cultures. We will utilize traditional biochemistry and state of the art confocal live cell imaging technology to address this aim.***Objective 2: Conditional Cre-lox mediated excision of Floxed cJun and Fra2 in mice is an ambitious goal to assess the role of AP-1 in embryonic muscle formation and also post-natal muscle maintenance and regeneration.***Objective 3: Characterization of the signalling pathway regulated control of Fra2 complex composition and stability will be carried out using an innovative state of the art interactome analysis pipeline that we have developed using affinity purification-mass spectrometry, along with antibody mediated detection of post-translational modification of Fra2.******Feasibility and Impact: ***This proposal, based on extensive data generated over several NSERC funding cycles, will address the fundamental regulation of AP-1 and its role in skeletal muscle. Personnel, reagents and technology are in place to address these objectives with high feasibility. These studies will result in impactful science in line with our other contributions in this field.

骨骼肌的发育、维持和再生是真后生动物（除了海绵和盘形动物以外的所有动物）的主要特征。肌肉作为专门用于收缩的组织，是神经系统的基本效应器，这些神经系统是诸如运动、逃跑、进食和防御等活动的基础。在以前的工作中，我们暗示AP-1转录复合物在骨骼肌基因调控的肌分化的基本过程中的控制。最近，我们报道AP-1不仅在胚胎成肌细胞中表达，而且在成人骨骼肌的驻留干细胞（卫星细胞）中也表达。我们还证明ERK信号稳定AP-1复合物的Fra 2亚基。在所有哺乳动物中，卫星细胞对于骨骼肌的重要产后维护和修复至关重要。在拟议的研究中，我们计划进一步评估骨骼肌发育和再生对AP-1的需求。为了做到这一点，我们将解决以下具体假设驱动的目标：*1）AP-1是成肌细胞和卫星细胞中Notch信号传导所必需的。2)小鼠中的条件性cjun/fosl 2缺失将破坏胚胎肌发生和/或出生后肌肉再生。3)生长因子信号传导控制Fra 2转录复合物的组成和肌生成。实验策略：* 目标1：先前的工作确定AP-1在转录上靶向肌肉中Notch信号通路的许多组分。下一步是采用AP 1功能分析的获得和丧失来评估这如何影响原代成肌细胞和单肌纤维培养物中Notch信号传导的组分和下游靶点。我们将利用传统的生物化学和最先进的共聚焦活细胞成像技术来实现这一目标。*目标二：小鼠中Floxed cJun和Fra 2的条件性Cre-lox介导的切除是评估AP-1在胚胎肌肉形成以及出生后肌肉维持和再生中的作用的雄心勃勃的目标。目标三：Fra 2复合物组成和稳定性的信号传导途径调节控制的表征将使用我们已经使用亲和纯化-质谱法开发的最先进的相互作用组分析管道的创新状态进行，沿着Fra 2的翻译后修饰的抗体介导的检测。**可行性和影响：* 这项提案基于NSERC几个资助周期产生的大量数据，将解决AP-1的基本调节及其在骨骼肌中的作用。人员、试剂和技术都已到位，以高度可行性实现这些目标。这些研究将产生与我们在该领域的其他贡献相一致的有影响力的科学。