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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=686804
• 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、维持和再生是真丝虫(除海绵和胎盘动物外的所有动物)的主要特征。肌肉作为专门用于收缩的组织，是神经系统基本活动的基本效应器，如运动、逃逸、摄食和防御。在以前的工作中，我们发现AP-1转录复合体在骨骼肌成肌分化的基本过程中参与了骨骼肌基因调控。最近，我们报道了AP-1不仅在胚胎成肌细胞中表达，而且在成人骨骼肌的常驻干细胞(卫星细胞)中也有表达，我们还发现ERK信号稳定了AP-1复合体的Fra2亚单位。卫星细胞对所有哺乳动物出生后骨骼肌的重要维持和修复至关重要。在拟议的研究中，我们计划进一步评估AP-1在骨骼肌发育和再生中的需求。为此，我们将解决以下特定假设驱动的目标：*1)在成肌细胞和卫星细胞中Notch信号传导需要AP-1。*2)小鼠有条件的cjun/fosl2缺失将扰乱胚胎和/或出生后肌肉的再生。*3)生长因子信号控制Fra2转录复合体和肌肉发生。*实验策略：*目标1：先前的工作发现，AP-1在转录上针对肌肉中Notch信号通路的许多成分。下一步是利用AP1的得失功能分析来评估这如何影响原代成肌细胞和单肌纤维培养中Notch信号的成分和下游目标。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解决这一目标。*目标2：条件Cre-lox介导的小鼠cJun和Fra2的条件切除是一个雄心勃勃的目标，目的是评估AP-1在胚胎肌肉形成和出生后肌肉维持和再生中的作用。*目标3：将使用我们开发的创新的相互作用组分析管道，利用亲和纯化-质谱仪来表征信号通路调控的Fra2复合体的组成和稳定性，以及抗体介导的Fra2翻译后修饰的检测。*可行性和影响：*这项建议基于NSERC几个资金周期产生的大量数据，将解决AP-1的基本调控及其在骨骼肌中的作用。人员、试剂和技术都已到位，以实现这些目标并具有很高的可行性。这些研究将产生与我们在该领域的其他贡献一致的有影响力的科学成果。