Understanding the Role of the Class II Activator, CIITA, in Skeletal Muscle

描述(申请人提供)：骨骼肌在一生中具有非凡的生长和修复能力。骨骼肌再生不足会导致正常的人体衰老和肌肉营养不良。了解肌肉感觉损伤的过程和肌肉修复的分子细节在人类生物学中都是至关重要的。我们认为，我们已经确定了炎症反应和骨骼肌生肌调节家族(MRF)介导的骨骼肌重构之间的新联系。炎症反应在启动骨骼肌再生中起着重要作用。参与炎症反应的细胞因子之一是抗纤维化药物干扰素？干扰素-？对肌肉生成有复杂的影响，但在体内有效的肌肉再生是必需的。我们已经确定II类反式激活因子CIITA是与肌生成素相互作用的一个因子，肌生成素是介导末端分化的MRF。在免疫细胞中，干扰素-？通过CIITA的作用刺激大量基因的激活和抑制，CIITA既是辅助激活因子又是辅助抑制因子。CITA由干扰素-？激活。通过JAK/STAT通路，导致STAT1直接激活CIITA。CITA既有构成成分，又有干扰素？刺激细胞中的角色。我们已经证实了CIITA在C2C12成肌细胞系中的表达，并表明CIITA作为肌肉特异性基因的转录激活因子，是肌肉生成素活性的特异性抑制因子。CITA的表达受干扰素-β的刺激，两者均受干扰素-β的刺激。CIITA的过度表达下调了肌肉特异性基因的表达，抑制了分化。因此，我们假设CITA是干扰素？？的介体。骨骼肌细胞中的信号。在这项提案中，我们试图了解CIITA如何有助于肌肉发生和肌肉修复。首先，我们建议通过证实我们在原代成肌细胞中的初步发现，以及通过过表达和敲除研究证实CIITA的抗分化作用，来扩展我们目前关于CIITA在成肌细胞中的作用的工作。我们还计划启动对干扰素的作用的研究。在正常的肌肉发生中通过表征干扰素-？由成肌细胞产生，并通过阻断干扰素？受体测定对干扰素-β丢失的影响发信号。接下来，我们通过对CIITA等位基因突变的小鼠进行肌肉损伤研究，试图了解CIITA对肌肉修复的贡献。最后，我们试图确定CIITA是否是干扰素-？和成肌细胞中的STAT1信号。我们的初步数据表明，CIITA及其对肌生成素的抑制与干扰素的许多作用有关。和STAT1对肌源性细胞的影响。我们将试图通过证实STAT1激活成肌细胞中的CIITA，并使用STAT1抑制剂来证实在干扰素？刺激。然后，我们将尝试通过过度表达CIITA来挽救这种影响。我们还将化验干扰素-？在Myog-/-成肌细胞中的作用，以确定是否需要肌生成素来介导干扰素？介导了肌肉特异性靶点的下调。 与公共健康相关：骨骼肌能够在一生中生长和修复。当肌肉由于年龄或肌肉营养不良而无法修复时，这会导致失去行动能力，最终导致死亡。这项提议试图描述肌肉损伤的炎症反应诱导的信号与修复肌肉纤维的因素之间的一种新的联系。我们在骨骼肌细胞中发现了一种因子，它既能对炎症释放的信号做出反应，又能抑制重建肌肉的最后步骤所需的特定因子的活性。我们认为，这个系统给了肌肉在进行最后的修复步骤之前启动修复所需的时间。这项建议旨在进一步确定肌肉细胞中这一因子的特征，并确定其在骨骼肌分化和修复中的细胞作用。这项工作对了解骨骼肌损伤后如何自我修复具有重要意义，并可能为如何在衰老或疾病情况下刺激肌肉再生提供新的见解。

DESCRIPTION (provided by applicant): Skeletal muscle has a remarkable ability to grow and repair throughout life. Deficiencies in skeletal muscle regeneration contribute to normal human aging and muscular dystrophies. Understanding both the process of how muscle senses damage and the molecular details of how muscle is repaired are of central importance in human biology. We believe that we have identified a novel connection between the inflammatory response and skeletal muscle remodeling mediated by the myogenic regulatory family (MRF). The inflammatory response plays an important role in initiating skeletal muscle regeneration. One of the cytokines involved in the inflammation response is the anti-fibrotic agent interferon gamma (IFN-?). IFN-? has complex effects on myogenesis, but is required for efficient muscle regeneration in vivo. We have identified the class II transactivator, CIITA, as a factor that interacts with myogenin, the MRF that mediates terminal differentiation. In immune cells, IFN-? stimulates both the activation and repression of a large set of genes through the action of CIITA, which serves as both a co-activator and co-repressor. CIITA is activated by IFN-? through the JAK/STAT pathway which leads to the direct activation of CIITA by STAT1. CIITA has both constitutive and IFN-? stimulated roles in the cell. We have confirmed that CIITA is expressed in the C2C12 myoblast line and shown that CIITA acts as a specific inhibitor of myogenin's activity as a transcriptional activator of muscle specific genes. CIITA expression is stimulated by IFN-?, and both stimulation with IFN-? and over expression of CIITA down regulate muscle specific gene expression and inhibit differentiation. Thus, we hypothesize that CIITA is the mediator of the IFN-? signal in skeletal muscle cells. In this proposal, we seek to understand how CIITA contributes to myogenesis and muscle repair. First, we propose to extend our current work on the role of CIITA in myoblasts by confirming our preliminary findings in primary myoblasts and confirming the anti-differentiation effect of CIITA with over expression and knock down studies. We also plan to initiate studies on the role of IFN-? in normal myogenesis by characterizing the level of IFN-? produced by myoblasts and by blocking the IFN-? receptor to assay the effects of loss of IFN-? signaling. Next, we seek to understand the contribution of CIITA to muscle repair by conducting muscle damage studies on mice with a disruption of the CIITA allele. Finally, we seek to determine if CIITA is the mediator of IFN-? and STAT1 signaling in myoblasts. Our preliminary data suggests that CIITA and its inhibition of myogenin are responsible for many of the effects of IFN-? and STAT1 on myogenic cells. We will attempt to support our hypothesis by confirming that STAT1 activates CIITA in myoblasts and by using STAT1 inhibitors to confirm a loss of anti differentiation effects following IFN-? stimulation. We will then attempt to rescue this effect by over expressing CIITA. We will also assay for IFN-? effects in Myog-/- myoblasts to determine if myogenin is required to mediate the IFN-? mediated down regulation of muscle specific targets. PUBLIC HEALTH RELEVANCE: Skeletal muscle is able to grow and repair throughout life. When muscle cannot be repaired due to age or muscular dystrophies, this leads to a loss of the ability to move and eventually leads to death. This proposal seeks to characterize a novel link between the signaling induced by the inflammatory response to muscle damage and the factors that can repair muscle fibers. We have discovered a factor in skeletal muscle cells that both responds to the signals released from inflammation and represses the activity of a specific factor required for the final steps in rebuilding muscle. We propose that this system gives muscle the time it needs to initiate repair before committing to the final steps of repair. This proposal seeks to further characterize this factor in muscle cells and determine its cellular role in skeletal muscle differentiation and repair. This work has important implications in understanding how skeletal muscle repairs itself after injury and may be able to offer novel insight into how to stimulate muscle regeneration in the case of aging or disease.