Role of STAT3 signaling in muscle stem cell activation

描述（由申请人提供）：成体肌肉干细胞（MuSC），也称为卫星细胞，是骨骼肌再生的主要来源。这是由于它们在体内自我更新的能力，即在细胞分裂时产生更多的自身拷贝，并产生承诺的后代，正如我们通过单次MuSC移植所最终证明的那样。MuSC在健康成人组织中处于静止状态，在受到应激或损伤时被激活增殖，产生大量祖细胞修复受损组织。虽然静止和激活之间的平衡是MuSC功能的一个关键开关，但调节这一转变的分子机制仍在很大程度上未知。更清楚地了解MuSC激活的调控网络可能会提供新的靶点，有助于开发改善肌肉萎缩疾病的治疗方法。我们的初步研究结果首次表明，STAT3在bHLH肌生成调节因子MyoD的转录激活中起直接作用，这是MuSC退出静止状态的关键事件。STAT3是一种转录因子，在多种干细胞的自我更新中发挥重要作用，包括胚胎干细胞、肠道干细胞和造血细胞。在细胞因子的刺激下，STAT3被JAK激酶磷酸化，同二聚体，易位到细胞核，结合DNA激活靶基因的转录。尽管先前已经证明STAT3在骨骼肌损伤时被激活，但其在MuSC激活中的作用及其相关的下游靶点仍然知之甚少。因此，本研究的重点是研究JAK/STAT3通路在MuSC激活中的作用及其与MyoD的功能相互作用。我们的研究将利用以下工具：(1)结合时间延迟显微镜的功能丧失研究，监测体外MuSC的激活、增殖和存活；(2)使用MyoD近端增强子缺失突变体的荧光素酶报告基因测定，验证STAT3在MyoD转录激活中的直接作用；(3)MuSC中STAT3的条件遗传消融，以评估其在完整动物体内MuSC功能中的作用。总之，这些研究将构成该领域概念上的进步，因为它们将确定JAK/STAT3和MyoD之间的直接功能相互作用，并进一步扩展我们对MuSC激活调控网络的了解。最后，这些发现将有助于制定旨在促进肌肉干细胞介导的组织再生的策略，以改善肌肉萎缩性疾病。

DESCRIPTION (provided by applicant): Adult muscle stem cells (MuSC), also known as satellite cells, are the main source skeletal muscle regeneration. This is due to their ability to self-renew in vivo, i.e. to generate upon cell division more copies of themselves as well as give rise to committed progeny, as we have conclusively demonstrated by single MuSC transplantation. MuSC exist in healthy adult tissues in a quiescent state, and upon stress or injury they are activated to proliferate and generate large numbers of progenitors to repair the damaged tissue. Although the balance between quiescence and activation is a critical switch in MuSC function, the molecular mechanisms regulating this transition are still largely unknown. A more clear understanding of the regulatory networks underlying MuSC activation might provide novel targets that would aid in the development of therapeutic approaches to ameliorate muscle-wasting diseases. Our preliminary findings indicate for the first time that STAT3 plays a direct role in the transcriptional activation of the bHLH myogenic regulatory factor MyoD, a key event as MuSC exit the quiescent state. STAT3 is a transcription factor that plays a major role in self-renewal of several types of stem cells, including embryonic, intestinal and hematopoietic compartments. Upon cytokine stimulation, STAT3 is phosphorylated by JAK kinases, it homodimerizes, translocates to the nucleus and binds DNA to activate the transcription of target genes. Although it has been previously shown that STAT3 is activated upon skeletal muscle injury, its role in MuSC activation as well as its relevant downstream targets are still poorly understood. Accordingly, the focus of this proposal is to investigate the role of the JAK/STAT3 pathway in MuSC activation and its functional interaction with MyoD. Our research will take advantage of the following tools: (1) Loss of function studies in conjunction with time-lapse microscopy, to monitor MuSC activation, proliferation and survival in vitro, (2) Luciferase reporter assay using deletion mutants of the MyoD proximal enhancer, to validate the direct role of STAT3 in MyoD transcriptional activation, (3) Conditional genetic ablation of STAT3 in MuSC, in order to evaluate its role in MuSC function in vivo in the intact animal. Together, these studie would constitute a conceptual advancement in the field, as they would identify a direct functional interaction between JAK/STAT3 and MyoD, and further extend our knowledge of the regulatory network in MuSC activation. Finally, these findings would aid in the development of strategies aimed at promoting muscle stem cell-mediated tissue regeneration to ameliorate muscle-wasting diseases.