Effects of HGF and Myosatin on HIF-1Alpha and Satellite Cell Activation

描述(申请人提供)：骨骼肌减少，与年龄相关的骨骼肌质量和力量的丧失，与老年人的生活质量下降、残疾发生率增加、丧失独立生活能力和疾病风险增加有关。卫星细胞的激活、增殖和分化是维持肌肉质量所必需的过程，随着年龄的增长，卫星细胞的功能下降，包括卫星细胞的激活。在这方面，卫星细胞激活是一个合适的靶点，用于治疗干预以改善骨质疏松症的有害影响，因此，长期目标是更好地了解卫星细胞激活。从静止到激活的过程以细胞周期进入增加和新陈代谢增加为特征。由于低氧诱导因子-1(HIF-1)能够反式激活糖酵解代谢的重要基因，一个合理的假设是HIF-1对卫星细胞激活的增加很重要，并且可能与随着年龄增长而发生的卫星细胞激活减少有关。为了验证这些假说，以激活卫星细胞而闻名的肝细胞生长因子(HGF)和卫星细胞激活的负调控因子myostatin将被用来确定HIF-1活性和细胞代谢增加在卫星细胞激活中的重要性，这些信息将被用来确定卫星细胞随年龄增长而延迟激活的机制。本研究的具体目的是：1)确定HIF-1在HGF诱导的卫星细胞激活增加中的作用；2)确定HIF-1在肌肉生长抑素诱导的卫星细胞激活减少中的作用；以及3)确定HIF-1在随着年龄增长而延迟的卫星细胞激活中的重要性。卫星细胞和单纤维培养，以及成年和老年动物的完整骨骼肌模型将被用来实现这些目标。HGF、myostatin、MAPK/ERK、Smad和HIF-1活性将结合HIF-1依赖的启动子活性、mRNA和蛋白质测量进行药理学和/或遗传学操作。由于之前尚未确定HIF-1在卫星细胞激活过程中的功能，因此除了染色质免疫沉淀外，还将测量关键HIF-1靶基因的mRNA水平。卫星细胞(成人肌肉干细胞)的激活在衰老过程中受到损害，可能是随着衰老而发生的骨骼肌块丢失的一个因素。这项研究旨在加深对两种生长因子HGF和myostatin如何影响卫星细胞激活的了解，以及这些生长因子是否通过转录因子缺氧诱导因子(HIF-1)发挥作用。

DESCRIPTION (provided by applicant): Sarcopenia, the age-associated loss of skeletal muscle mass and strength, is linked to a decreased quality of life for, an increased incidence of disability, loss of independent living, and increased risk for morbidity for the elderly. The activation, proliferation, and differentiation of satellite cells is a necessary progression for the maintenance of muscle mass, and decreases in satellite cell function, including satellite cell activation, have been reported with aging. In this regard, satellite cell activation is an appropriate target for therapeutic interventions to ameliorate the deleterious effects of sarcopenia, therefore, the long-term objectives are to better understand satellite cell activation. The progression from quiescence to activation is characterized by increased cell cycle entry and an increase in metabolism. Since hypoxia-inducible factor-1 (HIF-1) is known for its ability to transactivate genes important for glycolytic metabolism, a plausible hypothesis is that HIF-1 is important for increased in satellite cell activation, and may be involved in decreased satellite cell activation that occurs with aging. To test these hypotheses, hepatocyte growth factor (HGF), which is well-known for its ability to activate satellite cells, and myostatin, a negative regulator of satellite cell activation, will be used to determine the importance of HIF-1 activity and increased cell metabolism in satellite cell activation, and this information will be used to determine mechanisms underlying delayed satellite cell activation with aging. The specific aims of this proposal are to 1) Determine the role of HIF-1 during HGF-induced increases in satellite cell activation, 2) Determine the role of HIF-1 during myostatin-induced decreases in satellite cell activation, and 3) Determine the importance of HIF-1 in delayed satellite cell activation with aging. Satellite cell and single fiber cultures, and whole skeletal muscle models in adult and old animals will be used to accomplish these aims. HGF, myostatin, MAPK/ERK, Smad, and HIF-1 activities will be manipulated pharmocologically and/or genetically in conjunction with HIF-1 -dependent promoter activity, mRNA, and protein measurements. Since the function of HIF-1 during satellite cell activation has not previously been determined, mRNA levels of key HIF-1 target genes will be measured in addition to chromatin immunoprecipitatioin. Satellite cell (adult muscle stem cell) activation is impaired in aging and may be a contributor to the loss of skeletal muscle mass that occurs with aging. This research is directed towards increasing the understanding how two growth factors affect satellite cell activation, HGF and myostatin, and if these growth factors work through the transcription factor hypoxia-inducible factor (HIF-1).