蛋白激酶Pip5K1α在骨骼肌成肌细胞分化中的功能和机制研究

Protein kinases are responsible for substrate phosphorylation and control various biological processs, which makes protein kinases attractive drug targets. It is known that many kinases are involved in controlling skeletal msucle stem cell function. Howerver, there is no systematic study to look in to the function kinases and skeletal muscle myoblast. In our preliminary study, we employed an genome-wide scale RNA interference (RNAi)-based screens to look for the kinases which are essencial for myoblast differentiation. From the screening, we identified Pip5K1 as one of the positive hits. Pip5K1 family has three isoforms, α,βand γ. It is well known that Pip5K1 is involved in production of PtdIns(4,5)P2 and regulates cytoskeleton organization, endocytosis and apoptosis. Our work in this proposal show for the first time that the Pip5K1α was highly expressed in both mature skeletal and myoblast. Knockdown of Pip5K1α using specific siRNAs inhibited myogenic differentiation in both C2C12 myoblast and primary muscle satellite cell. In this proposal, we will employed microarry and siRNA to examine the downstream genes of Pip5K1α during muscle stem cell differentiation. Also, we'll use the mouse model to exaim the function of Pip5K1α in muscle development. We hope that our proposed study will allow us to gain a more complete view of how Pip5K1α is inivolved the myoblast differentiation. Such new knowledge will contributes to a more complete and deeper understanding of myogenic differentiation at the molecular levels and could be useful in the future in designing effective strategies to manipulate muscle stem cells in humans in order to speed up the muscle regeneration process after muscle injury/damage.

激酶通过磷酸化底物调控许多生理过程，也是药物研究的热点。肌肉干细胞负责骨骼肌发育和损伤修复，但目前尚没有对激酶在肌肉干细胞中的功能进行系统研究。通过全基因组激酶的RNAi筛选，我们发现激酶Pip5K1α在肌肉组织和肌肉干细胞中高表达，并调控C2C12和小鼠原代肌肉干细胞的成肌性分化，但机理尚不清楚。本研究将(1)采用基因干扰、免疫印迹和荧光素报告基因等方法在肌肉干细胞中检测Pip5K1α对MEF2,MyoD等重要转录因子表达水平、转录活性的影响；(2)研究Pip5K1α和对p38、ERK、Akt等蛋白的磷酸化水平的影响；(3)采用酵母双杂交方法，筛选Pip5K1α结合的新蛋白并确定其对肌肉干细胞分化的影响；(4)通过动物实验，明确Pip5K1α对小鼠骨骼肌发育的影响。该项目的实施将阐明Pip5K1α调控肌肉分化的分子机制，并为后续骨骼肌损伤修复和肌肉萎缩的研究治疗提供理论依据和靶点。

通过全基因组激酶的RNAi筛选，我们发现激酶Pip5K1α在肌肉组织和肌肉干细胞中高表达，并调控C2C12和小鼠原代肌肉干细胞的成肌性分化，但机理尚不清楚。本研究通过构建稳定表达荧光素报告基因的C1C12细胞株，应用蛋白激酶组siRNA文库筛选出调控成肌细胞分化的激酶Pip5K1α，发现在C2C12细胞中过表达PIP5K1a加速了肌肉细胞的分化。通过研究Pip5K1α在成肌性分化过程中对p38, ERK，AKT等重要信号分子活性的影响，发现Pip5K1α特异性的通过调节AKT通路的活化来促进骨骼肌成肌细胞的分化，并通过PIP2参与成肌细胞质钙离子释放。采用基因芯片技术，对受Pip5K1α调控的下游靶基因进行筛选后，我们发现Tagln 的表达水平在PIP5K1a抑制的C2C12 细胞中剧烈下降，而通过RNAi抑制Tagln的表达后，成肌细胞的分化也受到抑制。激酶通过磷酸化底物调控许多生理过程，也是药物研究的热点。肌肉干细胞负责骨骼肌发育和损伤修复，但目前尚没有对激酶在肌肉干细胞中的功能进行系统研究。本项目阐明了Pip5K1α调控肌肉分化的分子机制，并为后续骨骼肌损伤修复和肌肉萎缩的研究治疗提供了理论依据和靶点。