Investigation of the role of AMP-activated protein kinase alpha2 (AMPKa2) in bone

成骨受到多条信号通路的调节，其中几条信号通路起着关键作用。 信号通路。为了找出调节骨量的新因素，我们筛选了一名人类 蛋白激酶siRNA文库寻找与间充质成骨分化相关的蛋白 干细胞。小干扰RNA介导的腺苷α2催化亚基5‘- 骨髓间充质干细胞中一磷酸活化蛋白激酶(AMPK)活性显著增强 碱性磷酸酶是成骨分化的早期标志，这表明AMPKa2可能具有 在成骨细胞中发挥作用，可能在骨形成中起作用。AMPK是哺乳动物细胞的能量传感器，因为它 直接对燃料分子三磷酸腺苷的消耗做出反应。虽然AMPK在各种不同的 外周器官，包括肌肉、心脏和肝脏，已经被广泛研究，AMPK在 骨内环境平衡尚未被描述。在这里，我们建议阐明AMPKa2在骨骼中的作用 动态平衡。首先，我们将通过进行X射线检查来表征AMPKa2基因敲除小鼠的骨骼表型 X光照相、DXA、骨组织形态计量学和微型计算机断层扫描。雇佣各种不同的 生化和分子生物学方法，包括BrdU标记，von Kossa染色，定量 逆转录聚合酶链式反应，原位末端标记法和免疫印迹分析，AMPK在成骨细胞增殖中的作用， 成熟和凋亡也将在体内和体外进行研究。因为骨骼是一个充满活力的器官 容易受到全身和细胞能量平衡的影响，这项研究将为进一步的研究奠定基础 阐明能量动态平衡与骨代谢的关系。

Osteogenesis is regulated by multiple signaling pathways and kinases play a critical role in several of these signaling pathways. As an effort to uncover novel factors for bone mass regulation, we screened a human kinase siRNA library searching for kinases that are involved in osteogenic differentiation of mesenchymal stem cells. Small interfering RNA-mediated knockdown of alpha2 catalytic subunit of adenosine 5'- monophosphate-activated protein kinase (AMPK) in mesenchymal stem cells significantly increased activity of alkaline phosphatase, an early marker of osteogenic differentiation, suggesting that AMPKa2 may have a role in osteoblasts, probably in bone formation. AMPK is an energy sensor of mammalian cells because it directly responds to the depletion of the fuel molecule ATP. Although the roles of AMPK in various peripheral organs, including muscle, heart, and liver, have been extensively studied, implication of AMPK in bone homeostasis has not been described. Here, we propose to elucidate the role of AMPKa2 in bone homeostasis. First, we will characterize skeletal phenotype of AMPKa2 knockout mice by carrying out X-ray radiography, DXA, bone histomorphometry, and microcomputed tomography. Employing various biochemical and molecular biological approaches, including BrdU labeling, von Kossa staining, quantitative RT-PCR, TUNEL assay, and immunoblotting analyses, the role of AMPK in osteoblast proliferation, maturation, and apoptosis will also be investigated both in vivo and ex vivo. As bone is a dynamic organ that is susceptible to systemic and cellular energy balance, this study will be groundwork for the further elucidation of the association of energy homeostasis with bone metabolism.