Notch and Wnt signaling in protection by nandrolone against disuse atrophy

描述（由申请人提供）： 目前的建议来自于对诺龙保护神经横断引起的肌肉萎缩的分子机制的研究，并受到Notch和Wnt在肌肉损伤后的组织修复中发挥关键作用的研究结果的刺激。Notch刺激卫星细胞的增殖，卫星细胞是一种能够增殖以允许自我更新的间充质“干”细胞，并且能够产生能够定向于骨骼肌谱系、形成成肌细胞并融合以形成新的肌纤维或有助于现有肌纤维的子细胞。Wnt抑制Notch信号传导并刺激肌源性分化。我们的初步数据表明，去神经激活Notch和Wnt信号在去神经肌肉和诺龙终止Notch信号，同时进一步增加Wnt信号。诺龙终止Notch信号传导与Notch抑制剂Numb的表达增加相关。Numb启动子序列的初步分析表明，它含有转录因子（TCF/LEF）的结合位点，Wnt通过转录调节因子β-连环蛋白的激活和核转位调节基因表达，以及可能的雄激素受体反应元件;雄激素受体是一种配体激活的转录因子，与这些位点结合以上调基因表达。我们的假设是，诺龙诱导上调Numb和Wnt信号在卫星细胞，这是至关重要的保护免受萎缩诺龙，并依赖于上调Numb转录;上调是由雄激素受体和Wnt信号介导的。提出了以下具体目标：1。确定诺龙改变Numb的细胞类型，以及Notch和β-连环蛋白的信号传导。我们的假设是，诺龙调节Numb的表达和Notch和β-连环蛋白依赖的信号在卫星细胞和诺龙的Numb的表达和β-连环蛋白依赖的信号发生在相同的细胞中的影响。在去神经萎缩的小鼠模型中，我们将通过免疫组织化学确定诺龙上调Numb和β-连环蛋白信号的细胞类型。2.检查Numb在诺龙诱导的Notch信号传导抑制和保护去神经萎缩中的作用。我们的假设是Numb的上调终止Notch信号传导，并且是诺龙保护去神经萎缩的作用所必需的。我们将使用转基因小鼠，其中Numb基因可以在特定时间失活。实验将测试Numb基因的失活是否阻断诺龙对Notch信号传导的影响，并在去神经萎缩的小鼠模型中保护肌肉萎缩。3.表征诺龙上调Numb的分子机制。我们的假设是Numb启动子包含一簇雄激素反应元件，因此诺龙上调Numb需要雄激素受体与这些DNA序列结合。在动物模型中，我们将通过敲除β-连环蛋白或过表达Wnt或其抑制剂来测试Wnt信号在失神经肌肉中Numb表达中的作用。在细胞培养中，我们将确定是否：诺龙与雄激素受体（AR）的结合是诺龙上调Numb所必需的; Numb启动子功能内的战神上调其转录;诺龙改变Numb mRNA的稳定性。

DESCRIPTION (provided by applicant): The current proposal grew from studies of the molecular mechanisms by which nandrolone protects against muscle atrophy caused by nerve transection and were stimulated by findings that Notch and Wnt play critical roles in tissue repair after muscle injury. Notch stimulates proliferation of satellite cells, a mesenchymal 'stem' cell capable of proliferating to allow self-renewal, and of producing daughter cells able to commit to the skeletal muscle lineage, form myoblasts, and fuse to form new muscle fibers or contribute to existing ones. Wnt inhibits Notch signaling and stimulates myogenic differentiation. Our preliminary data suggest that denervation activates Notch and Wnt signaling in denervated muscle and that nandrolone terminates Notch signaling while further increasing Wnt signaling. Termination of Notch signaling by nandrolone is associated with increased expression of the Notch inhibitor Numb. A preliminary analysis of the sequence of the Numb promotor suggests that it contains binding sites for transcription factors (TCF/LEF) by which Wnts regulate gene expression through activation and nuclear translocation of the transcriptional regulator ss-catenin, as well as possible androgen receptor response elements; the androgen receptor is a ligand-activated transcription factor that binds to such sites to upregulate gene expression. Our hypothesis is that nandrolone induces upregulation of Numb and Wnt signaling in satellite cells, which is critical to protection against atrophy by nandrolone, and dependent upon upregulation of Numb transcription; upregulation is mediated by the androgen receptor and Wnt signaling. The following specific aims are proposed: 1. Determine the cell type(s) in which nandrolone alters Numb, and signaling by Notch and ss- catenin. Our hypothesis is that nandrolone regulates Numb expression and Notch and ss-catenin- dependent signaling in satellite cells and that effects of nandrolone on Numb expression and ss-catenin- dependent signaling occur in the same cells. In a mouse model of denervation atrophy, we will determine the cell type in which nandrolone upregulates Numb and signaling by ss-catenin by immunohistochemistry. 2. Examine the role of Numb in nandrolone-induced inhibition of Notch signaling and protection against denervation atrophy. Our hypothesis is that upregulation of Numb terminates Notch signaling and is necessary for effects of nandrolone to protect against denervation atrophy. We will use a transgenic mouse in which the Numb gene can be inactivated at a specific time. Experiments will test whether inactivation of the Numb gene blocks effects of nandrolone on Notch signaling and protection against muscle atrophy in a mouse model of denervation atrophy. 3. Characterize molecular mechanisms by which nandrolone upregulates Numb. Our hypothesis is that the Numb promotor contains a cluster of androgen response elements, and thus upregulation of Numb by nandrolone requires binding of the androgen receptor to these DNA sequences. In animal models, we will test the role for Wnt signaling in Numb expression in denervated muscle by either knocking out ss-catenin or overexpressing Wnts or their inhibitors. In cell culture, we will determine whether: binding of nandrolone to the androgen receptor (AR) is needed for upregulation of Numb by nandrolone; AREs within the Numb promotor function to upregulate its transcription; and nandrolone alters Numb mRNA stability.