Notch and Wnt signaling in protection by nandrolone against disuse atrophy

描述(由申请人提供)： 目前的建议源于对诺德隆预防神经切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启发。Noch刺激卫星细胞的增殖，卫星细胞是一种间质干细胞，能够增殖以允许自我更新，并产生能够致力于骨骼肌谱系的子代细胞，形成成肌细胞，并融合形成新的肌肉纤维或对现有的肌肉纤维做出贡献。WNT抑制Notch信号转导，刺激肌细胞分化。我们的初步数据表明，去神经支配激活失神经肌肉中的Notch和Wnt信号，而诺龙终止Notch信号，同时进一步增加Wnt信号。Nandroone终止Notch信号转导与Notch抑制物数表达增加有关。对Numb启动子序列的初步分析表明，它包含转录因子的结合位点(Tcf/Lef)，WNTs通过转录调节因子ss-catenin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，结合到这些位点上上调基因表达。我们的假设是，Nandroone诱导卫星细胞中Numb和Wnt信号的上调，这对防止Nandroone的萎缩至关重要，并依赖于Numb转录的上调；上调是由雄激素受体和Wnt信号介导的。具体目标如下：1.确定诺酮改变细胞数量的细胞类型(S)，并通过Notch和ss-catenin进行信号转导。我们的假设是，在卫星细胞中，诺孕酮调节Numb表达和依赖Notch和ss-catenin的信号转导，而对Numb表达和ss-catenin依赖的信号转导的影响发生在同一细胞中。在失神经萎缩的小鼠模型中，我们将通过免疫组织化学的方法来确定诺洛酮通过ss-catenin上调Numb和Signal信号的细胞类型。2.研究Numb在Nandroone抑制Notch信号通路和保护失神经萎缩中的作用。我们的假设是，Numb的上调终止了Notch信号，这是诺酮预防去神经萎缩所必需的。我们将使用一种转基因小鼠，在这种小鼠中，Numb基因可以在特定时间失活。实验将测试在失神经萎缩的小鼠模型中，Numb基因的失活是否能阻断Nandroone对Notch信号和肌肉萎缩的保护作用。3.研究南多龙上调NAMB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Numb的上调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ss-catenin或过度表达Wnts或其抑制物来测试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：诺洛酮是否需要与雄激素受体(AR)结合才能上调Numb；NumE启动子内的ARs是否具有上调其转录的功能；以及Nandroone是否改变NumRNA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.