Notch and Wnt signaling in protection by nandrolone against disuse atrophy

Notch 和 Wnt 信号传导在诺龙防止废用性萎缩中的保护作用

• 批准号: 8202501
• 负责人: CHRISTOPHER P CARDOZO
• 金额: --
• 依托单位: JAMES J PETERS VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8202501
• 关键词: Adult; Affect; Androgen Receptor; Androgen Response Element; Animal Model; Asthma; Atrophic; Binding; Binding Sites; Burn injury; Cell Culture Techniques; Cell Nucleus; Cell physiology; Cells; Chronic Obstructive Airway Disease; Commit; Communities; DNA; DNA Binding; DNA Sequence; Data; Denervation; Disease; Disuse Atrophy; Event; Fiber; Future; Gene Expression; Genes; Genetic Transcription; Hypertrophy; Immobilization; Immunohistochemistry; Injury; Knock-out; Knowledge; Ligand Binding; Ligands; Mediating; Medical; Mesenchymal Stem Cells; Messenger RNA; Molecular; Muscle; Muscle Fibers; Muscular Atrophy; Myoblasts; Nandrolone; Nerve; Nuclear Translocation; Paralysed; Pharmaceutical Preparations; Play; Proliferating; Proteolysis; Quality of life; Recovery; Response Elements; Role; Sequence Analysis; Signal Transduction; Site; Skeletal Muscle; Soldier; Speed; Spinal cord injury; Stroke; TCF Transcription Factor; Testing; Time; Transgenic Mice; Up-Regulation; Veterans; activating transcription factor; cell type; daughter cell; functional restoration; improved; inhibitor/antagonist; mRNA Stability; mouse model; muscle form; notch protein; overexpression; prevent; promoter; receptor; research study; restoration; satellite cell; self-renewal; tissue repair; transcription factor

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.

描述（由申请人提供）： 目前的提议源于对诺龙防止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 在肌肉损伤后组织修复中发挥关键作用的发现的刺激。 Notch 刺激卫星细胞的增殖，卫星细胞是一种间充质“干”细胞，能够增殖以实现自我更新，并产生能够分化为骨骼肌谱系、形成成肌细胞并融合形成新肌纤维或有助于现有肌纤维的子细胞。那些。 Wnt 抑制 Notch 信号传导并刺激生肌分化。我们的初步数据表明，去神经激活了去神经肌肉中的Notch和Wnt信号传导，诺龙终止了Notch信号传导，同时进一步增强了Wnt信号传导。诺龙终止 Notch 信号传导与 Notch 抑制剂 Numb 的表达增加相关。对 Numb 启动子序列的初步分析表明，它包含转录因子 (TCF/LEF) 的结合位点，Wnts 通过转录因子 ss-catenin 的激活和核易位以及可能的雄激素受体反应来调节基因表达元素；雄激素受体是一种配体激活的转录因子，它与这些位点结合以上调基因表达。我们的假设是，诺龙诱导卫星细胞中 Numb 和 Wnt 信号的上调，这对于诺龙防止萎缩至关重要，并且依赖于 Numb 转录的上调；上调由雄激素受体和 Wnt 信号传导介导。提出以下具体目标： 1. 确定诺龙改变 Numb 的细胞类型以及 Notch 和 ss-catenin 的信号传导。我们的假设是诺龙调节卫星细胞中的 Numb 表达以及 Notch 和 ss-catenin 依赖性信号传导，并且诺龙对 Numb 表达和 ss-catenin 依赖性信号传导的影响发生在相同的细胞中。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 ss-catenin 信号传导的细胞类型。 2. 检查 Numb 在诺龙诱导的 Notch 信号传导抑制和防止去神经萎缩中的作用。我们的假设是，Numb 的上调会终止 Notch 信号传导，并且对于诺龙防止去神经萎缩的作用是必要的。我们将使用一种转基因小鼠，其中 Numb 基因可以在特定时间失活。实验将测试 Numb 基因失活是否会阻止诺龙对去神经萎缩小鼠模型中 Notch 信号传导和防止肌肉萎缩的影响。 3. 表征诺龙上调 Numb 的分子机制。我们的假设是 Numb 启动子含有一组雄激素反应元件，因此诺龙对 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 或过表达 Wnt 或其抑制剂来测试 Wnt 信号传导在去神经肌肉 Numb 表达中的作用。在细胞培养中，我们将确定： 诺龙与雄激素受体 (AR) 的结合是否是诺龙上调 Numb 所必需的； Numb 启动子内的 ARE 具有上调其转录的功能；诺龙会改变 Numb mRNA 的稳定性。