Roles of Smurf1 in Cbfa1 Degradation and Bone Formation

Smurf1 在 Cbfa1 降解和骨形成中的作用

• 批准号: 6769237
• 负责人: DI CHEN
• 金额: $ 31.99万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-21 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6769237
• 关键词: biological signal transduction; bone development; bone morphogenetic proteins; cell differentiation; developmental genetics; gene expression; genetic promoter element; genetically modified animals; immunoprecipitation; laboratory mouse; mass spectrometry; osteoblasts; parathyroid hormones; phosphorylation; physiologic bone resorption; proteasome; protein degradation; protein protein interaction; protein structure function; transcription factor; transfection; ubiquitin

DESCRIPTION (provided by applicant): Osteoporosis is a metabolic bone disease characterized by low bone mass and deterioration of bone microarchitecture. An understanding of the mechanisms by which bone formation is controlled is critical to understanding the pathogenesis of osteoporosis and defining molecular targets for development of new therapies for the treatment of this debilitating disease. Several lines of evidence show that BMP signaling plays a critical role in bone formation in vivo. A major mechanism by which BMPs stimulate osteoblast differentiation is by activation of the bone-specific transcription factor Cbfa1 through its downstream signaling molecules, Smad1 and 5. Recently we have discovered for the first time that inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro. We also identified for the first time that the E3 ubiquitin ligase Smurf1 mediates Cbfa1 degradation in an ubiquitin-proteasome-dependent manner. The detailed molecular mechanisms by which Smurf1 mediates Cbfa1 degradation and the regulatory role of Smurf1 in bone formation in vivo remain undefined. In the proposed studies, we will determine the mechanisms by which Smurf1 mediates Cbfa1 degradation and the role of Smurf1 in bone formation in vivo. The underlying hypothesis is that Smurf1 is an important regulatory molecule for Smad1 and Cbfa1 function and plays a critical role in regulation of bone formation in vivo. In Specific Aim 1, we will determine whether Smurf1-mediated Cbfa1 degradation is dependent on activation of BMP signaling, phosphorylation of Smad1 and formation of a Smad1/Cbfa1 protein complex. We will also determine whether PTH induces Cbfa1 degradation and the effects of Smurf1 on PTH-induced Cbfa1 degradation. In Specific Aim 2, we will determine the effects of over-expression of wild-type and mutant Smurf1 on bone formation in intact and ovariectomized animals and osteoblast function and protein levels of Smad1 and Cbfa1 in Smurf1 transgenic mice. We will also determine the effects of bone growth regulatory factors on bone formation in Smurf1 transgenic mice. To investigate the specific role of Smurf1 in bone formation during specific developmental stages, such as postnatal and adult life, we will also generate tissue-specific transgenic mice in which transgene expression is inducible. Our working hypotheses are that (1) Smurf1-mediated Cbfa1 degradation is dependent on activation of BMP signaling; (2) Modulation of osteoblast Smurf1 activity in vivo will result in abnormalities in bone formation that cannot be compensated for by other members of the E3 ubiquitin ligase family; and (3) Smurf1 regulates bone formation in both postnatal and adult life. The proposed studies will lead to a better understanding of the molecular mechanisms by which Smurf1 mediates Cbfa1 degradation and the physiological role of Smurf1 in bone formation in vivo. This study will also help identify a new molecular target for drug development for the treatment of osteoporosis and other bone-loss associated diseases.

描述（申请人提供）：骨质疏松症是一种代谢性骨病，其特征是骨量低和骨微结构恶化。了解控制骨形成的机制对于了解骨质疏松症的发病机制和确定开发治疗这种衰弱疾病的新疗法的分子靶点至关重要。多项证据表明，BMP 信号传导在体内骨形成中发挥着关键作用。 BMP刺激成骨细胞分化的主要机制是通过其下游信号分子Smad1和5激活骨特异性转录因子Cbfa1。最近我们首次发现成骨细胞蛋白酶体抑制剂在体内和体外刺激骨形成。我们还首次发现 E3 泛素连接酶 Smurf1 以泛素蛋白酶体依赖性方式介导 Cbfa1 降解。 Smurf1 介导 Cbfa1 降解的详细分子机制以及 Smurf1 在体内骨形成中的调节作用仍不清楚。 在拟议的研究中，我们将确定 Smurf1 介导 Cbfa1 降解的机制以及 Smurf1 在体内骨形成中的作用。基本假设是Smurf1是Smad1和Cbfa1功能的重要调节分子，并且在体内骨形成的调节中发挥着关键作用。在具体目标 1 中，我们将确定 Smurf1 介导的 Cbfa1 降解是否依赖于 BMP 信号传导的激活、Smad1 的磷酸化以及 Smad1/Cbfa1 蛋白复合物的形成。我们还将确定 PTH 是否诱导 Cbfa1 降解以及 Smurf1 对 PTH 诱导的 Cbfa1 降解的影响。在具体目标 2 中，我们将确定野生型和突变型 Smurf1 的过度表达对完整和卵巢切除动物的骨形成以及 Smurf1 转基因小鼠中的成骨细胞功能和 Smad1 和 Cbfa1 蛋白水平的影响。我们还将确定骨生长调节因子对 Smurf1 转基因小鼠骨形成的影响。为了研究 Smurf1 在特定发育阶段（例如出生后和成年后）骨形成中的具体作用，我们还将生成可诱导转基因表达的组织特异性转基因小鼠。我们的工作假设是：(1) Smurf1 介导的 Cbfa1 降解依赖于 BMP 信号传导的激活； (2)体内成骨细胞Smurf1活性的调节会导致骨形成异常，而E3泛素连接酶家族的其他成员无法弥补这一异常； (3) Smurf1 调节出生后和成年后的骨形成。拟议的研究将有助于更好地了解 Smurf1 介导 Cbfa1 降解的分子机制以及 Smurf1 在体内骨形成中的生理作用。这项研究还将有助于确定治疗骨质疏松症和其他骨质流失相关疾病的药物开发的新分子靶点。