The Mechanism through which TGF-Beta Maintains Chondrocytes in Proliferating Stag

描述(申请人提供)：软骨内骨形成涉及间充质细胞凝聚和分化成软骨细胞，随后软骨细胞增殖、成熟、肥大分化和凋亡。软骨内骨形成过程的每一步都受到局部生长因子、信号分子和转录因子的精确调控，包括转化生长因子-b、b-连环蛋白和Runx2。转化生长因子-β促进软骨细胞增殖，但抑制软骨细胞分化和肥大。转化生长因子-b在软骨细胞中作用的分子机制尚不完全清楚。在初步研究中，我们有两个关键的新发现：1)转化生长因子-β以Smad5依赖的方式激活软骨细胞中的b-连环蛋白信号，并通过b-连环蛋白介导细胞周期蛋白D1的表达；2)细胞周期蛋白D1诱导Runx2的磷酸化和降解。在拟议的研究中，我们将使用综合的分子和遗传学方法来研究转化生长因子-β激活软骨细胞中的b-连环蛋白信号和抑制Runx2功能的分子机制。这一假设的基本假设是，转化生长因子-β通过增加细胞周期蛋白D1的表达来刺激软骨细胞的增殖，进而抑制Runx2的功能，从而防止软骨细胞分化的开始。在这项申请中提出了两个具体目标。在特定的目标1中，我们将确定转化生长因子-β诱导软骨细胞增殖过程中b-连环蛋白信号转导的机制。我们将1)确定b-catenin与Smad3结合的特定结构域，并检测Smad3相互作用是否阻止b-catenin降解；2)确定Smad3是否促进b-catenin核转位；3)研究Smad3和b-catenin协同激活软骨细胞周期蛋白D1基因转录的机制。在特定的目标2中，我们将确定细胞周期蛋白D1在增殖的软骨细胞中介导Runx2降解的分子机制。我们建议1)确定细胞周期蛋白D1是否诱导软骨细胞中Runx2的磷酸化和降解；2)确定与细胞周期蛋白D1诱导的Runx2泛素化有关的特定的E3泛素连接酶；3)确定CDKs和CDK抑制剂在Runx2降解和软骨细胞分化中的作用。拟议研究的结果将为了解转化生长因子-b在生长板软骨细胞发育中的作用和控制软骨内骨形成的机制提供新的见解。

DESCRIPTION (provided by applicant): Endochondral bone formation involves condensation and differentiation of mesenchymal cells into chondrocytes followed by chondrocyte proliferation, maturation, hypertrophic differentiation and apoptosis. Each step of the endochondral bone formation process is precisely regulated by local growth factors, signaling molecules and transcription factors, including TGF-b, b-catenin and Runx2. TGF-b promotes chondrocyte proliferation but prevents chondrocyte differentiation and hypertrophy. The molecular mechanisms of TGF-b action in chondrocytes are not fully understood. In preliminary studies, we have made two key novel discoveries: 1) TGF-b activates b-catenin signaling in chondrocytes in a Smad5-dependent manner and TGF-b-induced cyclin D1 expression is mediated by b-catenin; and 2) cyclin D1 induces Runx2 phosphorylation and degradation. In the proposed studies, we will use comprehensive molecular and genetic approaches to investigate the molecular mechanisms through which TGF-b activates b-catenin signaling and inhibits Runx2 function in chondrocytes. The underlying hypothesis for this proposal is that TGF-b stimulates chondrocyte proliferation by increasing cyclin D1 expression which in turn inhibits Runx2 function, thereby preventing onset of chondrocyte differentiation. Two specific aims are proposed in this application. In specific aim 1, we will determine the mechanisms of TGF-b-induced b-catenin signaling during chondrocyte proliferation. We will 1) identify the specific domain of b-catenin that binds to Smad3 and examine if Smad3 interaction prevents b-catenin degradation; 2) determine whether Smad3 enhances b-catenin nuclear translocation; and 3) investigate the mechanisms through which Smad3 and b-catenin cooperatively activate cyclin D1 gene transcription in chondrocytes. In specific aim 2, we will define the molecular mechanism through which cyclin D1 mediates Runx2 degradation in proliferating chondrocytes. We propose 1) to determine if cyclin D1 induces Runx2 phosphorylation and degradation in chondrocytes; 2) to identify the specific E3 ubiquitin ligase responsible for cyclin D1-induced Runx2 ubiquitination; and 3) to determine the role of Cdks and Cdk inhibitor in Runx2 degradation and chondrocyte differentiation. The findings from proposed studies will provide novel insights into the role of TGF-b in growth plate chondrocyte development and the mechanisms controlling endochondral bone formation.