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

描述（由申请方提供）：软骨内骨形成涉及间充质细胞浓缩和分化为软骨细胞，随后是软骨细胞增殖、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（包括TGF-β、β-连环蛋白和Runx 2）的精确调控。TGF-β促进软骨细胞增殖，但阻止软骨细胞分化和肥大。TGF-β在软骨细胞中作用的分子机制尚未完全了解。在初步研究中，我们有两个关键的新发现：1）TGF-β以Smad 5依赖的方式激活软骨细胞中的β-catenin信号传导，TGF-β诱导的cyclin D1表达由β-catenin介导; 2）cyclin D1诱导Runx 2磷酸化和降解。在拟议的研究中，我们将使用全面的分子和遗传方法来研究TGF-β激活β-连环蛋白信号传导和抑制软骨细胞中Runx 2功能的分子机制。该提议的基本假设是TGF-β通过增加细胞周期蛋白D1的表达刺激软骨细胞增殖，细胞周期蛋白D1反过来抑制Runx 2功能，从而阻止软骨细胞分化的发生。在本申请中提出了两个具体目的。在具体目标1中，我们将确定软骨细胞增殖过程中TGF-β诱导的β-连环蛋白信号转导机制。我们将1）鉴定与Smad 3结合的β-连环蛋白的特定结构域，并检查Smad 3相互作用是否阻止β-连环蛋白降解; 2）确定Smad 3是否增强β-连环蛋白核转位; 3）研究Smad 3和β-连环蛋白协同激活软骨细胞中细胞周期蛋白D1基因转录的机制。在具体目标2中，我们将定义细胞周期蛋白D1介导增殖软骨细胞中Runx 2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Runx 2的磷酸化和降解; 2）鉴定负责细胞周期蛋白D1诱导的Runx 2泛素化的特异性E3泛素连接酶; 3）确定Cdks和Cdk抑制剂在Runx 2降解和软骨细胞分化中的作用。拟议研究的结果将为TGF-b在生长板软骨细胞发育中的作用以及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.