MECHANISM OF CHONDROPROGENITOR CELL CONDENSATION

软骨祖细胞凝聚机制

基本信息

批准号：
6312009
负责人：
ROCKY S TUAN
金额：
$ 4.46万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-02-01 至 2004-01-31
项目状态：
已结题

项目摘要

The condensation of chondroprogenitor cells is one of the earliest steps in endochondral skeletal development. In the developing limb bud, the core of condensing mesenchymal cells subsequently differentiate into chondrocytes to give rise to the cartilage anlage. The cellular condensation step is crucial to chondrogenesis, since both in vivo and in vitro perturbations of condensation inhibit mesenchymal chondrogenesis. We have previously demonstrated that N-cadherin, a cell-cell adhesion protein, is functionally involved in limb mesenchymal condensation and chondrogenesis. Cadherins are membrane glycoproteins which mediate cell adhesion in a Ca2+-dependent, homotypic manner, and have been postulated to act as morphoregulatory molecules during development. Cadherins function as a complex with the cytosolic, alpha-, -beta- and gamma-catenins, and the complex is generally believed to function in adhesion-mediated signaling. In this proposal, we aim to further examine the mechanism and function of N-cadherin-mediated mesenchymal cell interactions in chondrogenesis, using two systems of mesenchymal chondrogenesis: 1) chick embryonic limb mesenchyme, which is primed to undergo chondrogenesis when cultured at high density; and 2) the murine multipotential cell line, C3H1OT1/2, which will undergo chondrogenesis when plated as high density micromass and treated with bone morphogenetic protein-2 (BMP-2). The high density requirement of both systems underscores the importance of intimate cell-cell interactions. The specific aims are: 1) To determine the subcellular site of N-cadherin-mediated activity by examining the effects of expressing structural variants of N-cadherin; 2) To examine the functional role of beta-catenin in cellular condensation and chondrogenesis by analyzing its subcellular distribution, phosphorylation status, and association with N-cadherin, and the effect of expressing its structural variants; 3) To demonstrate that N- cadherin-mediated cell adhesion is pivotally involved in the effects of chondroinductive influences, including the activity of TGF-beta superfamily members, on mesenchymal cells; and 4) To test the hypothesis that N-cadherin expression/activity is a functional marker of chondroprogenitor cells residing in non skeletal tissues. These proposed studies will provide valuable information on the early events of mesenchymal chondrogenesis, and the cellular and molecular mechanisms of cell-cell interactions in general. Investigating the basic mechanism of cartilage development is highly relevant to understanding the etiology of congenital skeletal deformities and the biology of skeletal repair.

软骨祖细胞的浓缩是最早的步骤之一在软骨内骨骼发育中。在发育中的肢芽中，浓缩间充质细胞的核心随后分化为软骨细胞产生软骨原基。蜂窝式冷凝步骤对于软骨形成至关重要，因为在体内和凝结的体外扰动抑制间充质软骨形成。我们之前已经证明，N-钙粘蛋白是一种细胞-细胞粘附蛋白，在功能上参与肢体间充质凝结和软骨形成。钙粘蛋白是膜糖蛋白以 Ca2+ 依赖性同型方式介导细胞粘附，并且已被假设在发展。钙粘蛋白作为与胞质、α-、 -β-和γ-连环蛋白，并且该复合物通常被认为是在粘附介导的信号传导中发挥作用。在本提案中，我们的目标是进一步研究N-cadherin介导的机制和功能软骨形成中的间充质细胞相互作用，使用两个系统间充质软骨形成：1）鸡胚肢体间充质，其中当高密度培养时，准备好进行软骨形成；和 2) 小鼠多能细胞系 C3H1OT1/2，将进行当镀成高密度微团并用骨形态发生蛋白-2 (BMP-2)。高密度要求这两个系统都强调了亲密细胞间的重要性互动。具体目标是： 1) 确定亚细胞通过检查 N-钙粘蛋白介导的活性位点的影响表达 N-钙粘蛋白的结构变体； 2）检查 β-连环蛋白在细胞凝结中的功能作用通过分析软骨形成的亚细胞分布，磷酸化状态、与 N-钙粘蛋白的关联以及效果表达其结构变体； 3）证明N- 钙粘蛋白介导的细胞粘附在以下作用中起着关键作用：软骨诱导影响，包括 TGF-β 的活性超家族成员，关于间充质细胞； 4）检验假设 N-钙粘蛋白表达/活性是功能标记软骨祖细胞存在于非骨骼组织中。这些拟议的研究将提供有关早期事件的宝贵信息间充质软骨形成及其细胞和分子机制细胞与细胞相互作用的一般情况。研究基本机制软骨发育的过程与了解软骨高度相关先天性骨骼畸形的病因学和骨骼生物学维修。