MECHANISM OF CHONDROPROGENITOR CELL CONDENSATION

软骨祖细胞的凝聚是最早的步骤之一 在软骨内骨骼发育中的作用 在发育中的肢芽中， 浓缩的间充质细胞的核心随后分化为 软骨细胞以产生软骨原基。 蜂窝 冷凝步骤对于软骨形成是至关重要的，因为在体内和体外， 体外干扰抑制间充质细胞凝结 软骨形成 我们以前已经证明，N-钙粘蛋白， 细胞-细胞粘附蛋白，在功能上参与肢体间充质 冷凝和软骨形成。钙粘蛋白是膜糖蛋白 其以Ca 2+依赖性同型方式介导细胞粘附， 已经被假定为作为形态调节分子， 发展钙粘蛋白作为与细胞溶质，α-， - β-和γ-连环蛋白，并且该复合物通常被认为 在粘附介导的信号传导中起作用。 在本建议中，我们的目标是 进一步研究N-钙粘蛋白介导的 间充质细胞在软骨形成中的相互作用，使用两种系统， 间充质软骨发生：1）鸡胚肢体间充质， 当以高密度培养时，引发经历软骨形成;并且 2)小鼠多能细胞系C3 H1 OT 1/2，其将经历 当作为高密度微团铺板并用 骨形态发生蛋白-2（BMP-2）。 的高密度要求， 这两个系统都强调了亲密细胞间的重要性 交互.具体目的是：1）确定亚细胞 通过检查N-钙粘蛋白介导的活性的影响， 表达N-钙粘蛋白的结构变体; 2）为了检查 β-连环蛋白在细胞凝聚中的功能作用， 通过分析其亚细胞分布， 磷酸化状态，与N-钙粘蛋白的关系，以及 表达其结构变体; 3）为了证明N- 钙粘蛋白介导的细胞粘附是关键参与的影响， 软骨诱导影响，包括TGF-β的活性 超家族成员，间充质细胞;和4）为了检验假设 N-钙粘蛋白表达/活性是一种功能性标记物， 存在于非骨骼组织中的软骨祖细胞。 这些 拟议中的研究将提供有关早期事件的宝贵信息， 间充质软骨形成的细胞和分子机制， 细胞与细胞之间的相互作用。 调查基本机制 软骨发育与理解 先天性骨骼畸形的病因学和骨骼生物学 修复.

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.