MECHANISM OF CHONDROPROGENITOR CELL CONDENSATION

软骨前体细胞的凝聚是最早的步骤之一 在软骨内骨骼发育中。在发育中的肢芽中， 凝聚间充质细胞的核心随后分化为 软骨细胞形成软骨原基。蜂窝手机 凝聚步骤对软骨形成至关重要，因为在体内和 凝结抑制间充质形成的体外扰动 软骨生成。我们之前已经证明了N-钙粘附素，一种 细胞-细胞黏附蛋白参与肢体间充质的功能 凝结和软骨生成。钙粘附素是一种膜糖蛋白 它以依赖于钙离子的同型方式调节细胞黏附，以及 被认为是作为形态调节分子在 发展。钙粘附素的功能是与胞浆、α-、 -β-和伽马-连环蛋白，该复合体通常被认为 在黏附介导的信号转导中起作用。在这项建议中，我们的目标是 进一步研究N-钙粘附素介导的机制和功能 软骨形成中间充质细胞的相互作用，使用两个系统 间充质软骨形成：1)鸡胚胎肢体间充质， 当高密度培养时，已经准备好进行软骨形成；以及 2)小鼠多潜能细胞系C3H1OT1/2 当以高密度微团块形式电镀时的软骨形成 骨形态发生蛋白2(BMP-2)。的高密度要求 这两种系统都强调了亲密细胞的重要性 互动。具体目标是：1)确定亚细胞 N-钙粘附素介导的活性部位的检测 表达N-钙粘附素的结构变体；2)检测 β-连环蛋白在细胞凝聚和释放中的作用 通过分析软骨形成的亚细胞分布， 磷酸化状态、与N-钙粘附素的关联及其影响 表达其结构变体；3)证明N- 钙粘附素介导的细胞黏附关键参与了 软骨诱导的影响，包括转化生长因子-β的活性 超家族成员，间充质细胞；以及4)检验这一假设 N-钙粘蛋白的表达/活性是一种功能性标志物 软骨前体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有价值的信息 间充质软骨形成及其细胞分子机制的研究进展 细胞与细胞之间的相互作用。探寻其基本机制 对软骨发育的研究与理解 先天性骨骼畸形的病因与骨骼生物学 修理。

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.