Woodward W A, Tuan R S
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Dev Genet. 1999;24(1-2):178-87. doi: 10.1002/(SICI)1520-6408(1999)24:1/2<178::AID-DVG16>3.0.CO;2-M.
Cellular condensation is a requisite step in the initiation of mesenchymal chondrogenesis in the embryonic limb bud. We have previously shown that cellular condensation of limb chondroprogenitor mesenchymal cells is accompanied by elevated expression of N-cadherin during chondrogenesis both in vivo and in vitro. N-Cadherin-mediated cell-cell interaction is also functionally required for proper mesenchymal chondrogenesis both in vivo and in vitro. In this report, we have further analyzed the functional importance of N-cadherin in the cellular condensation-chondrogenesis pathway by examining N-cadherin expression and related activities in high density micromass cultures of chick limb mesenchymal cells in which chondrogenesis is being stimulated with the cationic polymer, poly-L-lysine (PL). The chondrogenesis-promoting action of PL is thought to involve the clustering of cells via ionic cross-linking, perhaps mimicking the action of an endogenous matrix component. Immunohistochemistry, immunoblotting, and Northern blot analysis all show that PL treatment results in a time-dependent increase in N-cadherin expression at both the protein and mRNA levels. In addition, inhibition of N-cadherin function with a neutralizing monoclonal antibody directed to its extracellular domain inhibits the chondrogenesis-stimulating effect of PL. PL treatment also alters the tyrosine-phosphorylation state of the N-cadherin associated signaling protein, beta-catenin. These results suggest that N-cadherin-mediated cell adhesion is a requisite regulatory component of the limb mesenchymal chondrogenic differentiation program, involving at least in part beta-catenin tyrosine phosphorylation as a signaling step.
细胞凝聚是胚胎肢芽中间充质软骨形成起始过程中的一个必要步骤。我们之前已经表明，在体内和体外软骨形成过程中，肢软骨祖细胞间充质细胞的细胞凝聚伴随着N-钙黏蛋白表达的升高。N-钙黏蛋白介导的细胞间相互作用在体内和体外适当的间充质软骨形成中在功能上也是必需的。在本报告中，我们通过检测在阳离子聚合物聚-L-赖氨酸（PL）刺激软骨形成的鸡肢间充质细胞高密度微团培养物中N-钙黏蛋白的表达及相关活性，进一步分析了N-钙黏蛋白在细胞凝聚-软骨形成途径中的功能重要性。PL促进软骨形成的作用被认为涉及通过离子交联使细胞聚集，可能模拟了内源性基质成分的作用。免疫组织化学、免疫印迹和Northern印迹分析均表明，PL处理导致蛋白质和mRNA水平上N-钙黏蛋白表达随时间增加。此外，用针对其细胞外结构域的中和单克隆抗体抑制N-钙黏蛋白功能可抑制PL的软骨形成刺激作用。PL处理还改变了与N-钙黏蛋白相关的信号蛋白β-连环蛋白的酪氨酸磷酸化状态。这些结果表明，N-钙黏蛋白介导的细胞黏附是肢间充质软骨分化程序的一个必要调节成分，至少部分涉及β-连环蛋白酪氨酸磷酸化作为一个信号步骤。
