DeLise Anthony M, Tuan Rocky S
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Cell Biochem. 2002;87(3):342-59. doi: 10.1002/jcb.10308.
Cartilage formation in the embryonic limb is presaged by a cellular condensation phase that is mediated by both cell-cell and cell-matrix interactions. N-Cadherin, a Ca(2+)-dependent cell-cell adhesion molecule, is expressed at higher levels in the condensing mesenchyme, followed by down-regulation upon chondrogenic differentiation, strongly suggesting a functional role in the cellular condensation process. To further examine the role of N-cadherin, we have generated expression constructs of wild type and two deletion mutants (extracellular and intracellular) of N-cadherin in the avian replication-competent, RCAS retrovirus, and transfected primary chick limb mesenchymal cell cultures with these constructs. The effects of altered, sustained expression of N-cadherin and its mutant forms on cellular condensation, on the basis of peanut agglutinin (DNA) staining, and chondrogenesis, based on expression of chondrocyte phenotypic markers, were characterized. Cellular condensation was relatively unchanged in cultures overexpressing wild type N-cadherin, compared to controls on all days in culture. However, expression of either of the deletion mutant forms of N-cadherin resulted in decreased condensation, with the extracellular deletion mutant demonstrating the most severe inhibition, suggesting a requirement for N-cadherin mediated cell-cell adhesion and signaling in cellular condensation. Subsequent chondrogenic differentiation was also affected in all cultures overexpressing the N-cadherin constructs, on the basis of metabolic sulfate incorporation, the presence of the cartilage matrix proteins collagen type II and cartilage proteoglycan link protein, and alcian blue staining of the matrix. The characteristics of the cultures suggest that the N-cadherin mutants disrupt proper cellular condensation and subsequent chondrogenesis, while the cultures overexpressing wild type N-cadherin appear to condense normally, but are unable to proceed toward differentiation, possibly due to the prolonged maintenance of increased cell-cell adhesiveness. Thus, spatiotemporally regulated N-cadherin expression and function, at the level of both homotypic binding and linkage to the cytoskeleton, is required for chondrogenesis of limb mesenchymal cells.
胚胎肢体中的软骨形成以细胞凝聚阶段为前奏，该阶段由细胞间和细胞与基质的相互作用介导。N-钙黏蛋白是一种依赖钙离子的细胞间黏附分子，在凝聚的间充质中表达水平较高，随后在软骨分化时下调，这强烈表明其在细胞凝聚过程中发挥功能作用。为了进一步研究N-钙黏蛋白的作用，我们构建了野生型以及N-钙黏蛋白的两个缺失突变体（细胞外和细胞内）在具有复制能力的禽RCAS逆转录病毒中的表达载体，并用这些载体转染原代鸡肢体间充质细胞培养物。基于花生凝集素（DNA）染色，对N-钙黏蛋白及其突变形式的改变的、持续的表达对细胞凝聚的影响进行了表征，基于软骨细胞表型标志物的表达，对软骨形成的影响进行了表征。与培养各天的对照相比，过表达野生型N-钙黏蛋白的培养物中的细胞凝聚相对未发生变化。然而，N-钙黏蛋白的任何一种缺失突变体形式的表达均导致凝聚减少，细胞外缺失突变体表现出最严重的抑制作用，这表明细胞凝聚需要N-钙黏蛋白介导的细胞间黏附和信号传导。基于代谢性硫酸盐掺入、软骨基质蛋白II型胶原蛋白和软骨蛋白聚糖连接蛋白的存在以及基质的阿尔新蓝染色，过表达N-钙黏蛋白构建体的所有培养物中的后续软骨分化也受到影响。培养物的特征表明，N-钙黏蛋白突变体破坏了正常的细胞凝聚和随后的软骨形成，而过表达野生型N-钙黏蛋白的培养物似乎正常凝聚，但无法进行分化，这可能是由于细胞间黏附性增加的持续维持。因此，肢体间充质细胞软骨形成需要在同型结合和与细胞骨架连接水平上时空调节的N-钙黏蛋白表达和功能。
