EXON-SPECIFIC FIBRONECTIN ISOFORMS AND CHONDROGENESIS

纤维连接蛋白是组织细胞外基质中的一种连接分子，在发育中的胚胎肢体中，纤维连接蛋白的结构在软骨形成、间充质分化为软骨的过程中发生变化。纤维连接蛋白亚型的改变是由涉及纤维连接蛋白基因中至少三个外显子(IIIB、IIIA和V)的mRNA选择性剪接事件引起的。在软骨形成过程中，纤维连接蛋白基因的剪接模式由前软骨间充质中的B+A+V+转变为软骨中的B+A-V+。外显子IIIA编码的区域在软骨形成过程中发生凝结事件后，从间充质纤维连接蛋白中消失。这种结构变化可能反过来又决定了功能的变化。我们最新的研究表明，在高密度肢体间充质微团培养中加入针对外显子IIIA编码区域的单抗可以抑制软骨形成，可能是通过干扰细胞凝聚的形成和/或维持。我们计划验证这样一种假设，即由mRNA选择性剪接产生的特定纤维连接蛋白亚型对于软骨发育的不同阶段是必要的。前4个目标将集中在间充质纤维连接蛋白的结构，特别是外显子IIIA编码的区域，是否是软骨形成过程中发生的间质凝聚事件的某些方面所必需的。目标1中的实验将在存在和不存在外显子特异性抗体和多肽的情况下，在细胞黏附分析中研究间充质细胞与各种纤维连接蛋白亚型的相互作用。目的2将通过迁移分析和聚集分析确定间充质纤维连接蛋白是否支持间充质细胞迁移和/或参与软骨凝聚的聚集事件。目的3将评估纤维连接蛋白亚型对间充质软骨形成的影响，特别是与细胞密度的依赖以及与N-钙粘附素介导的细胞-细胞相互作用的可能的功能串扰有关。目的4通过研究纤维连接蛋白与转化生长因子-β1软骨调节活性的功能关系，探讨纤维连接蛋白异构体对间充质软骨形成的调控作用。最终目的(目标5)将通过研究纤维连接蛋白与分化的软骨细胞的相互作用及其对软骨细胞表型维持的影响，开始研究软骨纤维连接蛋白(B+A+V+)外显子IIIA缺失的生物学意义。这些实验所得到的信息将进一步加深我们对纤维连接蛋白在软骨形成不同阶段的功能的理解，并为在软骨发育过程中以组织特异性的方式展示纤维连接蛋白基因中不同的剪接事件的目的提供有用的见解。

The structure of fibronectin, a connecting molecule in the extracellular matrices of tissues, changes during chondrogenesis, the differentiation of mesenchyme into cartilage, in the developing embryonic limb. Fibronectin isoform-changes result from mRNA alternative splicing events involving at least three exons in the fibronectin gene (IIIB, IIIA, and V). During chondrogenesis, the fibronectin mRNA splicing patterns change from B+A+V+ in precartilage mesenchyme to B+A-V+ in cartilage. The region encoded by exon IIIA disappears from mesenchymal fibronectin just after the condensation event that occurs during chondrogenesis. This structural change may in turn dictate a change in function. Our most recent studies show that addition of a monoclonal antibody specific for the region encoded by exon IIIA to high-density limb mesenchymal micromass cultures inhibits chondrogenesis, possibly by interfering with the formation and/or maintenance of cellular condensations. We plan to test the hypothesis that a specific fibronectin isoform resulting from mRNA alternative splicing is necessary for different stages of cartilage development. The first 4 aims will focus on whether the structure of mesenchymal fibronectin, specifically the region encoded by exon IIIA, is required for some aspect of the mesenchymal condensation event that occurs during chondrogenesis. Experiments in Aim 1 will investigate the interactions of mesenchymal cells with various fibronectin isoforms in cell adhesion assays in the presence and absence of exon-specific antibodies and peptides. Aim 2 will determine whether mesenchymal fibronectin supports mesenchymal cell migration and/or aggregation events involved in chondrogenic condensation using migration assays and aggregation assays. Aim 3 will assess the effect of fibronectin isoforms on mesenchymal condrogenesis, particularly related to the dependence on cell density and possible functional cross-talk with N-cadherin mediated cell-cell interactions. Aim 4 will examine the regulation of fibronectin isoform-specific effects on mesenchymal chondrogenesis by characterizing the functional relationship with the chondro- regulatory activities of TGF-beta1. The final aim (Aim 5) will begin to investigate the biological significance of the loss of exon IIIA in cartilage fibronectin (B+A+V+) by characterizing fibronectin interactions with differentiated chondrocytes and their effects on the maintenance of the chondrocyte phenotype. The information resulting from these experiments will further our understanding of the function of fibronectin at different stages of chondrogenesis and provide useful insights into the purpose for alternative splicing events in the fibronectin gene that are exhibited in a tissue-specific manner during cartilage development.