EXON-SPECIFIC FIBRONECTIN ISOFORMS AND CHONDROGENESIS

外显子特异性纤连蛋白异构体和软骨形成

• 批准号: 6043234
• 负责人: ROCKY S TUAN
• 金额: $ 25.42万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6043234
• 关键词: RNA splicing; cartilage development; cell migration; chick embryo; fibronectins; mesenchyme; protein isoforms; protein structure function; transforming growth factors; vertebrate embryology

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.

纤连蛋白是组织细胞外基质中的一种连接分子，在胚胎肢体发育过程中，软骨形成（即间充质分化为软骨）过程中会发生变化。 纤连蛋白亚型变化是由涉及纤连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，纤连蛋白 mRNA 剪接模式从软骨前间质中的 B+A+V+ 变为软骨中的 B+A-V+。软骨形成过程中发生凝结事件后，外显子 IIIA 编码的区域立即从间充质纤连蛋白中消失。 这种结构变化可能反过来决定功能的变化。 我们最近的研究表明，将外显子 IIIA 编码区域特异的单克隆抗体添加到高密度肢体间充质微团培养物中，可能通过干扰细胞凝聚的形成和/或维持来抑制软骨形成。 我们计划测试以下假设：由 mRNA 选择性剪接产生的特定纤连蛋白亚型对于软骨发育的不同阶段是必需的。 前 4 个目标将重点关注间充质纤连蛋白的结构，特别是由外显子 IIIA 编码的区域，是否是软骨形成过程中发生的间充质浓缩事件的某些方面所必需的。目标 1 中的实验将研究在存在和不存在外显子特异性抗体和肽的情况下，在细胞粘附测定中间充质细胞与各种纤连蛋白亚型的相互作用。 目标 2 将使用迁移测定和聚集测定确定间充质纤连蛋白是否支持软骨形成凝结中涉及的间充质细胞迁移和/或聚集事件。 目标 3 将评估纤连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性以及与 N-钙粘蛋白介导的细胞间相互作用可能的功能性串扰有关。 目标 4 将通过表征与 TGF-β1 软骨调节活性的功能关系来检查纤连蛋白亚型特异性对间充质软骨形成的调节作用。 最终目标（目标 5）将通过表征纤连蛋白与分化软骨细胞的相互作用及其对维持软骨细胞表型的影响，开始研究软骨纤连蛋白 (B+A+V+) 中外显子 IIIA 丢失的生物学意义。这些实验产生的信息将进一步我们对纤连蛋白在软骨形成不同阶段的功能的理解，并为了解纤连蛋白基因中在软骨发育过程中以组织特异性方式表现出的选择性剪接事件的目的提供有用的见解。