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: 6511911
• 负责人: Noreen J Hickok
• 金额: $ 26.87万
• 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/6511911
• 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.

纤连蛋白的结构是组织外部细胞外基质中的一种连接分子，在软骨发生过程中发生了变化，在发育中的胚胎肢体中，间充质分化为软骨。 纤连蛋白的同工型变换是由mRNA替代剪接事件引起的，涉及纤连蛋白基因中至少三个外显子（IIIB，IIIA和V）。 在软骨生成过程中，纤连蛋白mRNA剪接模式从前后间充质中的B+A+V+变为软骨中的B+A-V+。外显子IIIA编码的区域在软骨发生期间发生的凝结事件后从间充质纤连蛋白中消失。 这种结构变化反过来可能决定了功能的变化。 我们最近的研究表明，添加了对外显子IIIA编码的区域特异性的单克隆抗体，用于高密度的肢体中充质微瘤培养物，可能会抑制软骨的发生，这可能是通过干扰细胞冷凝的形成和/或维持的。 我们计划检验以下假设：MRNA替代剪接产生的特定纤连蛋白同工型对于软骨发育的不同阶段是必需的。 前4个目标将集中于间充质纤连蛋白的结构，特别是外显子IIIA编码的区域，是在软骨发生过程中发生的间充质凝结事件的某些方面是否需要的。 AIM 1中的实验将研究间充质细胞与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与各种纤连蛋白同工型的相互作用。 AIM 2将确定间充质纤连蛋白是否支持使用迁移分析和聚集分析参与软骨凝结的间充质细胞迁移和/或聚集事件。 AIM 3将评估纤连蛋白同工型对间充质良孔发生的影响，尤其是与对细胞密度的依赖性以及与N-钙粘着蛋白介导的细胞相互作用的依赖性相关的。 AIM 4将通过表征与TGF-BETA1的软骨调节活性的功能关系来检查纤连蛋白同工型特异性对间充质软骨发生的调节。 最终目标（AIM 5）将开始研究软骨纤连蛋白（B+A+V+）外显子IIIA损失的生物学意义，通过表征纤连蛋白相互作用与分化软骨细胞的纤连蛋白相互作用及其对维持软骨细胞表型的影响。这些实验产生的信息将进一步了解纤连蛋白在软骨发生的不同阶段的功能，并为在软骨发育过程中以组织特异性方式表现出纤连蛋白基因中替代剪接事件的目的提供了有用的见解。