TYPE III COLLAGEN GENE EXPRESSION IN CARTILAGE

肢体间充质中存在III型胶原mRNA和蛋白，但 在正常生长的软骨中没有发现；因此合成了III型 在软骨形成过程中，胶原蛋白必须停止。然而，有少量的 在成人关节软骨中发现了III型胶原，其含量 在骨关节炎关节软骨中似乎有所增加。小鸡的类型 III型胶原基因含有两个功能启动子，分别显示 不同的表达模式在胚胎发育和 开始制作具有非常不同结构和 功能。负责生产的上游推动者 在III型胶原mRNA中，在皮肤成纤维细胞中是活跃的，但不活跃 在软骨细胞中。一种内部的软骨细胞特异性启动子已经被 确定谁负责生产替代产品 将外显子1-23替换为先前未描述的外显子 外显子。备选文字记录在以下方面显示了复杂的结构 蛋白质编码潜力，至少包含三个可能的开放 阅读框架，其中一个是截断的胶原蛋白；因此 另一种转录本不能编码III型胶原。三个具体的 目的是为了研究III型的复杂调控 胶原基因在软骨形成过程中的表达。1)原地 杂交和免疫组织化学将被用来定义 III型胶原mRNA和蛋白的时空表达 以及在发育中的肢体中的另一种转录。2) 调控III型胶原转录的分子机制 软骨形成过程中来自双启动子的基因将被描述。 3)将利用几个互补的战略来确定潜力 备选文字记录的功能。过度表达的影响 以及体内选择性转录本的表达减少 软骨生成将被确定。此外，如果替代方案 转录本似乎可能在肢体中起到蛋白质编码功能 间充质，将对该蛋白质进行鉴定和表征。这些 实验将定义防止类型的产生的机制 软骨中的III胶原蛋白，并提供了对其机制的洞察 导致软骨细胞III型胶原合成重新激活 在软骨内骨折修复和骨关节炎方面。这些战略将 也增加了我们对潜在的附加功能的理解 另一种转录本介导的III型胶原基因。

Type III collagen mRNA and protein are present in limb mesenchyme, but are not found in normal growth cartilages; thus synthesis of type III collagen must cease during chondrogenesis. However, a small amount of type III collagen is found in adult articular cartilage, and the amount appears to increase in osteoarthritic articular cartilage. The chick type III collagen gene contains two functional promoters which display different patterns of expression during embryonic development and initiate production of transcripts with very different structures and functions. The upstream promoter, which is responsible for production of the type III collagen mRNA, is active in skin fibroblasts, but inactive in chondrocytes. An internal, chondrocyte-specific promoter has been identified which is responsible for production of an alternative transcript in which exons 1-23 are replaced by a previously undescribed exon. The alternative transcript displays a complex structure in terms of protein coding potential, containing at least three possible open reading frames, one of which is a truncated collagen; thus the alternative transcript cannot encode type III collagen. Three specific aims are proposed to investigate the complex regulation of type III collagen gene expression during chondrogenesis. 1) In situ hybridization and immunohistochemistry will be used to define the spatial and temporal expression of type III collagen mRNA and protein, as well as the alternative transcript, in the developing limb. 2) The molecular mechanisms that regulate transcription of the type III collagen gene from dual promoters during chondrogenesis will be characterized. 3) Several complementary strategies will be utilized to define potential functions of the alternative transcript. The effects of overexpression and reduced expression of the alternative transcript on in vivo chondrogenesis will be determined. In addition, if the alternative transcript seems likely to serve a protein coding function in limb mesenchyme, the protein will be identified and characterized. These experiments will define the mechanisms that prevent production of type III collagen in cartilage and provide insight into the mechanisms resulting in reactivation of type III collagen synthesis in chondrocytes in endochondral fracture repair and osteoarthritis. These strategies will also increase our understanding of potential additional functions of the type III collagen gene mediated by the alternative transcript.