TYPE III COLLAGEN GENE EXPRESSION IN CARTILAGE

III型胶原mRNA和蛋白存在于肢体间质中，但 在正常生长的软骨中没有发现;因此III型软骨的合成 胶原蛋白在软骨形成过程中必须停止。 然而，少量的 III型胶原蛋白存在于成人关节软骨中， 似乎在骨关节炎关节软骨中增加。 小鸡型的 III型胶原基因含有两个功能启动子， 胚胎发育过程中的不同表达模式， 启动具有非常不同结构的转录物的产生， 功能协调发展的 上游启动子，负责生产 在皮肤成纤维细胞中有活性，但无活性 在软骨细胞中。 一个内部的软骨细胞特异性启动子已经被 确定负责生产替代品的机构 其中外显子1-23被先前未描述的 外显子。 另一种转录本显示了一个复杂的结构， 蛋白质编码潜力，包含至少三个可能的开放 阅读框，其中之一是截短的胶原蛋白;因此， 替代转录物不能编码III型胶原。 三个具体 目的是研究复杂的调节类型III 软骨形成过程中胶原基因的表达。 1)原位 将使用杂交和免疫组织化学来确定 III型胶原mRNA和蛋白时空表达， 在发育中的肢体中也有替代转录物。 2)的 调节III型胶原蛋白转录的分子机制 将表征在软骨形成期间来自双启动子的基因。 3)将利用几种补充战略来确定潜在的 替代性转录本的功能。 过度表达的影响 并降低体内替代转录物的表达 将确定软骨形成。 此外，如果替代方案 转录物似乎在肢体中起着蛋白质编码的作用 间充质中，蛋白质将被鉴定和表征。 这些 实验将确定防止类型产生的机制， III软骨中的胶原蛋白，并提供深入的机制 导致软骨细胞中III型胶原合成的再活化 软骨内骨折修复和骨关节炎。 这些战略将 也增加了我们对潜在的额外功能的理解， III型胶原基因介导的替代转录。

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.