TYPE III COLLAGEN GENE EXPRESSION IN CARTILAGE

软骨中 III 型胶原蛋白基因表达

• 批准号: 6171677
• 负责人: SHERRILL L. ADAMS
• 金额: $ 24.84万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6171677
• 关键词: RNase protection assay; cartilage development; chick embryo; chondrocytes; collagen; gene expression; genetic promoter element; immunocytochemistry; in situ hybridization; mass tissue /cell culture; mesenchyme; messenger RNA; protein biosynthesis; transcription factor

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.

III型胶原mRNA和蛋白质存在于肢体中，但 在正常生长软骨中找不到；因此III型的合成 在软骨发生过程中，胶原蛋白必须停止。 但是，少量 在成人关节软骨中发现了III型胶原蛋白，数量 骨关节炎关节软骨似乎增加了。 小鸡类型 III胶原蛋白基因包含两个功能启动子，显示 胚胎发育过程中的不同表达方式和 引发具有不同结构的成绩单的生产，并且 功能。 上游启动子，负责生产 III型胶原mRNA的活性在皮肤成纤维细胞中，但不活跃 在软骨细胞中。 内部软骨细胞特异性启动子已经 确定的是负责替代品的生产 外显子1-23被先前未描述的成绩单取代 外显子。 替代成绩单以术语显示复杂的结构 蛋白质编码潜力，至少包含三个可能的开放 读取框架，其中之一是截短的胶原蛋白；因此 替代成绩单不能编码III型胶原蛋白。 三个具体 提出了目的来研究III型的复杂调节 软骨发生过程中的胶原基因表达。 1）原位 杂交和免疫组织化学将用于定义 III型胶原mRNA和蛋白质的空间和时间表达， 以及替代成绩单，在发育中的肢体中。 2） 调节III型胶原蛋白转录的分子机制 在软骨发生过程中，双启动子的基因将被表征。 3）将利用几种补充策略来定义潜力 替代成绩单的功能。 过表达的影响 并减少了体内替代转录本的表达 将确定软骨发生。 另外，如果另外 成绩单似乎很可能在肢体中发挥蛋白质编码功能 间充质，将鉴定和表征蛋白质。 这些 实验将定义防止产生类型的机制 软骨中的III胶原蛋白，并洞悉机制 导致软骨细胞中III型胶原蛋白合成的重生 在软骨骨折修复和骨关节炎中。 这些策略会 还可以提高我们对潜在的其他功能的理解 III型胶原蛋白基因由替代转录本介导。