Regulation of Gene Expression in Cartilage

描述(由申请人提供)：了解控制软骨生成的机制对于改进组织工程策略具有重要的临床意义，例如，组织工程策略可用于骨关节炎期间的软骨修复。资助更新期的研究集中在基因转录、前体信使RNA(Pre-mRNA)选择性剪接和蛋白质合成水平上破译软骨形成的调控。以下三个特定目标将直接涉及软骨发育的这些方面：1)研究转录因子在软骨形成过程中的作用，特别是AP-2、DEF-1和C/EBP；2)研究软骨形成过程中发生的II型前胶原前mRNA开关的调节；3)确定发育中表达的IIA型前胶原蛋白亚型在体内的功能。来自这些特定目标的综合数据将为软骨形成的调控提供独特的见解，因为转录、RNA剪接和翻译过程显然是紧密协调的。我们还计划研究第四个特定目标中描述的对软骨维持和修复重要的机制：4)研究IL-1b和TNF-a诱导成人软骨细胞BMP-2的机制。软骨形成过程被认为在软骨退化条件下的组织修复过程中被重演。因此，最终的具体目标与前三个专注于软骨形成的具体目标相结合，将为维持软骨健康提供宝贵的信息。这些课题的主要实验方法将是使用体外软骨形成系统，利用ATDC5细胞系和从脂肪组织中分离的成人干细胞来研究特定状态的分化事件。调节因子将通过过度表达和抑制研究进行测试，并与体内表达相关。

DESCRIPTION (provided by applicant): Understanding the mechanisms that control chondrogenesis is of major clinical importance for improving tissue engineering strategies that can be applied to repair cartilage during osteoarthritis, for example. Studies proposed for the grant renewal period are focused on deciphering the regulation chondrogenesis at the level of gene transcription, precursor messenger RNA (pre-mRNA) alternative splicing and protein synthesis. The following three Specific Aims will directly address these aspects of cartilage development: 1) Investigate the role of transcription factors during chondrogenesis, specifically AP-2, dEF-1 and C/EBP; 2) Investigate the regulation of the type II procollagen pre-mRNA switch that occurs during chondrogenesis and 3) Determine the function of the developmentally-expressed type IIA procollagen protein isoform in vivo. Combined data from these Specific Aims will provide unique insight into the regulation of chondrogenesis as it is becoming apparent that processes of transcription, RNA splicing and translation are tightly coordinated. We also plan to study mechanisms important for cartilage maintenance and repair as described in the fourth Specific Aim: 4) Investigate the mechanism of IL-1b and TNF-a induction of BMP-2 in adult chondrocytes. Processes that occur during chondrogenesis are believed to be recapitulated during tissue repair under conditions of cartilage degradation. Therefore, the final Specific Aim in combination with the first three Specific Aims focused on chondrogenesis will provide invaluable information into the maintenance of cartilage health. The predominant experimental approach to these topics will be to use in vitro systems of chondrogenesis utilizing the ATDC5 cell line and adult stem cells isolated from adipose tissue to investigate state-specific differentiation events. Regulatory factors will be tested by over expression and inhibition studies and correlated with expression in vivo.