FUNCTION AND REGULATION OF CD-RAP

项目摘要/摘要 到2020年，骨性关节炎将影响5000万美国人。疾病的过程是 以基因表达异常和软骨细胞无力为特征的 修复导致软骨退变的细胞外基质。CD-RAP是一种小型的 软骨特有的蛋白，在软骨中与II型胶原和 阿格利坎。它是一个紧凑的基因，与IIB型胶原共表达。 软骨形成，使其成为研究软骨细胞特异性基因的优秀模型 表情。在过去的四年里，我们扩展了对单个基因的了解 对软骨细胞表型的转录调控进行分析。 采用计算和实验相结合的方法，分析了几组不同的协同效应。 表达的基因和定义表达的基因的共同调控结构域 关节软骨。目前的提案将侧重于扩展这些分析，以定义 主要在关节中运行并定义关节的转录调控网络 软骨发育(与生长板相反，生长板导致骨形成)。 当新的转录或信号因子出现时，特定的活动机制将是 使用我们的CD-RAP和COL2A1基因模型或细胞培养破译。具体的 该提案的目的是：(1)确定转录调控机制 通过E-box蛋白EF-1/USF1/USF2、C/EBPss和HMGA对CD-RAP基因进行编码。(2) 确定转录因子网络在软骨和干细胞分化中的作用 细胞(3)确定转录调节和信号在早期模型中的作用 骨关节炎和(4)我们在特定目标的初步实验中产生的3， 研究EGF和成纤维细胞生长因子信号的细胞内调节因子Sprouty-4在血管内皮细胞生长中的作用 软骨生成和骨关节炎。实验方法将重点放在(1)使用 全基因组表达分析以确定共调控基因，(2)筛选 使用1700寡核苷酸阵列表达转录因子，(3)计算 共调控基因启动子转录因子结合域分析， 以及(4)对特定软骨基因CD-RAP的机械作用的确认 和COL2A1。这些研究将应用强大的技术，将实验和 超越候选基因以阐明调控的计算方法 制定控制软骨形成战略的关键机制 在组织工程和修复方面的发展，并最终帮助寻找生物 方法控制骨性关节炎。

Project Summary/Abstract Osteoarthritis will affect 50 million Americans by 2020. The disease process is characterized by the aberrant gene expression and the inability of cartilage chondrocytes to repair the extracellular matrix leading to cartilage degeneration. CD-RAP is a small cartilage-characteristic protein that is co-expressed in cartilage with type II collagen and aggrecan. It is a compact gene and co-expressed with type IIB collagen in chondrogenesis, making it an excellent model for study of chondrocyte-specific gene expression. Over the past four years, we have extended our knowledge of a single gene to the analysis of the transcriptional regulation of the chondrocyte phenotype. Computational and experimental methods were combined to analyze groups of co- expressed genes and define common regulatory domains of genes expressed in articular cartilage. The current proposal will focus on extending these analyses to define transcriptional regulatory networks that predominantly operate in and define articular cartilage development (as opposed to the growth plate which results in bone formation). As new transcription or signaling factors arise, specific mechanisms of activity will be deciphered using our CD-RAP and COL2A1 gene models or cell culture. The specific aims of the proposal are: (1) Determine the mechanism of transcriptional regulation of the CD-RAP gene by the E-box proteins EF-1/USF1/USF2, C/EBPss, and HMGA. (2) Define the role of transcription factor networks in differentiation of cartilage from stem cells (3) Define the role of transcriptional regulation and signaling in a model for early osteoarthritis and (4) Arising from our preliminary experiments in Specific Aim 3, investigate the role of the intracellular regulator of EGF and FGF signaling, Sprouty-4, in chondrogenesis and osteoarthritis. The experimental approach will focus on (1) using genome-wide expression analyses to determine co-regulated genes, (2) screening for expression of transcription factors using a 1700 oligonucleotide array, (3) computational analyses for transcription factor binding domains of the promoters of co-regulated genes, and (4) confirmation of mechanistic function on the specific cartilage genes, CD-RAP and COL2A1. These studies will apply powerful techniques combining experimental and computational approaches to look beyond candidate genes to elucidate regulatory mechanisms critical for developing strategies to control chondrogenesis during development, in tissue engineering and repair, and ultimately to help find biological methods to control osteoarthritis.