Cis Regulatory Motifs in Adult Articular Chondrocytes

描述(由申请人提供)： 骨关节炎是最常见的关节炎形式，全世界有数千万有症状的骨关节炎患者，还有数亿人患有骨关节炎相关的病变。虽然在发育过程中关于基因调控的大量信息正在积累，但调控成年软骨细胞基因表达的机制实际上是未知的。分析成人关节软骨细胞及其对选定的生长因子和细胞因子的反应，将为研究骨性关节炎的软骨退变提供必要的基础科学信息。理解这些机制很重要，因为当软骨受到创伤或骨关节炎的破坏时，随着细胞对损伤的反应，全局基因表达会发生变化，导致生长因子或细胞因子的产生，最终增加新陈代谢活动。这种代谢活动表现为基质基因表达的变化以及分解代谢酶的增加，最终会破坏软骨。随着大量基因分析的到来，现在可以分析细胞对生长因子和细胞因子的全局基因反应，并从简单的实验中检索大量信息。虽然对这些数据的解释一直很繁琐，但最近的创新计算方法促进了这一过程。我们的策略将是确定受各种细胞因子和生长因子上调或下调的基因转录本，这些基因转录本对关节软骨的发育或维持至关重要，或出现在疾病过程中；然后分析无关基因中的转录反应元件。这一建议的具体好处是：1)确定受特定细胞因子和生长因子协调调控的共同基因转录本，这些基因转录本是已知影响成熟软骨细胞表型的。接受检测的细胞因子将是IL-1β、肿瘤坏死因子-α、IL-4和IL-17。接受检测的生长因子将是BMP-2、IGF-1和FGF-18。2)确定对这些细胞因子和生长因子有反应的基因中常见的和新的调控基序。这项研究的总体目标是了解成人关节软骨细胞表型的表达规律，并最终应用于损伤或病变组织。该实验方法将包括在单层培养和三维基质中对人类和成人关节软骨细胞进行不同处理的DNA微阵列分析。然后将利用一种结合了系统发育足迹和计算算法的新技术来分析编码对这些因素有反应的转录本的基因。然后，这些调控基序将在正常和患病的软骨细胞中进行实验测试，使用标准的体外转基因分析来验证它们的功能。这些研究将有助于阐明调控软骨细胞基因转录的机制，并将为制定控制软骨细胞在发育、组织工程和修复以及骨关节炎等疾病中基因表达的策略提供必要的基础科学数据。该提案中概述的策略和技术也可能在许多其他实验系统和组织中得到更广泛的应用。

DESCRIPTION (provided by applicant): OA is the most common form of arthritis, with tens of millions of symptomatic OA sufferers worldwide, and hundreds of millions of people with OA-related pathological changes. While a great deal of information is accumulating regarding gene regulation during development, the mechanisms that regulate gene expression in adult chondrocytes are virtually unknown. Analysis of adult articular chondrocytes and their response to selected growth factors and cytokines will provide basic science information that is necessary for the study of cartilage degeneration as seen in OA. It is important to understand these mechanisms because when cartilage is damaged, by traumatic injury or osteoarthritis, global gene expression changes are elicited as the cells respond to the insult, resulting in growth factor or cytokine production and, ultimately, increased metabolic activity. This metabolic activity is manifested by changes in matrix gene expression as well as increases in catabolic enzymes that can eventually destroy the cartilage. With the advent of high-volume gene analysis, it is now possible to analyze a cell's global gene response to growth factors and cytokines and retrieve an enormous amount of information from simple experiments. While the interpretation of this data has been cumbersome, recent innovative computational approaches have facilitated this process. Our strategy will be to determine sets of genes transcripts that are up or down regulated by various cytokines and growth factors known to be important for development or maintenance of articular cartilage or appear during disease processes; then to analyze the transcriptional response elements in the irrespective genes. The specific alms of this proposal are: 1) Identify common sets of gene transcripts that are coordinately regulated by specific cytokines and growth factors known to affect the mature chondrocyte phenotype. The cytokines to be tested will be IL-1beta, TNF-alpha, IL-4 and IL-17. The growth factors to be tested will be BMP-2, IGF-1 and FGF-18. 2) Identify common known and novel regulatory motifs in the genes responsive to these cytokines and growth factors. The overall goal of this research is to understand the regulation of expression of the adult articular chondrocyte phenotype with the eventual application to injured or diseased tissue. The experimental approach will include DNA microarray analysis of human, adult articular chondrocytes with various treatments, both in monolayer cultures and in a three dimensional matrix. The genes encoding the transcripts with demonstrated response to these factors will then be analyzed utilizing a novel technique combining phylogenetic foot printing with computational algorithms. These regulatory motifs will then be experimentally tested in normal and diseased chondrocytes using standard in vitro transfection assays to validate their functionality. These studies will help to elucidate the mechanism for regulating gene transcription in chondrocytes and will provide the basic science data necessary to develop strategies to control chondrocyte gene expression during development, in tissue engineering and repair, and in diseases such as osteoarthritis. The strategies and techniques outlined in this proposal may also have broader applications to many other experimental systems and tissues.