Cis Regulatory Motifs in Adult Articular Chondrocytes

描述（由申请人提供）： OA是关节炎最常见的形式，全世界有数千万有症状的OA患者，数亿人患有OA相关的病理变化。虽然大量的信息正在积累有关基因调控在发展过程中，调节基因表达的机制，在成人软骨细胞几乎是未知的。分析成人关节软骨细胞及其对所选生长因子和细胞因子的反应，将为研究OA中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性损伤或骨关节炎损伤时，随着细胞对损伤的反应，引起整体基因表达的变化，导致生长因子或细胞因子的产生，并最终增加代谢活性。这种代谢活动表现为基质基因表达的变化以及最终破坏软骨的分解代谢酶的增加。随着大量基因分析的出现，现在可以分析细胞对生长因子和细胞因子的整体基因反应，并从简单的实验中检索大量信息。虽然这些数据的解释一直很麻烦，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由各种细胞因子和生长因子上调或下调的基因转录本，这些细胞因子和生长因子已知对关节软骨的发育或维持很重要，或者在疾病过程中出现;然后分析无关基因中的转录反应元件。该建议的具体贡献是：1）鉴定由已知影响成熟软骨细胞表型的特定细胞因子和生长因子协调调节的常见基因转录物组。待检测的细胞因子为IL-1 β、TNF-α、IL-4和IL-17。待测生长因子为BMP-2、IGF-1和FGF-18。 2)确定常见的已知和新的调控基序的基因对这些细胞因子和生长因子的反应。本研究的总体目标是了解成人关节软骨细胞表型表达的调控，并最终应用于受伤或患病组织。实验方法将包括在单层培养物和三维基质中对具有各种处理的人类成人关节软骨细胞进行DNA微阵列分析。然后将利用一种新的技术，结合系统发育足迹与计算算法的基因编码的转录本与这些因素的反应进行分析。然后，这些调控基序将在正常和患病的软骨细胞中使用标准的体外转染测定进行实验测试，以验证其功能。这些研究将有助于阐明调节软骨细胞中基因转录的机制，并将提供必要的基础科学数据，以制定在发育过程中控制软骨细胞基因表达的策略，在组织工程和修复，以及在骨关节炎等疾病中。本提案中概述的策略和技术也可能对许多其他实验系统和组织具有更广泛的应用。

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.