Cis Regulatory Motifs in Adult Articular Chondrocytes

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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.