Understanding the role of inflammatory mediators in the pathogenesis of cartilage degeneration in osteoarthritis (OA) is increasingly recognized as a strategy for the development of mechanism-based therapeutics aimed to retard disease progression. While the catabolic effects of IL-1-beta, TNF-alpha and nitric oxide are well described, the consequences of the excess production of eicosanoids, a feature of progressive osteoarthritis, are poorly understood. This lack of clarity is, in part, because diverse eicosanoid end-products, poorly delineated in OA, act via distinct surface receptors, intracellular signaling pathways, and transcription factors to exert highly dissimilar effects on cellular functions. In this grant request, we will perform a comprehensive determination of the predominant eicosanoids produced by OA chondrocytes in order to clarify the effects of specific end-products on selected gene expression and cartilage homeostasis. Using enzyme immunoassay and LC/MS-MS, we will characterize the profile and enzymatic source of the eicosanoids produced by OA cartilage. Based upon our Preliminary Studies, a central focus will be upon the regulation and action of PGD2, and its metabolites PGJ2 and 15-deoxydelta-12,14 PGJ2. Since subcellular segregation of individual proteins may reflect functional coupling, we will utilize immunostaining and GFP transfection techniques to determine the spatiotemporal subcellular localization of key enzymes of arachidonate metabolism and analyze the eicosanoids produced by individual cellular compartments. Following the identification of OA predominant eicosanoids, we will assess their effects on differential gene expression of OA and normal chondrocytes. Confirmation of effects on selected gene product expression will be made by Northern and reverse Northern hybridization and immunoblot analysis. Finally, we will determine the effects of PGD2 and other OA predominant eicosanoids on chondrocyte metabolism (proteoglycan synthesis and degradation), inflammatory mediator production and apoptosis. Based upon preliminary data, we will place a significant focus on an analysis of the PGD synthase pathway and the consequences of PPAR-g activation. These studies will provide new insights into the role of eicosanoids in osteoarthritis and elucidate the potential consequences of chronic pharmacologic COX-2 inhibition on the structural integrity of articular cartilage.
