GTPase Activating Proteins in Aging and Osteoarthritis

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DESCRIPTION (provided by applicant): Project Summary: During aging and in osteoarthritis (OA), articular cartilage exhibits diminished responsiveness to the anabolic growth factor insulin-like growth factor-l (IGF-I). We have shown that IGF-I suppresses activation of the small G-protein Cdc42 (cell-division-cycle 42) in chondrocytes by upregulation of GTPase activating protein (GAP) activity, and that this signaling mechanism is linked to chondrocyte phenotypic expression. We have also documented loss of IGF-I induced regulation of Cdc42 activity during aging in chondrocytes. The premise of this proposal is that elucidating the signaling underlying IGF~>Cdc42- -> phenotype during aging and in OA is critical toward understanding the pathogenesis of OA, and may provide insight into novel approaches for the prevention or treatment of OA. The broad objectives of this proposal are to examine the regulation of Cdc42 in chondrocytes by IGF-I during aging and OA. The hypothesis for this proposal is: "The IGF-I regulated GAP will be identified, and retaining normal regulation of this GAP's activity will be critical for maintenance of a normal chondrocyte phenotype during aging and OA". In Specific Aim 1, we will identify and characterize GAPs in chondrocytes. We will use immunoprecipitation, affinity column purification, and yeast 2-hybrid approaches to attract GAPs which bind to active Cdc42. Putative GAPs will be sequenced using mass spectrometry. For proteins with GAP-homology domains, we will confirm functional GAP activity and determine which GAPs are regulated by IGF-I using [?-32P]GTP hydrolysis assays. In Specific Aim 2, we will determine the impact of GAPs on cartilage matrix homeostasis through gain and loss of function assays. We will transiently express wild-type, active, and dominant negative GAP mutants in young and aged chondrocytes and then test for resulting mRNA expression of the cartilage molecules aggrecan, collagen type II, and matrix metalloproteinases 3 &13 using quantitative PCR. We will then express GAP mutants in aged and OA chondrocytes, treat the cells with IGF-I, and measure [35S]sulfate incorporation into proteoglycans to determine if GAP expression can rescue chondrocytes from an IGF-I unresponsive state. Relevance of this research to Public Heath: These studies will further our understanding of the IGF-I~>GAP- ->Cdc42 signaling pathway in chondrocytes in pursuit of identifying the molecular mechanism of IGF-I unresponsiveness in aging articular cartilage. We anticipate that these findings will facilitate identification of target molecules for development of therapeutics modalities for OA.