ROLE OF RHO PROTEINS IN CHONDROCYTE DIFFERENTIATION

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A MCSDA would allow me as a veterinary surgeon and Ph.D., to expand my knowledge of cartilage biology and further understand chondrocyte metabolism in the search of preventive or treatment modalities for osteoarthritis. The mentor I have chosen is internationally recognized in intracellular signaling and has a well documented record of training scientists to become independent researchers. The studies outlined in this proposal examine the role of Rho-subfamily GTP-binding protein signaling in chondrocyte differentiation. Rho-binding proteins (Cdc42, Rac, RhoA) regulate actin cytoskeletal architecture and are involved in interleukin-1 (IL-1) signaling which induces chondrocyte dedifferentiation and cartilage degradation. Insulin-like growth factor-I (IGF-I) promotes the phenotypic expression of differentiated chondrocytes and can alleviate the degradative effects of IL-1 and tumor necrosis factor (TNF). Thus, the maintainance of a differentiated chondrocytes phenotype, and the key to prevention of osteoarthritis, may lie in IGF- I-regulated cytokine signaling through Cdc42, Rac, or RhoA. The broad objective of this study is to define the role IGF-I plays in the regulation of IL-1/TNF-induced dedifferentiation. During Phase I training, I will gain experience in biochemical and molecular-based approaches to study Rho-protein signaling pathways in chondrocytes. Aim 1 will examine how IGF-I, IL-1 and TNF influence the activation and localization of Cdc42, Rac, and RhoA. Aim 2 will employ microinjection methods to determine if mutated (constitutively active or inactive) Rho-proteins can prompt morphologic dedifferentiation. And Aim 3 will investigate the ability of vaccinia virus-expressed, mutated (constitutively active or inactive) Rho-proteins to incite phenotypic dedifferentiation. In Phase II, I will begin to develop an independent research program which will further extend my knowledge of Rho-proteins signaling in chondrocytes with emphasis on identifying Rho-target proteins involved in chondrocyte differentiation. I will focus on Rho kinase, a specific target for RhoA, and PAK-2 (p21-activated kinase), a specific target for Cdc42 and Rac. The vaccinia virus system will be used to express constitutively active or kinase-dead mutants of Rho kinase an PAK-2 to determine the effects of these kinases on chondrocyte differentiation. The expectation is that these studies will provide important insights into the regulation of chondrocyte differentiation while the ultimate goal is to use the knowledge gained to identify target molecules for the development of drug intervention or gene therapy modalities for osteoarthritis. This training would allow me to pursue an academic career as a clinician scientist with the unique qualifications to expand the interactions between basic and clinical research.