The overall goal of this proposal is to determine the biological effects of a novel F-box-containing protein (F-bx07) that we identified recently, with an emphasis on its role in the regulation of proliferation of rheumatoid synovial fibrobiasts (RASF). This goal will be achieved through three specific aims, centered on three objectives. The first objective is to determine if F-bx07 protein regulates the cell cycle of synovial fibroblasts. The second objective is to identify the protein(s) that F-bx07 targets leading to the attenuation of proliferation of RASF. The third objective is to determine if expression of F-bx07 protein is essential for prevention of the development of arthritis in vivo. The first objective is strongly supported by our initial observations that hyperproliferating RASFs lack of expression of F-bx07 mRNA, and transfection with AAVCMVF-bx07 attenuates RASFs proliferation. We will determine if inhibition of RASF proliferation of F-bx07 protein is controlled through regulation of the cell cycle. Cell cycle kinetics will be analyzed by flow cytometry analysis of cells stained with propidium iodide to determine the cell cycle stage controlled by F-bx07.1n the second aim, we will identify the molecules targeted by F-bx07 that directly and/or indirectly regulate cell cycle. Our preliminary data indicate that F-bx07 causes degradation of phosphorylated AKT and other investigators have shown that AKT participates in cell cycle regulation by controlling cell cycle kinase activity. To determine if additional cell cycle regulated proteins, especially those associated with the ubiquitin pathway are potential targets for F-bx07 degradation, the RASFs will be transfected with AAVCMVF-bx07 in the presence or absence of proteasome inhibitors and co-immunoprecipitated. Novel proteins will be identified by mass spectrometer analysis. Finally, we will validate our findings in vivo. Brdu labeling to track RASF proliferation in rheumatoid synovium organ culture, and immuno-histostaining of F-bx07 and cell cycle regulated proteins including AKT will determine the biological roles of F-bx07 in vivo. Antiproliferative effects of F-bx07 protein on of RASF will be demonstrated by gene therapy in an RASF implanted SClD mouse model. The findings will have practical applications in the potential treatment of human arthritis. At a more basic level, these experiments will test the hypothesis that F-bx07 regulates synovial fibroblast proliferation and that loss of F-bx07 protein activity triggers synovial fibroblast hyperproliferation in RA. This will lead to a better understanding of the mechanisms underlying hyperproliferation of RASFs.
