Functional dissection and medical application of the 4 modules in the chondrocyte-derived growth factor, ecogenin/CTGF.

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1. Establishment of the production and detection system with specific antibodies of each module of ecogenin/CTGF : Firstly, each module was independently prepared by a Brevibacillus production s stem, and the epitopes of anti-CTGF antibodies were located. As a result at least one antibody against each module, IGFBP, VWC, TSP1 or CT, was obtained Among the ELISA systems we established, the most sensitive one is already in use for subsequent research. Additionally, the other ELISA systems to quantify CTGF subfragments were also established.2. Investigation of the roles of modular structure in cell growth and differentiation : Utilizing each purified module, we examined if the signal transduction cascades in chondrocytic HCS-2/8 cells were activated. As a result, a few different pathways were activated by specific modules. Since different results were obtained with other cell types, CTGF is supposed to use multiple signaling pathways differentially. Of note, proteoglycan synthesis by HCS-2/8 was rather enhanced by a few module-specific antibodies, whereas proliferation was inhibited by those antibodies. These findings suggest the utility of the antibodies in therapeutics as well as the CTGF derivatives. Next, we evaluated the effects of the modules on cell adhesion and found the promoting effects of all of the modules. Among the four, CT was the most effective. Finally, effects of overexpression of the modules on cell cycle was evaluated. Then, dimodular TSP-CT and single CT were suggested to arrest the cell cycle at M-phase. As such, particular care should be taken for CT module in considering therapeutic application.3. Experimental therapeutics with animal models : Prior to the application of modular mutants, wild type CTGF was applied to the experimental rat cartilage defect models and the effects were evaluated. Then CTGF was found to regenerate damaged articular cartilage. This methodology is going to be applied to the evaluation of modular mutants.