Investigation of the intracellular function of human ecogenin/CTGF, a chondrocyte-derived growth factor.

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1) Evaluation of the cell-cycle modification effects of overexpressed CTGF : We overexpressed CTGF in a monkey kidney-derived Cos-7 cell line. Twelve hours after DNA transfection, accumulation of CTGF was observed at a particular perinuclear spot. Double staining of the cells with an anti-α-tubulin antibody indicated that it might be centrosome. Afterwards, CTGF accumulation became more prominent at 24 h posttransduction, which was accompanied by abnormal cell morphology with losing attachment and drastic increase of DNA content. These characteristics corresponded to those of cells in G2-M phases of cell cycle. Indeed, such findings were quite similar to those induced by colchicine, which halts mitosis at the M-phase. Since cell proliferation was rather retarded, CTGF was thought to arrest, or delay the cell cycle. Next, we examined the intracellular distribution of CTGF in vivo by immunohistochemical analysis of growth cartilage. Then, it was observed that CTGF accumulated in the same … More spot of hypertrophic chondrocytes which had stopped proliferation. We are going to transduce a cell line by a CTGF expression plasmid and analyze its gene expression pattern by a macro array system.2) Relationship between the modular structure and cell cycle modification effects of CTGF : CTGF consists of 4 conserved modules. In order to clarify which module is responsible for the findings above, a variety of plasmids that express CTGF deletion mutants were constructed. Using these plasmids, it has been uncovered that IGFBP module at the N-terminus is dispensable for the cell cycle modification effect, and that VWC plays a crucial role in the perinuclear accumulation of CTGF. Successful production of independent modular proteins was also carried out.3) Pursuit of intracellular target/receptor of CTGF : By means of CTGF-affinity column chromatography, we purified a CTGF-binding protein from cytosolic extract, determined a partial amino acid sequence, and identified it as a cytoskeletal protein. Less