Cloning and functional analysis of novel genes related to chondrocyte differentiation using the human chondrocytic cell lines

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To isolate new functional and regulatory molecules, which play an important role in the process of endochondral ossification, we first characterized the newly established human chondrosarcoma cell lines (HCS) and established a model culture system on the proliferation and differentiation of rabbit growth cartilage cells. We then analyzed mRNAs expressed in HCS cell lines, normal rabbit chondrocytes and other types of cells using differential display-PCR.Consequently, we obtained 30 species of chondrocyte- of HCS-specific DNA fragments. Nucleotide sequences of 17 of 30 species derived from HCS cells were determined. Comparison of the base sequences revealed seven novel sequence tags and a few sequence tags showing homology with known DNA sequences. One of the sequence tags (tag no.24) showed high structural homology with the nucleotide sequence of connective tissue growth factor (CTGF) and the corresponding gene (hcs24) was selectively expressed in HCS cells and rabbit growth cartilage cells in culture but was not expressed in osteoblastic cells of osteosarcoma cells in culture. The expression of hcs24 in HCS cells was up-regulated by the addition of TGF-beta or BMP-2. During in vitro culture of rabbit growth cartilag cells, its expression reached a maximum at the stage corresponding to early hypertrophic chondrocytes in vivo. In situ hybridization revealed that hcs24 was expressed only in the hypertrophic chondrocytes of costal cartilage and the vertebral column in embryonic mice. Anti-sense oligonucleotides strongly inhibited the proliferation of HCS cells and increased their proteoglycan synthesis. The anti-sense oligomer also increased alkaline phosphatase activity in rabbit growth cartilage cells in culture. These results suggest that Hcs24 protein is produced by hypertrophic chondrocytes and that it promotes the proliferation of growth cartilage cells and suppresses their differentiation toward endochondral ossification.