Cloning of Mouse Chondromodulin-I cDNA and Localization of the Gene Transcripts

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Studies of chondrocytes have been carried out by using primary cultures of chondrocytes that were isolated from mammalian cartilaginous tissue. However, it is theoretically not feasible to study the mechanism of chondrogenic differentiation using primary cultures of differentiated chondrocytes. Moreover, passages of primary chondrocytes result in a rapid loss of differentiated phenotype of cells. Therefore, we attempted to construct an in vitro chondrogenic culture system using a clonal EC cell line ATDC5. We demonstrated that the culture of ATDC5 cells kept track of the early-phase and late-phase differentiation that includes formation of type II collagen expressing proliferating chondrocytes and the subsequent cellular hypertrophy and mineralization.Then, we isolated a full length cDNA for mouse chondromodulin-I (ChM-I) from cDNA prepared from the differentiated ATDC5 cell culture by a PCR cloning method. The nucleotide sequence of mouse ChM-I cDNA revealed that mouse ChM-I precursor … More protein contained 334 amino acid residues. The Ch-SP domain of the mouse ChM-I precursor exhibited about 90% sequence identity, compared to the human counterpart. The mature ChM-I domain contained 120 amino acid residues as the human mature ChM-I did. The sequence identity in this mature domain was 86%. Most substitutions of amino acids were found in the N-terminal domain (40 residues). However, the N-linked oligosaccharide attachment site (Asn29) was concerved. In contrast, the C-terminal domain (about 80 residues from Phe42 to Val120) was completely conserved, except for the substitution from Ilel 16 to VaIl 16.Expression of ChM-I mRNA was induced along with the early-phase chondrogenic differentiation of ATDC5 cells and withdrawn as the late-phase differentiation proceeded. In situ hybridization in the ATDC5 cultures revealed the localized expression of ChM-I mRNA in cartilage nodules. In mouse embryo, cartilage formation was first observed on day 11. ChM-I transcripts were specifically detected at the sites of cartilage formation. On day 16, ChM-l expression was specifically abolished in the late-hypertrophic and mineralizing cartilage. This pattern of expression was well compatible with the functional role of ChM-I protein as "angioinhibin." Less