Abstract
During endochondral bone formation, cartilage cells show increased matrix synthesis and rapid proliferation. We found that cartilage matrix contains at least two types of heparin binding growth-promoting components. One, with a higher affinity to heparin, was identified as chondromodulin I (Hiraki, Y., Tanaka, H., Inoue, H. , Kondo, J., Kamizono, A., and Suzuki, F. (1991) Biochem. Biophys. Res. Commun. 175, 871-977). In this study, we isolated a novel growth-promoting component, chondromodulin II, which has a lower heparin affinity, from the dissociative extracts of fetal bovine epiphyseal cartilage. Chondromodulin II stimulated the proteoglycan synthesis in rabbit cultured growth plate chondrocytes, an expression of the differentiated phenotype of chondrocytes. It also stimulated DNA synthesis in chondrocytes in both the absence and the presence of fibroblast growth factor-2. The apparent molecular mass of chondromodulin II on SDS-polyacrylamide gel electrophoresis was 16 kDa. Its complete amino acid sequence was determined by overlapping sequences of the peptides released by endopeptidase digestion and CNBr cleavage. Chondromodulin II consists of 133 amino acids (calculated Mr = 14,548). The sequence was unique but homologous to the repeats 1 and 2 of the deduced amino acid sequence of the chicken mim-1 gene, which is specifically transactivated by the v-Myb oncogene product in promyelocytes. We also found a minor component with a higher heparin affinity, chondromodulin III, in cartilage extracts. Chondromodulin III stimulated DNA synthesis in chondrocytes in vitro, and its N-terminal sequence was identical with ribosomal protein L31 lacking the N-terminal three amino acids. These findings suggest that the growth and differentiation of chondrocytes are regulated by multiple components in the cartilage matrix.
Highlights
During endochondral bone formation, cartilage cells show increased matrix synthesis and rapid proliferation
We found a minor component with a higher heparin affinity, chondromodulin III, in cartilage extracts
The following fractionated cartilage extract materials were added to confluent culture of rabbit growth plate chondrocytes in the presence or absence of Recombinant human FGF-2 (rhFGF-2) (0.4 ng/ml): 1 M guanidine extract containing materials of 10 –50 kDa (10 –50 kDa extract, 200 g/ml) and the fractions obtained by heparin affinity chromatography of 10 –50 kDa extract: unbound-fraction, the first protein peak eluted with 0.5 M NaCl, the second protein peak eluted with 0.5 M NaCl, and the protein peak eluted with 1.2 M NaCl
Summary
Yuji Hiraki‡, Hiroyuki Inoue, Jun Kondo§, Akihito Kamizono§, Yoshino Yoshitake¶, Chisa Shukunami, and Fujio Suzuki. Chondromodulin III stimulated DNA synthesis in chondrocytes in vitro, and its N-terminal sequence was identical with ribosomal protein L31 lacking the N-terminal three amino acids These findings suggest that the growth and differentiation of chondrocytes are regulated by multiple components in the cartilage matrix. As we have reported previously, some proteinaceous components in cartilage synergistically stimulated DNA synthesis and the growth of cultured chondrocytes in vitro as well as stimulating proteoglycan synthesis in chondrocytes [6, 7]. Chondromodulin II has an apparent molecular mass of 16 kDa on SDS-PAGE, and it stimulated proteoglycan synthesis in the cells It stimulated DNA synthesis in rabbit growth plate chondrocytes in vitro in both the absence and the presence of FGF-2. The complete amino acid sequence of ChM-II is reported here
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