Molecular cloning of human chondromodulin‐I, a cartilage‐derived growth modulating factor, and its expression in Chinese hamster ovary cells

Abstract

Bovine chondromodulin‐I (ChM‐I) purified from fetal cartilage stimulated the matrix synthesis of chondrocytes, and inhibited the growth of vascular endothelial cells in vitro. The human counterpart of this bovine growth regulating factor has not been identified. We report here the cloning of human ChM‐I precursor cDNA and its functional expression in Chinese hamster ovary (CHO) cells. We first identified a genomic DNA fragment which encoded the N‐terminus of the ChM‐I precursor, and then isolated human ChM‐I cDNA from chondrosarcoma tissue by PCR. The deduced amino acid sequence revealed that mature human ChM‐I consists of 120 amino acids. In total, 16 amino acid residues were substituted in the human sequence, compared to the bovine counterpart. Almost of all the substitutions were found in the N‐terminal hydrophilic domain. In the C‐terminal hydrophobic domain (from Phe42 to Val120), the amino acid sequence was identical except for Tyr90, indicating a functional significance of the domain. Northern blotting and in situ hybridization indicated a specific expression of ChM‐I mRNA in cartilage. We also successfully determined the cartilage‐specific localization of ChM‐I protein, using a specific antibody against recombinant human ChM‐I. Multiple transfection of the precursor cDNA into CHO cells enabled us to isolate the mature form of human ChM‐I from the culture supernatant. Purified recombinant human ChM‐I stimulated proteoglycan synthesis in cultured chondrocytes. In contrast, it inhibited the tube morphogenesis of cultured vascular endothelial cells in vitro and angiogenesis in chick chorioallantoic membrane in vivo.

Cartilaginous features in matrix-producing carcinoma of the breast: four cases report with histochemical and immunohistochemical analysis of matrix molecules

“…Type II collagen deposition in matrix-producing carcinoma was limited to lacunar formation regions. Although ChM-I is also a cartilage-specific noncollagenous matrix molecule, 7,8 this molecule was weakly and focally deposited in the matrix of matrix-producing carcinoma. Such findings suggested that the matrix of matrix-producing carcinoma of the breast was similar to that of normal cartilage, but had not matured to that of the hypertrophic zone of growth plate cartilages.…”

“…3 This macromolecule deposits in the hypertrophic zones of growth plate cartilage, 4 although it was reported to deposit in the salivary pleomorphic adenoma, which frequently showed mesenchyme-like elements, including myxoid and chondroid tissues. 5,6 Type II collagen, which is a cartilage-specific major collagen, and chondromodulin-I (ChM-I), which is cartilage-specific noncollagenous matrix protein, 4,7,8 are deposited in the normal cartilaginous matrix. These molecules can be useful markers of cartilaginous differentiation.…”

Expression Profiles Provide Insights into Early Malignant Potential and Skeletal Abnormalities in Multiple Endocrine Neoplasia Type 2B Syndrome Tumors

“…1A, Table 1). CHM1 is a known regulator of cartilage and bone growth and is normally expressed in proliferating and prehypertrophic chondrocytes of the growth plate (21). In bone explants, its overex- pression promotes cartilage deposition but inhibits bone deposition, whereas studies of CHM1 mutant mice show that its deficiency results in increased bone deposition (33,34).…”

Anti-angiogenic action of the C-terminal domain of tenomodulin that shares homology with chondromodulin-I

“…We isolated and purified the factor that promoted this growth inhibition, and subsequent amino acid sequencing identified it as chondromodulin-I (ChM-I) (Hiraki et al, 1991). Transfection of ChM-I precursor cDNAs into either COS7 cells or CHO cells resulted in secretion of mature ChM-I (120 amino acids) as a 25 kDa glycoprotein following processing at the furin cleavage site of the type II membrane ChM-1 precursor protein (Azizan et al, 2001;Hiraki et al, 1997;Hiraki et al, 1999). In situ hybridization and immunohistochemistry indicated that ChM-I is specifically localized in the avascular zone of cartilage during endochondral bone formation Shukunami et al, 1999).…”

“…All eight cysteine residues in the mature ChM-I protein are present in this domain and form four intramolecular disulfide bonds (Hiraki et al, 1991;Neame et al, 1990). These bonds are critical for the bioactivity of ChM-I, which is abrogated by their reduction Hiraki et al, 1999).…”