Molecular cloning and biological activity of a novel lysyl oxidase-related gene expressed in cartilage

Abstract

We cloned a cDNA encoding a novel lysyl oxidase-related protein, named LOXC, by suppression subtractive hybridization between differentiated and calcified ATDC5 cells, a clonal mouse chondrogenic EC cell line. The deduced amino acid sequence of mouse LOXC consists of 757 amino acids and shows 50% identity with that of mouse lysyl oxidase. Northern blot analysis showed a distinct hybridization band of 5.4 kilobases, and Western blot analysis showed an immunoreactive band at 82 kilodaltons. Expression of LOXC mRNA was detected in osteoblastic MC3T3-E1 cells and embryonic fibroblast C3H10T1/2 cells, whereas none of NIH3T3 fibroblasts and myoblastic C2C12 cells expressed LOXC mRNA in vitro. Moreover, the LOXC mRNA and protein levels dramatically increased throughout a process of chondrogenic differentiation in ATDC5 cells. In vivo, LOXC gene expression was localized in hypertrophic and calcified chondrocytes of growth plates in adult mice. The conditioned media of COS-7 cells transfected with the full-length LOXC cDNA showed the lysyl oxidase activity in both type I and type II collagens derived from chick embryos, and these activities of LOXC were inhibited by beta-aminopropionitrile, a specific inhibitor of lysyl oxidase. Our data indicate that LOXC is expressed in cartilage in vivo and modulates the formation of a collagenous extracellular matrix.