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

We cloned a cDNA encoding a novel lysyl oxidase-related protein, named LOXC , by suppression subtractive hybridization between differentiated and calci fied ATDC5 cells, a clonal mouse chondrogenic EC cell line. The deduced ami no acid sequence of mouse LOXC consists of 757 amino acids and shows 50% id entity with that of mouse lysyl oxidase. Northern blot analysis showed a di stinct hybridization band of 5.4 kilobases, and Western blot analysis showe d an immunoreactive band at 82 kilodaltons. Expression of LOXC mRNA was det ected in osteoblastic MC3T3-E1 cells and embryonic fibroblast C3H10T1/2 cel ls, whereas none of NIH3T3 fibroblasts and myoblastic C2C12 cells expressed LOXC mRNA in vitro. Moreover, the LOXC mRNA and protein levels dramaticall y increased throughout a process of chondrogenic differentiation in ATDC5 c ells. In vivo, LOXC gene expression was localized in hypertrophic and calci fied chondrocytes of growth plates in adult mice. The conditioned media of COS-7 cells transfected with the full-length LOXC cDNA showed the lysyl oxi dase activity in both type I and type II collagens derived from chick embry os, and these activities of LOXC were inhibited by P-aminopropionitrile, a specific inhibitor of lysyl oxidase. Our data indicate that LOXC is express ed in cartilage in vivo and modulates the formation of a collagenous extrac ellular matrix.