Molecular cloning and biological activity of a novel lysyl oxidase-related gene expressed in cartilage.
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.
Ito
H
H
Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Sakyo, Kyoto 606-8507, Japan.
Akiyama
H
H
Iguchi
H
H
Iyama
K
K
Miyamoto
M
M
Ohsawa
K
K
Nakamura
T
T
D000594:Amino Acid Oxidoreductases; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D002356:Cartilage / Q000201:enzymology*; D002460:Cell Line; D003001:Cloning, Molecular; D006132:Growth Plate / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014162:Transfection
D012333:RNA, Messenger; D000594:Amino Acid Oxidoreductases; C445682:LOXL4 protein, human; C432408:Loxl4 protein, mouse; D008249:Protein-Lysine 6-Oxidase