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J. Biol. Chem., Vol. 278, Issue 48, 48259-48266, November 28, 2003

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Cystatin 10, a Novel Chondrocyte-specific Protein, May Promote the Last Steps of the Chondrocyte Differentiation Pathway^*

Yu Koshizuka, Takashi Yamada, Kazuto Hoshi, Toru Ogasawara, Ung-il Chung, Hirotaka Kawano, Yusuke Nakamura¶, Kozo Nakamura, Shiro Ikegawa||, and Hiroshi Kawaguchi**

From the Departments of Orthopedic Surgery and Tissue Engineering, Faculty of Medicine, and the ^¶Laboratory of Molecular Medicine, Institute of Medical Science, University of Tokyo, Tokyo 113-8655 and the ^||Laboratory of Bone and Joint Diseases, SNP Research Center, Institute of Physical and Chemical Research, Tokyo 108-8639, Japan

This study attempts to characterize cystatin 10 (Cst10), which we recently identified as a novel protein implicated in endochondral ossification. Expression of Cst10 was specific to cartilage, localized in the cytosol of prehypertrophic and hypertrophic chondrocytes of the mouse growth plate. In the mouse chondrogenic cell line ATDC5, Cst10 expression preceded type X collagen expression and increased in synchrony with maturation. When we compared ATDC5 cells transfected with Cst10 cDNA with cells transfected with a mock vector, hypertrophic maturation and mineralization of chondrocytes were promoted by Cst10 gene overexpression in that type X collagen expression was observed earlier, and alizarin red staining was stronger. On the other hand, type II collagen expression and Alcian blue staining, both of which are markers of the early stage of chondrocyte differentiation, were similar in both cells. Overexpression of the Cst10 gene also caused fragmentation of nuclei, the appearance of annexin V, a change in the mitochondrial membrane potential, and activation of caspases. These results strongly suggest that Cst10 may play an important role in the last steps of the chondrocyte differentiation pathway as an inducer of maturation, followed by apoptosis of chondrocytes.

Received for publication, November 14, 2002 , and in revised form, September 15, 2003.

^* This work was supported by Grant-in-aid for Scientific Research 14370454 from the Japanese Ministry of Education, Culture, Science, Sports, and Technology and by a grant-in-aid from the Investigation Committee on the Ossification of Spinal Ligaments, Japanese Ministry of Public Health and Welfare. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence and reprint requests should be addressed: Dept. of Orthopedic Surgery, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan. Tel.: 81-3-5800-8656; Fax: 81-3-3818-4082; E-mail: kawaguchi-ort{at}h.u-tokyo.ac.jp.

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