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J. Biol. Chem., Vol. 282, Issue 44, 32158-32167, November 2, 2007

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Functional Gene Screening System Identified TRPV4 as a Regulator of Chondrogenic Differentiation^*

Shuji Muramatsu¹, Makoto Wakabayashi, Takeshi Ohno, Katsuhiko Amano, Rika Ooishi, Toshinori Sugahara, Satoshi Shiojiri, Kosuke Tashiro^¶, Yutaka Suzuki^||, Riko Nishimura, Satoru Kuhara^¶, Sumio Sugano^||, Toshiyuki Yoneda, and Akio Matsuda

From the Laboratory for Drug Discovery, Research Center, Asahi Kasei Pharma Corp., 2-1 Samejima, Fuji, Shizuoka 416-8501, Japan, the ^¶Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, Fukuoka 812-8581, Japan, the ^||Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan, and the Department of Molecular and Cellular Biochemistry, Graduate School of Dentistry, Osaka University, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan

Sox9 is a transcription factor that is essential for chondrocyte differentiation and chondrocyte-specific gene expression. However, the precise mechanism of Sox9 activation during chondrogenesis is not fully understood. To investigate this mechanism, we performed functional gene screening to identify genes that activate SOX9-dependent transcription, using full-length cDNA libraries generated from a murine chondrogenic cell line, ATDC5. Screening revealed that TRPV4 (transient receptor potential vanilloid 4), a cation channel molecule, significantly elevates SOX9-dependent reporter activity. Microarray and quantitative real time PCR analyses demonstrated that during chondrogenesis in ATDC5 and C3H10T1/2 (a murine mesenchymal stem cell line), the expression pattern of TRPV4 was similar to the expression patterns of chondrogenic marker genes, such as type II collagen and aggrecan. Activation of TRPV4 by a pharmacological activator induced SOX9-dependent reporter activity, and this effect was abolished by the addition of the TRPV antagonist ruthenium red or by using a small interfering RNA for TRPV4. The SOX9-dependent reporter activity due to TRPV4 activation was abrogated by both EGTA and a calmodulin inhibitor, suggesting that the Ca²⁺/calmodulin signal is essential in this process. Furthermore, activation of TRPV4 in concert with insulin activity in ATDC5 cells or in concert with bone morphogenetic protein-2 in C3H10T1/2 cells promoted synthesis of sulfated glycosaminoglycan, but activation of TRPV4 had no effect alone. We showed that activation of TRPV4 increased the steady-state levels of SOX9 mRNA and protein and SOX6 mRNA. Taken together, our results suggest that TRPV4 regulates the SOX9 pathway and contributes to the process of chondrogenesis.

Received for publication, July 26, 2007 , and in revised form, September 4, 2007.

^* 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.

¹ To whom correspondence should be addressed. Tel.: 81-545-62-3220; Fax: 81-545-62-3329; E-mail: muramatsu.sb{at}om.asahi-kasei.co.jp.