title: Functional gene screening system identified TRPV4 as a regulator of chondrogenic differentiation.
abstract: 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.
authors.affiliation: Laboratory for Drug Discovery, Research Center, Asahi Kasei Pharma Corp., 2-1 Samejima, Fuji, Shizuoka 416-8501, Japan. muramatsu.sb@om.asahi-kasei.co.jp
authors.forename: Shuji
authors.initials: S
authors.lastname: Muramatsu
authors.forename: Makoto
authors.initials: M
authors.lastname: Wakabayashi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ohno
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Amano
authors.forename: Rika
authors.initials: R
authors.lastname: Ooishi
authors.forename: Toshinori
authors.initials: T
authors.lastname: Sugahara
authors.forename: Satoshi
authors.initials: S
authors.lastname: Shiojiri
authors.forename: Kosuke
authors.initials: K
authors.lastname: Tashiro
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Riko
authors.initials: R
authors.lastname: Nishimura
authors.forename: Satoru
authors.initials: S
authors.lastname: Kuhara
authors.forename: Sumio
authors.initials: S
authors.lastname: Sugano
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Yoneda
authors.forename: Akio
authors.initials: A
authors.lastname: Matsuda
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D020219:Chondrogenesis / Q000235:genetics*; D018076:DNA, Complementary; D015723:Gene Library; D016678:Genome; D006609:High Mobility Group Proteins / Q000378:metabolism; D051379:Mice; D055758:SOX9 Transcription Factor; D050916:TRPV Cation Channels / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism