Functional Gene Screening System Identified TRPV4 as a Regulator of Chondrogenic Differentiation
| Autor: | Satoshi Shiojiri, Riko Nishimura, Sumio Sugano, Katsuhiko Amano, Kosuke Tashiro, Satoru Kuhara, Shuji Muramatsu, Makoto Wakabayashi, Toshiyuki Yoneda, Toshinori Sugahara, Rika Ooishi, Takeshi Ohno, Akio Matsuda, Yutaka Suzuki |
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| Rok vydání: | 2007 |
| Předmět: |
Small interfering RNA
DNA Complementary Calmodulin Cell Culture Techniques TRPV Cation Channels Biology Biochemistry Cell Line Mice Chondrocytes Transcription (biology) Gene expression Animals SOX9 Transcription Factor Molecular Biology Transcription factor Gene Library Genome Activator (genetics) High Mobility Group Proteins Cell Biology Chondrogenesis Molecular biology embryonic structures biology.protein Transcription Factors |
| Zdroj: | Journal of Biological Chemistry. 282:32158-32167 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m706158200 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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