Functional gene screening system identified TRPV4 as a regulator of chondrogenic differentiation
Functional gene screening system identified TRPV4 as a regulator of chondrogenic differentiation
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DOI：
10.1074/jbc.m706158200
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发表时间：
2007-11-02
影响因子：
4.8
通讯作者：
Matsuda, Akio
中科院分区：
文献类型：
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作者：
Muramatsu, Shuji;Wakabayashi, Makoto;Matsuda, Akio
Sox 9是软骨细胞分化和软骨细胞特异性基因表达所必需的转录因子。然而，软骨形成过程中Sox 9激活的确切机制尚未完全了解。为了研究这一机制，我们进行了功能基因筛选，以确定激活SOX 9依赖性转录的基因，使用全长cDNA文库产生的小鼠软骨细胞系，ATDC 5。筛选显示TRPV 4（（t）在bar受体下（r）在bar受体下（p）在bar电位下（v）在bar苯胺样蛋白4下），一种阳离子通道分子，显著提高了SOX 9依赖性报告子活性。微阵列和定量真实的时间PCR分析表明，在ATDC 5和C3 H10 T1/2（小鼠间充质干细胞系）的软骨形成过程中，TRPV 4的表达模式与软骨形成标记基因的表达模式相似，如II型胶原和聚集蛋白聚糖。通过药理学激活剂激活TRPV 4诱导SOX 9依赖性报告活性，并且通过加入TRPV拮抗剂钌红或通过使用TRPV 4的小干扰RNA来消除这种作用。由于TRPV 4激活的SOX 9依赖性报告活性被EGTA和钙调蛋白抑制剂消除，表明Ca 2 +/钙调蛋白信号在此过程中是必不可少的。此外，在ATDC 5细胞中TRPV 4的激活与胰岛素活性或在C3 H10 T1/2细胞中TRPV 4的激活与骨形态发生蛋白-2的激活一起促进硫酸化糖胺聚糖的合成，但TRPV 4的激活单独没有影响。我们发现TRPV 4的激活增加了SOX 9 mRNA和蛋白以及SOX 6 mRNA的稳态水平。总之，我们的结果表明TRPV 4调节SOX 9通路并有助于软骨形成过程。
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 ( (t) under bar ransient (r) under bar eceptor (p) under bar otential (v) under bar anilloid 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 ( 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.