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Investigation of molecular mechanisms of Sox9 transcriptional factory during enchandral ossification

掌骨骨化过程中Sox9转录工厂的分子机制研究

基本信息

批准号：
17209059
负责人：
YONEDA Toshiyuki
金额：
$ 29.95万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17209059/
关键词：
chondrocyte transcriptional factory Sox9

项目摘要

Tb investigate the molecular mechanism by which Sox9 plays an essential role in chondrocyte differentiation, we established a gene screening system for identifying the members of Sox9 transcriptional factory. Using this system, we could identify a calcium ion channel, TRPV_4, as a member of Sox9 transcriptional factory, and found that TRPV4 upregulated expression and function of Sox9, thereby stimulating chondrocyte differentiation. We further screened cDNA library generated from chondrogenic cell line, ATDC5, and isolated the Znf219 cDNA. Whole mount in situ and RT-PCR analyses indicated that Znf219 is specifically expressed in limb bud of E12.5 embryo mice and chondrocytes. Co-immunoprecipitation experiments indicated that Znf219 physically interacts with Sox9. Moreover, we found that Znf219 co-localized with Sox9 in the nucleus. These results indicated that Znf219 forms transcriptional factory in the nucleus. To understand the functional role of Znf219 m chondrocyte differentiation, we next examined the effect of overexpression of Zof219 on chondrocyte differentiation. We found that overexpression of Znf219 stimulated the chondrogenic activity of Sox9. In contrast, overexpression of a dominant negative Znf219 markedly inhibited chondrocyte differentiation induced by Sox9. Furthermore, knockdown of Znf219 using micro RNA system suppressed chondrocyte differentiation. Collectively, our results demonstrated that Znf219 might play an important role in Sox9-regulated chondrocyte differentiation.

为了研究Sox9在软骨细胞分化中发挥重要作用的分子机制，我们建立了一个基因筛选系统来鉴定Sox9转录工厂的成员。利用该系统，我们可以识别钙离子通道TRPV_4作为Sox9转录工厂的成员，并发现TRPV4上调Sox9的表达和功能，从而刺激软骨细胞分化。我们进一步筛选了从软骨形成细胞系 ATDC5 生成的 cDNA 文库，并分离了 Znf219 cDNA。原位整体安装和RT-PCR分析表明Znf219在E12.5胚胎小鼠的肢芽和软骨细胞中特异性表达。免疫共沉淀实验表明 Znf219 与 Sox9 存在物理相互作用。此外，我们发现 Znf219 与 Sox9 在细胞核中共定位。这些结果表明Znf219在细胞核中形成转录工厂。为了了解 Znf219 m 软骨细胞分化的功能作用，我们接下来检查了 Zof219 过表达对软骨细胞分化的影响。我们发现 Znf219 的过度表达刺激了 Sox9 的软骨形成活性。相反，显性失活 Znf219 的过度表达显着抑制 Sox9 诱导的软骨细胞分化。此外，使用 micro RNA 系统敲低 Znf219 可抑制软骨细胞分化。总的来说，我们的结果表明 Znf219 可能在 Sox9 调节的软骨细胞分化中发挥重要作用。