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Expression Cloning of Joint Regenerative Genes

关节再生基因的表达克隆

基本信息

批准号：
17591549
负责人：
MATSUBARA Takehiro
金额：
$ 2.18万
依托单位：
Saitama Medical University (2006)The University of Tokyo (2005)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591549/
关键词：
Cartilage Metabolism Joint Development Transcription Factor 転写制御 WNT9A GDF5 HMG ATX

项目摘要

To understand molecular mechanism of joint development, transcriptional regulation of three joint marker genes, GDF5, WNT9A and autototaxin were analyzed by luciferase-reporter assay. We identified a highly conserved 42 by core enhancer in the proximal promoter of WNT9A and named it JSE (joint specific enhancer). To identify common regulatory signal of cartilage development including articular cartilage, we searched the promoter region of SOX6, a key transcription factor for chondrogenesis. By combining 5'RACE, comparative genomics and luciferase-reporter assay, we identified the human embryonic SOX6 promoter and a 46 by core enhancer highly conserved among species and named it CES6 (core enhancer of SOX6). To identify regulatory transcription factors of cartilage and joint development, we performed southwestern screening by combining phage display libraries derived from cartilage tissues and mouse embryos and bait DNA sequences of 4x JSE and 4x CES6, and identified two transcription f … More actors HMGB2 and p63 that transactivated both WNT9A and SOX6 promoter. Further analysis revealed that p63 directly bound proximal promoters of GDF5, SOX6 and WNT9A and activated them. The binding sequences of p63 in these three genes were 1-3 by different from the known p53 family binding motif "RRRCWWGYYY". We speculated that p63 would recognize looser motifs in wide varieties of genes and regulate successive steps of chondrogenesis including joint formation. On the other hand, retroviral overexpression of p63 in mouse teratocarcinoma derived chondrogenic cell line ATDC5 induced Sox6 and Col2a1 mRNA strongly and rarely induced Gdf5 and Wnt9a. Joint specific transcription factors should be needed for optimal induction of joint marker genes. We established the technique to transfer adenoviral vectors in ex-vivo organn cultured developmental limbs derived from mouse embryos. WNT9A and p63 overexpressions by adenoviral vectors in developmental mouse limbs failed to induce joint space formations, however. To establish a more specific and physiological ex-vivo joint formation system, the information on more joint specific transcription factors for joint formations will be needed. We believe that applying results of this study for further investigations will reveal sufficient signals for joint formations. Less

为了解关节发育的分子机制，应用荧光素酶报告基因分析了GDF5、WNT9A和自体趋化蛋白3个联合标记基因的转录调控。我们在WNT9A的启动子近端发现了一个高度保守的42位核心增强子，命名为JSE(联合特异性增强子)。为了确定包括关节软骨在内的软骨发育的共同调控信号，我们搜索了软骨形成的关键转录因子SOX6的启动子区域。通过5‘RACE、比较基因组学和荧光素酶报告基因分析，我们鉴定了人胚胎SOX6启动子和一个物种间高度保守的46位核心增强子，命名为CES6(SOX6的核心增强子)。为了鉴定软骨和关节发育的调控转录因子，我们结合来自软骨组织和小鼠胚胎的噬菌体展示文库和4xJSE和4xCES6的诱饵dna序列进行了西南筛选，鉴定了两个…转录因子。更多的HMGB2和P63同时反式激活了WNT9A和SOX6启动子。进一步分析表明，p63直接与GDF5、SOX6和WNT9A的近端启动子结合并激活它们。与已知的P53家族结合基序“RRRCWWGYYY”不同，这三个基因中p63的结合序列均为1-3。我们推测，p63将识别多种基因中较为松散的基序，并调控包括关节形成在内的软骨形成的连续步骤。反转录病毒p63在小鼠畸胎瘤软骨细胞系ATDC5中的过表达可强烈诱导SOX6和COL2a1基因的表达，而对Gdf5和WNT9a的诱导作用较弱。联合标记基因的最佳诱导需要联合特异性转录因子。我们建立了在体外器官培养的小鼠胚胎发育肢体中转移腺病毒载体的技术。然而，腺病毒载体在发育中的小鼠肢体中过表达WNT9A和P63并不能诱导关节间隙的形成。为了建立更具特异性和生理性的体外关节形成系统，需要更多关节特异性转录因子的信息。我们相信，将这项研究的结果应用于进一步的研究，将为联合构造揭示足够的信号。较少

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

骨・軟骨の再生医療.

骨和软骨再生医学。

DOI：
发表时间：
2005
期刊：
「老年医学update 2005-2006」(編集:日本老年医学会雑誌編集委員会)
影响因子：
0
作者：
緒方直史;川口浩
通讯作者：
川口浩

S100A1 and B, transcriptional targets of SOX trio, inhibit terminal differentiation of chondrocvtes

S100A1 和 B，SOX 三重奏的转录靶标，抑制软骨细胞的终末分化

DOI：
发表时间：
2007
期刊：
EMBO Rep Epub
影响因子：
0
作者：
Kashimura H.;Kashimura H.;Hiroshi Kashimura;Hiroshi Kashimura;Wada T.;Wada T.;Yoshitaka Kubo;Wada T.;Inoue T.;Komoribayasi N.;Wada T.;Inoue T.;Komoribayasi N.;Komoribayasi N.;Misaki T.;Beppu T.;Saito T.;Ikeda T.;Saito T et al.;Saito T.
通讯作者：
Saito T.

SOX

萨班斯

DOI：
发表时间：
2006
期刊：
骨粗鬆症治療 5 (1)
影响因子：
0
作者：
Kashimura H.;Kashimura H.;Hiroshi Kashimura;Hiroshi Kashimura;Wada T.;Wada T.;Yoshitaka Kubo;Wada T.;Inoue T.;Komoribayasi N.;Wada T.;Inoue T.;Komoribayasi N.;Komoribayasi N.;Misaki T.;Beppu T.;Saito T.;Ikeda T.;Saito T et al.;Saito T.;Ikeda T.;斎藤琢
通讯作者：
斎藤琢

S100A1 and B, transcriptional targets of SOX trio, inhibit terminal differentiation of chondrocytes

SOX trio 的转录靶标 S100A1 和 B 抑制软骨细胞的终末分化

DOI：
发表时间：
2007
期刊：
EMBO Rep (Epub)
影响因子：
0
作者：
Kashimura H.;Kashimura H.;Hiroshi Kashimura;Hiroshi Kashimura;Wada T.;Wada T.;Yoshitaka Kubo;Wada T.;Inoue T.;Komoribayasi N.;Wada T.;Inoue T.;Komoribayasi N.;Komoribayasi N.;Misaki T.;Beppu T.;Saito T.
通讯作者：
Saito T.

Identification and characterization of the human SOX6 promoter