The analysis of genome evolution in the MHC class III region and function of extracellular matrix tenascin-X

MHC III类区域基因组进化及细胞外基质tenascin-X功能分析

• 批准号: 10640603
• 负责人: MATSUMOTO Ken-ichi
• 金额: $ 2.11万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10640603/
• 关键词: extracellular matrix; tenascin family; VEGF-B; Sp1; two-hybrid

To identify proteins that interact with extracellular matrix tenascin-X (TNX) in the extracellular environment, we searched for TNX-binding proteins using a yeast two-hybrid system. We used mouse TNX-specific fibronectin type III repeats (mTNX/FNIIIィイD213-25ィエD2) as a bait. We found that vascular endothelial growth factor B (VEGF-B) binds to mTNX/FNIIIィイD213-25ィエD2. This interaction was confirmed by pull-down assays and coimmunoprecipitation assays. mTNX/FNIIIィイD213 -25ィエD2 specifically interacted with both altenative splice isoforms, VEGF-BィイD2186ィエD2 and VEGF-BィイD2167ィエD2, but not with other family members. Likewise, the full-length TNX could also bind to both VEGF-B isoforms. The minimal region of TNX that interacts with VEGF-B was mapped to the FNIII repeats (mTNX/FNIIIィイD213-25ィエD2) that were used for a bait but not to other characteristic domains of TNX. The TNX-binding site of VECJF-B was located in the N-terminal 115-amino-acids region. mTNX/FNIIIィイD213-25ィエD2 did not prevent t … More he interaction of VEGF-B with VEGFR-1 (VEGF receptor 1), and VEGF-B could simultaneously bind to both mTNX/FNIIIィイD213-25ィエD2 and VEGFR-1. A conditioned medium from transfected 293T cells coexpressing full-length TNX and VEGF-B can enhance DNA synthesis in ECV304 cells. These findings suggest that TNX is responsible for proliferation of the cells.To elucidate the molecular basis of the TNX gene expression, the promoter region of the mouse TNX gene (mTnx) has been characterized. The two adjacent transcription initiation sites were identified at 68 and 67 bp upstream of the previously known 5'- untranslated exon. Transient transfection of L and 293T cells with 5'-deletion constructs of the promoter region linked to the luciferase reporter revealed that the region (-141 to -136) containing a transcription factor Sp1-binding element contributes to the expression of mTnx. Site-directed mutagenesis of the Sp1-binding region confirmed this result. Electrophoretic mobility shift analysis using nuclear extracts obtained from the cells demonstrated that a distinct Sp1-DNA complex is formed at the element. Our results show that Sp1 plays a critical role in the gene expression of mTnx. Less

为了鉴定在细胞外环境中与细胞外基质腱生蛋白-X（TNX）相互作用的蛋白质，我们使用酵母双杂交系统搜索TNX结合蛋白。我们使用小鼠TNX特异性纤连蛋白III型重复序列（mTNX/FNIII重复序列D213-25重复序列D2）作为诱饵。我们发现血管内皮生长因子B（VEGF-B）与mTNX/FNIII受体D213-25受体D2结合。这种相互作用通过下拉试验和免疫共沉淀试验证实。mTNX/FNIII剪接体D213 - 25剪接体D2与VEGF-B剪接体D2186剪接体D2和VEGF-B剪接体D2167剪接体D2特异性相互作用，但不与其他家族成员相互作用。同样，全长TNX也可以结合两种VEGF-B亚型。与VEGF-B相互作用的TNX的最小区域被定位到用于诱饵的FNIII重复序列（mTNX/FNIII重复序列D213-25重复序列D2），但不定位到TNX的其它特征结构域。VECJF-B的TNF结合位点位于N端115个氨基酸的区域。mTNX/FNIII抗体D213-25和D2不能阻止细胞凋亡。 ...更多信息 VEGF-B与VEGFR-1（VEGF受体1）相互作用，VEGF-B可同时与mTNX/FNIII受体D213-25亚型D2和VEGFR-1结合。来自共表达全长TNX和VEGF-B的转染的293 T细胞的条件培养基可以增强ECV 304细胞中的DNA合成。为了阐明TNX基因表达的分子基础，对小鼠TNX基因启动子区（mTnx）进行了分析。两个相邻的转录起始位点被鉴定为位于先前已知的5 '-非翻译外显子上游的68和67 bp处。用与荧光素酶报告基因连接的启动子区的5 '缺失构建体瞬时转染L和293 T细胞，揭示了含有转录因子Sp1结合元件的区域（-141至-136）有助于mTnx的表达。Sp1结合区的定点突变证实了这一结果。电泳迁移率变化分析，使用从细胞中获得的核提取物表明，一个独特的Sp1-DNA复合物形成的元素。我们的研究结果表明，Sp1在mTnx的基因表达中起着关键作用。少

项目成果

Tomoki Ikuta, Norio Sogawa, Hiroyoshi Ariga, Toshimichi Ikemura, and Ken-ichi Matsumoto: "Structural analysis of mouse tenascin-X : Evolutionary aspects of reduplication of FNIII repeats in the tenascin gene family."Gene. 217. 1-13 (1998)

Tomoki Ikuta、Norio Sogawa、Hiroyoshi Ariga、Toshimichi Ikemura 和 Ken-ichi Matsumoto：“小鼠腱蛋白-X 的结构分析：腱蛋白基因家族中 FNIII 重复重复的进化方面。”基因。

Mitsuaki Fujimoto, Ken-ichi Matsumoto, Sanae, M. M. Iguchi-Ariga, Hiroyoshi Ariga: "Structures and comparison of genomic and complernentary DNAS of mouse MMSP, a c-Myc binding protein."Int. J. Onc.. 16. 245-251 (2000)

Mitsuaki Fujimoto、Ken-ichi Matsumoto、Sanae、M. M. Iguchi-Ariga、Hiroyoshi Ariga：“小鼠 MMSP（一种 c-Myc 结合蛋白）的基因组和互补 DNAS 的结构和比较。”Int。

松本健一: "細胞外マトリックス・テネイシンファミリー"道薬誌 北海道薬剤師会. 16. 4-11 (1999)

Kenichi Matsumoto：“细胞外基质腱蛋白家族”北海道制药杂志 16. 4-11 (1999)。

松本健一、小原政信: "間充織に存在する細胞外マトリックス"「細胞外マトリックス-基礎と臨床-」 林利彦、小出輝編 愛智出版「. 146-161 (2000)

Kenichi Matsumoto、Masanobu Ohara：“间充质中存在的细胞外基质”“细胞外基质 - 基础和临床”由 Toshihiko Hayashi 和 Teru Koide 爱知出版编辑“。146-161（2000）

松本健一、小原政信: "間充織に存在する細胞外マトリックス"「細胞外マトリックス-基礎と臨床-」林利彦、小出輝編 愛智出版. 146-161 (2000)

Kenichi Matsumoto、Masanobu Ohara：“间充质中存在的细胞外基质”“细胞外基质 - 基础和临床”由 Toshihiko Hayashi 和 Teru Koide Aichi Publishing 编辑 146-161 (2000)。