Investigation of the regulatory mechanism of gene expression of human ecogenin/CTGF, a chondrocyte-derived growth factor.

软骨细胞源性生长因子人 Ecogenin/CTGF 基因表达调控机制的研究。

• 批准号: 11671841
• 负责人: OHYAMA Kazumi
• 金额: $ 2.3万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671841/
• 关键词: CTGF; chondrocyte; gene expression; untranslated region; cis-element

1) Research on transcriptional control of ecogenin/CTGF gene expression : We constructed a series of chimeric reporter gene constructs, in which the human CTGF promoter and its deletion mutants were linked upstream of firefly luciferase genes. Using these constructs, we comparatively analyzed their promoter activities via transient expression assay in chondrocytic HCS-2/8 cells. Then, we found a 110-bp DNA segment located at 88 bp-upstream of the transcription initiation site as a critical determinant of the enhanced CTGF gene expression in HCS-2/8 cells. Moreover, we found two enhancer elements that were active in HCS-2/8 cells. One of them was a known TGF-β response element, whereas the other was a novel one discovered in this study. Mutation of either element resulted in drastic decrease of the promoter activity in HCS-2/8 cells. It is especially interesting that binding counterpart(s) of the latter latter element was found to be present specifically in HCS-2/8 cells.2) Research on … More post-transcriptional control of ecogenin/CTGF gene expression : We uncovered the strong repressive effect of the 1 kb-long 3'-untranslated region (UTR) of the ecogenin/CTGF gene on gene expression by comparatively evaluating the luciferase gene expression with or without the cis-linked 3'-UTR. Furthermore, we could identify an 84 base repressive cis-element by deletion analysis based on computer-associated structural prediction. Since this RNA element formed a stable secondary structure in solution, and the repressive function was highly dependent on the secondary structure forming potential, we entitled this element "cis-acting element of structure-anchored repression (CAESAR). Also recently, multiple stem-loop structure has been observed to be the structural determinant of CAESAR function. CAESAR did not display any effect outside of the transcribed region, and it did not affect the intracellular distribution of mRNA linked in cis. Therefore, CAESAR is thought to act at a step of mRNA translation without affecting the nuclear export of mRNA. Less

1) ecogenin/CTGF基因表达的转录调控研究：构建了一系列嵌合报告基因构建体，将人类CTGF启动子及其缺失突变体连接在萤火虫荧光素酶基因上游。利用这些构建体，我们通过在软骨细胞HCS-2/8细胞中的瞬时表达实验比较分析了它们的启动子活性。然后，我们发现位于转录起始位点上游88 bp处的110 bp DNA片段是HCS-2/8细胞中CTGF基因表达增强的关键决定因素。此外，我们发现两个增强子元件在HCS-2/8细胞中有活性。其中一个是已知的TGF-β反应元件，而另一个是本研究中发现的新元件。在HCS-2/8细胞中，任何一种元素的突变都导致启动子活性急剧降低。特别有趣的是，后者的结合对偶物被发现特异性地存在于HCS-2/8细胞中。2)对ecogenin/CTGF基因表达的更多转录后调控研究：通过比较有无3′-UTR的荧光素酶基因表达，我们发现了ecogenin/CTGF基因1 kb-long 3′- untranslation region （UTR）对基因表达的强烈抑制作用。此外，我们可以通过基于计算机相关结构预测的缺失分析识别出84碱基抑制性顺式元件。由于该RNA元件在溶液中形成稳定的二级结构，且抑制功能高度依赖于二级结构形成电位，因此我们将其命名为“顺式作用结构锚定抑制元件（CAESAR）”。最近，多茎环结构也被观察到是CAESAR函数的结构决定因素。CAESAR在转录区之外没有表现出任何作用，也不影响顺式连接的mRNA在细胞内的分布。因此，CAESAR被认为在mRNA翻译的一个步骤中起作用，而不影响mRNA的核输出。少

项目成果

Satoshi Kubota et al.: "Novel intracellular effects of human connective tissue growth factor expressed in Cos-7 cells"FEBS Letters. 474. 58-62 (2000)

Satoshi Kubota 等人：“Cos-7 细胞中表达的人结缔组织生长因子的新颖细胞内效应”FEBS Letters。

Kondo, S., et al.: "Characterization of a mouse ctgf 3'-UTR segment that mediates repressive regulation of gene expression."Biochem. Biophys. Res. Commun.. 278. 119-124 (2000)

Kondo, S. 等人：“介导基因表达抑制性调节的小鼠 ctgf 3-UTR 片段的表征。”Biochem。

Takanori Eguchi et al.: "Regulatory mechanism of human connective tissue growth factor (CTGF/Hcs24) gene expression in a human chondrocytic cell line, HCS-2/8"Journal of Biochemistry. 130. 79-87 (2001)

Takanori Eguchi 等人：“人软骨细胞系 HCS-2/8 中人结缔组织生长因子 (CTGF/Hcs24) 基因表达的调节机制”生物化学杂志。

Tsuyoshi Shimo et al.: "Connective tissue growth factor as a major angiogenic agent that is induced by hypoxia in a human breast cancer cell line"Cancer Letters. 174. 57-64 (2001)

Tsuyoshi Shimo 等人：“结缔组织生长因子是人乳腺癌细胞系缺氧诱导的主要血管生成剂”《癌症快报》。

Seiji Kondo et al.: "Characterization of a mouse ctgf3'-UTR segment that mediates repressive regulation of gene expression"Biochemical and Biophysical Research Coommunucations. 278. 119-124 (2000)

Seiji Kondo 等人：“介导基因表达抑制性调节的小鼠 ctgf3-UTR 片段的表征”生物化学和生物物理研究通讯。