Transcriptional regulation of arylhydrocarbon receptor (AhR), Arnt and E2F genes on proliferation process in A549 cells by dioxin and its mechanism.

二恶英对芳烃受体(AhR)、Arnt和E2F基因转录调控A549细胞增殖过程及其机制

• 批准号: 17590109
• 负责人: TEZUKA Masakatsu
• 金额: $ 2.24万
• 依托单位: Nihon University
• 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-17590109/
• 关键词: dioxin; AhR; Arnt; E2F; transcription factor; A549 cells; 薬学; 環境; 癌; 遺伝子

Arylhydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a spectrum of toxicological and bio-logical effects of 2,3,7,8-tetrachlorodiben.zo-p-dioxine (TCDD) and related compounds. AhR is included in a family of basic-helix-loop-helix/PAS domain-containing transcription factors. AhR expression is associated with the development of human lung carcinoma. In fact, we found that the overexpression of AhR accelerated the cell proliferation of A549 cells, established from a human alveolar cell carcinoma. We also demonstrated that the increased cell proliferations in A549 cells were associated with the activation of E2F gene expression, a transcription factor related in carcinogenesis, by the expression of heterodimer complex of AhR and AhR nuclear translocator (Arnt). In this study, we investigated the mechanisms of relationship of E2F gene activation and expression of AhR/Arnt complex.We obtained the deleted cell lines of AhR, Arnt and E2F genes expression by s … More iRNA transfection technique in A549 cells. E2F-dependent transcription activity was decreased by the suppression of AhR, Arnt and E2F expression. To identify the domains of AhR and Arnt genes responsible for E2F-dependent transcription activity, a series of deletion constructs linked the luciferase activity were transfected into A549 cells. The PAS regions of AhR and Arnt sequences were required for the activation of E2F transcription. Next, an effect of E2F expression to the transcription activity of AhR and Arnt was determined by using of reporter assay contained XRE sequences, as AhR/Arnt-binding regions. The AhR/Arnt complex-dependent transcription activity on XRE sequences was increased by the suppression of E2F expression. The Rb protein-binding regions in E2F sequences were required on the interaction of AhR/Arnt complex. Rb protein is a repressive factor of carcinogenesis. Furthermore, we demonstrated that AhR/Arnt complex was complexed with E2F protein using a immuno-precipitation assay. These results suggested that AhR/Arnt and E2F complex binding on E2F-binding sequences was accelerated the cell proliferation, while the complex binding on XRE sequences was affected as a repressor in gene transcription. Less

芳烃受体（Arylhydrocarbon receptor, AhR）是一种配体激活的转录因子，介导2,3,7,8-四氯二苯的一系列毒理学和生物学效应。邻对二恶英（TCDD）及其相关化合物。AhR是一个包含碱基-螺旋-环-螺旋/PAS结构域的转录因子家族。AhR的表达与人肺癌的发生发展有关。事实上，我们发现AhR的过表达加速了从人肺泡细胞癌中建立的A549细胞的增殖。我们还通过表达AhR和AhR核转运子（Arnt）的异源二聚体复合体，证明了A549细胞增殖的增加与致癌相关的转录因子E2F基因表达的激活有关。本研究探讨了E2F基因激活与AhR/Arnt复合物表达的关系机制。我们在A549细胞中采用s. More iRNA转染技术获得了AhR、Arnt和E2F基因表达缺失的细胞株。通过抑制AhR、Arnt和E2F的表达，E2F依赖性转录活性降低。为了鉴定负责e2f依赖性转录活性的AhR和Arnt基因结构域，将一系列与荧光素酶活性相关的缺失构建体转染到A549细胞中。AhR和Arnt序列的PAS区是激活E2F转录所必需的。接下来，利用含有XRE序列作为AhR/Arnt结合区域的报告基因实验，确定E2F表达对AhR和Arnt转录活性的影响。抑制E2F的表达增加了XRE序列上AhR/Arnt复合物依赖的转录活性。E2F序列中的Rb蛋白结合区是AhR/Arnt复合物相互作用所必需的。Rb蛋白是一种抑制癌变的因子。此外，我们通过免疫沉淀实验证明AhR/Arnt复合物与E2F蛋白络合。这些结果表明，AhR/Arnt和E2F复合物结合在E2F结合序列上加速了细胞的增殖，而XRE序列上的复合物结合作为基因转录的抑制因子受到影响。少