Weiss C, Kolluri S K, Kiefer F, Göttlicher M
Research Center Karlsruhe, Institute of Genetics, Karlsruhe, D-76021, Germany.
Exp Cell Res. 1996 Jul 10;226(1):154-63. doi: 10.1006/excr.1996.0214.
The Ah receptor (AhR) is a ligand-dependent transcription factor subunit that heterodimerizes with the AhR nuclear translocator (Arnt) and mediates the predominant biological effects of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD activates target genes in xenobiotica metabolism in many cell lines and, more specifically, delays G1-S progression of 5L hepatoma cells. Here we describe transient and stable AhR-expression analysis in AhR-deficient subclones of the TCDD-sensitive 5L cells. We tested the integrity of the AhR-signaling system beyond the lack of the receptor in the variant subclone and analyzed the role of AhR in cell cycle regulation. Transiently expressed AhR has a high basal activity on promoters containing AhR-binding sites, so-called XREs, when transfected into receptor-deficient variant cells compared to wild-type cells. Single- and double-hybrid analysis dissociates AhR ligand responsiveness, transactivation, and heterodimerization with Arnt from receptor binding to an XRE. Hybrid receptors also show the high basal activity in the absence of exogenous TCDD in AhR-deficient variant cells, indicating that the endogenous AhR-activating signal acts directly on the receptor rather than XRE-dependent promoters or DNA binding of the receptor. Stable expression of AhR in variant cell clones by retroviral infection fully reconstitutes TCDD responsiveness, including target-gene induction and delay of cell cycle progression. These AhR-reconstituted cells, like AhR-containing wild-type cells, show low basal activity of the transiently expressed AhR hybrid. Thus, the increased basal activity in AhR-deficient cells suggests a negative feedback control of AhR activity. In vitro ligand-binding assays are compatible with the idea that the increased basal activity is due to the accumulation of an AhR-binding endogenous ligand. In conclusion, AhR is causally responsible for TCDD-dependent cell cycle regulation and feedback control of AhR activity.
芳烃受体（AhR）是一种依赖配体的转录因子亚基，它与芳烃受体核转运蛋白（Arnt）形成异二聚体，并介导2,3,7,8-四氯二苯并对二恶英（TCDD）的主要生物学效应。TCDD可激活许多细胞系中异生物质代谢的靶基因，更具体地说，可延迟5L肝癌细胞从G1期到S期的进程。在此，我们描述了在对TCDD敏感的5L细胞的AhR缺陷亚克隆中进行的瞬时和稳定AhR表达分析。我们测试了变异亚克隆中除缺乏受体外AhR信号系统的完整性，并分析了AhR在细胞周期调控中的作用。与野生型细胞相比，当瞬时表达的AhR转染到受体缺陷的变异细胞中时，其对含有AhR结合位点（即所谓的XREs）的启动子具有较高的基础活性。单杂交和双杂交分析将AhR的配体反应性、反式激活以及与Arnt的异二聚化与受体与XRE的结合区分开来。在AhR缺陷的变异细胞中，杂交受体在没有外源性TCDD的情况下也显示出较高的基础活性，这表明内源性AhR激活信号直接作用于受体，而不是依赖于XRE的启动子或受体的DNA结合。通过逆转录病毒感染在变异细胞克隆中稳定表达AhR可完全恢复TCDD反应性，包括靶基因诱导和细胞周期进程的延迟。这些AhR重组细胞，与含有AhR的野生型细胞一样，瞬时表达的AhR杂交体显示出较低的基础活性。因此，AhR缺陷细胞中基础活性的增加表明存在对AhR活性的负反馈控制。体外配体结合试验与以下观点一致，即基础活性的增加是由于AhR结合内源性配体的积累。总之，AhR是TCDD依赖性细胞周期调控和AhR活性反馈控制的原因。
