Use of alpha-, beta-Estrogen Receptor as a "new tool" for detection of specific small molecule activity.
Use of alpha-, beta-Estrogen Receptor as a "new tool" for detection of specific small molecule activity.
复制标题
使用α-、β-雌激素受体作为检测特定小分子活性的“新工具”。
DOI：
10.1007/s11105-015-0879-5
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发表时间：
2015
影响因子：
2.1
通讯作者：
Littleton,John
中科院分区：
文献类型：
--
作者：
Gunjan,SamirKumar;Rogers,DennisTrent;Zhang,Jingxian;Yun,Kil-Young;Falcone,DeaneL;Littleton,John
使用表达单个人核受体（NR）的转基因植物细胞的核受体配体的基于细胞的筛选方法可能比表达多个NR的其他真核系统具有优势。例如，信噪比可能得到改善，因为配体将不太可能与其他NR结合和/或不太可能在植物细胞中引起混淆的功能变化。作为实现这一目标的第一步，我们已经在植物中表达了截短的人雌激素受体（ER）结构连接到报告或选择性标记，如荧光素酶，绿色荧光蛋白（GFP），潮霉素。然后测试了ER的各种配体（包括雌二醇和已知的植物雌激素）过表达连接的标记基因的能力，所述标记基因可以被测量（荧光素酶活性）、在荧光显微镜下观察（GFP活性）或在含抗生素的培养基上选择（潮霉素B）。我们的研究结果表明，ER配体在转基因植物根部的作用和它们对哺乳动物细胞中天然ER的作用之间存在密切联系。随着ER α-GFP配体检测系统在拟南芥中的稳定表达，雌激素受体部分激动剂（他莫昔芬）和拮抗剂（氟维司群）在与乳腺癌相关的浓度下抑制了转基因根中雌二醇介导的反应。我们的结论是，有可能在植物中表达人NR的形式，可以报告外源性或内源性ER配体，这些结构具有药理学相关的天然NR在人类细胞中的配体。
Cell-based screening methods for nuclear receptor ligands that use transgenic plant cells expressing a single human nuclear receptor (NR) may have advantages over other eukaryotic systems which express multiple NRs. For example, signal-to-noise ratio might be improved because ligands would be less likely to bind to other NRs and/or less likely to cause confounding functional changes in plant cells. As a first step toward this aim, we have expressed in plants truncated human estrogen receptor (ER) constructs linked to reporters or selective markers such as luciferase, green fluorescent protein (GFP), and hygromycin. A variety of ligands for the ER (including estradiol and known phytoestrogens) have then been tested for their ability to overexpress the linked marker gene(s) which could be measured (luciferase activity), visualized under fluorescent microscopy (GFP activity), or selected on antibiotic-containing media (hygromycin B). Our results show a close association between the effects of ER ligands in the transgenic plant roots and their effects on native ERs in mammalian cells. With the stable expression of an ERalpha-GFP ligand detection system inArabidopsis thaliana, the estradiol-mediated response in transgenic roots is inhibited by an ER partial agonist (tamoxifen) and an antagonist (fulvestrant) at concentrations relevant to their use in breast cancer. We conclude that it is possible to express human NRs in plants in a form that can report on exogenous or endogenous ER ligands and that these constructs have a pharmacology which is relevant to ligands for the native NRs in human cells.