Abbott Derek A, Mancini Maureen G, Bolt Michael J, Szafran Adam T, Neugebauer Kaley A, Stossi Fabio, Gorelick Daniel A, Mancini Michael A
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA.
GCC Center for Advanced Microscopy and Image Informatics, Houston, TX, USA.
Heliyon. 2023 Dec 8;10(1):e23119. doi: 10.1016/j.heliyon.2023.e23119. eCollection 2024 Jan 15.
In this study we present an inducible biosensor model for the Estrogen Receptor Beta (ERβ), GFP-ERβ:PRL-HeLa, a single-cell-based high throughput (HT) assay that allows direct visualization and measurement of GFP-tagged ERβ binding to ER-specific DNA response elements (EREs), ERβ-induced chromatin remodeling, and monitor transcriptional alterations via mRNA fluorescence in situ hybridization for a prolactin (PRL)-dsRED2 reporter gene. The model was used to accurately (Z' = 0.58-0.8) differentiate ERβ-selective ligands from ERα ligands when treated with a panel of selective agonists and antagonists. Next, we tested an Environmental Protection Agency (EPA)-provided set of 45 estrogenic reference chemicals with known ERα activity and identified several that activated ERβ as well, with varying sensitivity, including a subset that is completely novel. We then used an orthogonal ERE-containing transgenic zebrafish (ZF) model to cross validate ERβ and ERα selective activities at the organism level. Using this environmentally relevant ZF assay, some compounds were confirmed to have ERβ activity, validating the GFP-ERβ:PRL-HeLa assay as a screening tool for potential ERβ active endocrine disruptors (EDCs). These data demonstrate the value of sensitive multiplex mechanistic data gathered by the GFP-ERβ:PRL-HeLa assay coupled with an orthogonal zebrafish model to rapidly identify environmentally relevant ERβ EDCs and improve upon currently available screening tools for this understudied nuclear receptor.
在本研究中，我们提出了一种用于雌激素受体β（ERβ）的诱导型生物传感器模型，即GFP-ERβ:PRL-HeLa，这是一种基于单细胞的高通量（HT）检测方法，可直接可视化并测量绿色荧光蛋白（GFP）标记的ERβ与ER特异性DNA反应元件（ERE）的结合、ERβ诱导的染色质重塑，并通过对催乳素（PRL）-dsRED2报告基因进行mRNA荧光原位杂交来监测转录变化。当用一组选择性激动剂和拮抗剂处理时，该模型能够准确地（Z' = 0.58 - 0.8）区分ERβ选择性配体和ERα配体。接下来，我们测试了美国环境保护局（EPA）提供的一组45种已知具有ERα活性的雌激素参考化学物质，并鉴定出了几种也能激活ERβ的物质，其敏感性各不相同，包括一部分全新的物质。然后，我们使用了一种含正交ERE的转基因斑马鱼（ZF）模型，在生物体水平上交叉验证ERβ和ERα的选择性活性。通过这种与环境相关的ZF检测方法，可以确认一些化合物具有ERβ活性，从而验证了GFP-ERβ:PRL-HeLa检测方法可作为筛选潜在ERβ活性内分泌干扰物（EDC）的工具。这些数据证明了通过GFP-ERβ:PRL-HeLa检测方法结合正交斑马鱼模型收集的敏感多重机制数据的价值，该方法能够快速识别与环境相关的ERβ EDC，并改进目前针对这种研究不足的核受体的可用筛选工具。
