Regular Article
New Screening Methods for Chemicals with Hormonal Activities Using Interaction of Nuclear Hormone Receptor with Coactivator

https://doi.org/10.1006/taap.1998.8557Get rights and content

Abstract

The endocrine system exerts important functions in a multitude of physiological processes including embryogenesis, differentiation, and homeostasis. Xenobiotics may modify natural endocrine function and so affect human health and wildlife. It is necessary, therefore, to understand the degree to which xenobiotics can disrupt endocrine systems. The key targets of endocrine disruptors are nuclear hormone receptors, which bind to steroid hormones and regulate their gene transcription. We have developed relevant assay systems based on the ligand-dependent interaction between nuclear hormone receptor and coactivator. The coactivators used in this study contained CBP, p300, RIP140, SRC1, TIF1, and TIF2. By two hybrid assay in yeast, the interactions of estrogen receptor with RIP140, SRC1, TIF1, and TIF2 were detected and they were completely dependent on the presence of estrogen. Specificity of this assay was assessed by determining the effect of steroids, known estrogen receptor agonists, and phytoestrogens. The pattern of response to chemicals were consistent with estrogenic activity measured by other assay systems, indicating that this assay system is reliable for measuring estrogenic activity. In addition, we carried outin vitrobinding studies: GST pull-down assay and surface plasmon resonance analysis. The estrogen receptor also bound to coactivator in response to chemicals depending on their estrogenic activityin vitro.These data demonstrate that the measurement of interaction between steroid hormone receptor and coactivator serves as a useful tool for identifying chemicals that interact with steroid receptors.

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      Many assay systems characterized by cell type, such as ER-positive human breast cancer cells (MCF-7 and T-47D cells), ER-negative/GPER-positive human breast cancer cells (SKBR3 cells), human endometrial cancer cells (Ishikawa cells) or cells from other tissues (brain, bone and muscle cells) and species (yeast cells), or by reporter genes, such as those encoding Xenopus vitellogenin A2, firefly luciferase or green fluorescent protein, were developed to increase the sensitivity, specificity and reproducibility. Yeast two-hybrid assays quantitate the ligand-dependent interaction between the receptor and the transcriptional activator, where artificial (GAL4-based assay) and more natural (whole hERα-based assay) systems [148,149] have been developed. Transcription assays quantitate transcription of ER or marker genes and include DNA microarray assay, Northern blotting, RNA-seq and reverse transcription-polymerase chain reaction (RT-PCR) (comprehensively summarized in Kiyama and Zhu [135]).

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