New screening methods for chemicals with hormonal activities using interaction of nuclear hormone receptor with coactivator.
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 out in vitro binding 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 activity in 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.
Nishikawa
J
J
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka, 565-0871, Japan. nishihara@phs.osaka-u.ac.jp
Saito
K
K
Goto
J
J
Dakeyama
F
F
Matsuo
M
M
Nishihara
T
T
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry*; D004268:DNA-Binding Proteins; D004958:Estradiol / Q000494:pharmacology; D004968:Estrogens, Non-Steroidal / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D005656:Fungal Proteins / Q000235:genetics; D051548:Histone Acetyltransferases; D006728:Hormones / Q000819:agonists*; D006801:Humans; D007529:Isoflavones; D008297:Male; D009687:Nuclear Proteins / Q000378:metabolism; D056920:Nuclear Receptor Coactivator 1; D051958:Nuclear Receptor Coactivator 2; D000078723:Nuclear Receptor Interacting Protein 1; D048789:Phytoestrogens; D028321:Plant Preparations; D051381:Rats; D011960:Receptors, Estrogen / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011987:Receptors, Steroid / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D015262:Xenobiotics / Q000378:metabolism / Q000494:pharmacology*
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; D004968:Estrogens, Non-Steroidal; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D006728:Hormones; D007529:Isoflavones; C498001:NCOA2 protein, human; C000627127:NRIP1 protein, human; C498003:Ncoa2 protein, rat; D009687:Nuclear Proteins; D051958:Nuclear Receptor Coactivator 2; D000078723:Nuclear Receptor Interacting Protein 1; D048789:Phytoestrogens; D028321:Plant Preparations; D011960:Receptors, Estrogen; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D015262:Xenobiotics; C093517:transcriptional intermediary factor 1; D004958:Estradiol; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1