A genetically encoded indicator for assaying bioactive chemicals that induce nuclear transport of glucocorticoid receptor.
Glucocorticoids, the adrenal steroid hormones secreted during stress, are essential to homeostasis and metabolism in the human body. An impaired glucocorticoid signaling due to dysfunction of the glucocorticoid receptor (GR) by synthetic chemicals can cause diseases and disruptions of the homeostasis and metabolism. Here we demonstrate the development of a method for screening endocrine-disrupting chemicals and potent risk factors of human diseases based on the nuclear trafficking of the GR. We constructed a new assay using a pair of genetic indicators with the full length of the GR, split Renilla luciferase (RLuc), and split DnaE (a protein splicing element). The GR-containing fusion protein with C-terminal halves of DnaE and RLuc is localized in cytosol due to the cytosolic character of the GR, whereas the fusion protein with N-terminal halves of DnaE and RLuc stays in the nucleus due to the cofused nucleus localization signal. On being stimulated with a ligand, the GR is translocated into the cellular nucleus. Thus, a protein splicing occurs in the nucleus by an interaction between the splicing junctions of each DnaE fragment. The enzymatic activities from the reconstituted RLuc allow the ligand-dependent luminescence intensities. The feasibility of the method was evaluated by quantifying the hormonal activities of 20 different kinds of steroids and synthetic chemicals using the NIH 3T3 cells carrying the pair of indicators. The hormonal activities of tested ligands are discussed based on the chemical structure-activity relationship. We found that androgens, testosterone, and 19-nortestosterone weakly induce the nuclear transport of the GR. The current assay allows high-throughput screening of risk chemicals and drug candidates influential to a signal transduction pathway of the GR.
Kim
Sung Bae
SB
Department of Chemistry, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-0033, Japan.
Ozawa
Takeaki
T
Umezawa
Yoshio
Y
D021581:Active Transport, Cell Nucleus / Q000187:drug effects*; D000818:Animals; D002623:Chemistry Techniques, Analytical; D052244:Endocrine Disruptors / Q000494:pharmacology; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D049410:Luciferases, Renilla / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000235:genetics; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013256:Steroids / Q000494:pharmacology
D052244:Endocrine Disruptors; D007202:Indicators and Reagents; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D013256:Steroids; D049410:Luciferases, Renilla