Development of an in vitro Reporter Gene Assay for Screening the Ecdysone Receptor Agonists in Mysid Crustacean

One of steroid hormones, ecdysteroid is a key regulatory factor, controlling the development and molting in arthropod. Molecular target of ecdysteroids is a receptor complex, composed of the ecdysone receptor (EcR) and ultraspiracle (USP). To characterize the transactivation potentials of crustacean EcR by environmental pollutants, we established an in vitro reporter gene assay using insect cell line transiently transfected with mysid EcR and USP cDNA expression vectors and a reporter vector containing the synthesized ecdysone response element (EcRE). Comparison of amino acid residues in the ligand-binding domain of mysid EcR with those of other arthropod EcRs showed less similarities (36–45%), implying a functional divergence of the EcR across species. The in vitro reporter gene assay showed that treatments of insect cell line with an arthropod molting hormone, 20-hydroxyecdysone, and a plant ecdysteroid, ponasterone A, induced EcR-mediated transcriptional activities in a dose-dependent manner. These results suggest that this in vitro reporter gene assay with the mysid EcR/USP complex can be a useful tool to screen candidate compounds that may potentially affect the crustacean EcR signaling cascade.