Identification of ligands and coligands for the ecdysone-regulated gene switch

Abstract

The ecdysone-inducible gene switch is a useful tool for modulating gene expression in mammalian cells and transgenic animals. We have identified inducers derived from plants as well as certain classes of insecticides that increase the versatility of this gene regulation system. Phytoecdysteroids share the favorable kinetics of steroids, but are inert in mammals. The gene regulation properties of one of these ecdysteroids have been examined in cell culture and in newly developed strains of ecdysone-system transgenic mice. Ponasterone A is a potent regulator of gene expression in cells and transgenic animals, enabling reporter genes to be turned on and off rapidly. A number of nonsteroidal insecticides have been identified that also activate the ecdysone system. Because the gene-controlling properties of the ecdysone switch are based on a heterodimer composed of a modified ecdysone receptor (VgEcR) and the retinoid X receptor (RXR), we have tested the effect of RXR ligands on the VgEcR/RXR complex. Used alone, RXR ligands display no activity on the ecdysone switch. However, when used in combination with a VgEcR ligand, RXR ligands dramatically enhance the absolute levels of induction. This property of the heterodimer has allowed the development of superinducer combinations that increase the dynamic range of the system.

Figure 1

Identification of phytoecdysteroid ecdysone-system inducers. (A) Dose–response curve for several ecdysteroids. (B) Potency comparison between murA and 14-deoxymuristerone A. (C) Chemical structure of some ecdysteroids tested. MurA and ponA are good inducers, whereas 20-hydroxyecdysone and inokosterone are very poor activators.

Figure 2

In vitro characterization of ponA. (A) Comparison of the potency and kinetics of induction of ponA and murA. (B) Kinetics of induction and shutoff of the ecdysone system with 1 μM ponA as the inducer.

Figure 3

In vivo characterization of ponA. (A) Schematic of the transgenic approach used to test the ability of ponA to regulate gene expression in animals. Transactivator (K5-VgEcR/RXR) and reporter (E/GRE₄-ΔMTV-luciferase) mice were bred, and samples of blood, dorsal skin, and tail were obtained from bigenic offspring before and at various times after inducer administration. (B) Comparison of ponA and murA in vivo. Luciferase levels in skin and tail biopsies from bigenic animals injected with ponA or murA were measured and normalized to protein content. Shown is the analysis of tail samples from mice injected with 5 mg of inducer. (C) Reproducibility of ponA-inducible gene regulation: pattern of gene induction in tail samples from two different mice injected with 5 mg of ponA. (D) Inductions generated by 3 or 5 mg of ponA. Tail samples are shown.

Figure 4

Nonsteroidal ecdysone-system inducers. (A) Dose–response curve with selected nonsteroidal agonists. (B) Structure of RH 5992 (Tebufenozide), showing the basic features of this family of ecdysone receptor agonists.

Figure 5

Effect of RXR ligands on the transcriptional activity VgEcR/RXR complex. (A) Dose–response for ponA, an RXR ligand, or ponA plus a constant concentration of RXR ligand (100 nM). Increasing concentrations of the RXR ligand LG268 had no effect on the VgEcR/RXR heterodimer. Combination of 100 nM LG268 with increasing amounts of ponA resulted in considerably greater inductions than when ponA was used alone. (B) Combined effect of ecdysteroids and RXR ligands on VgEcR/RXR activation. Cells were treated with 10 μM murA, ponA, or inokosterone, alone or in combination with one of three RXR ligands present at 100 nM concentration. (C) LG268 potentiates the inducing properties of nonsteroidal VgEcR agonists. Cells were treated with 10 μM nonsteroidal ecdysone receptor ligand, alone or in combination with LG268 at 100 nM concentration. These mixtures induced higher levels of reporter activity than 10 μM murA.