Rapid Assessment of Insect Steroid Hormone Entry Into Cultured Cells.

Mitchell Masterson,Riyan Bittar,Sachiko Haga-Yamanaka,Naoki Yamanaka,Hannah Chu

doi:10.3389/fphys.2021.816058

Mitchell Masterson, Riyan Bittar + Show 3 more

Open Access

https://doi.org/10.3389/fphys.2021.816058

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Abstract

Steroid hormones control development and homeostasis in a wide variety of animals by interacting with intracellular nuclear receptors. Recent discoveries in the fruit fly Drosophila melanogaster revealed that insect steroid hormones or ecdysteroids are incorporated into cells through a membrane transporter named Ecdysone Importer (EcI), which may become a novel target for manipulating steroid hormone signaling in insects. In this study, we established an assay system that can rapidly assess EcI-mediated ecdysteroid entry into cultured cells. Using NanoLuc Binary Technology (NanoBiT), we first developed an assay to detect ligand-dependent heterodimerization of the ecdysone receptor (EcR) and retinoid X receptor (RXR) in human embryonic kidney (HEK) 293T cells. We also developed HEK293 cells that stably express EcI. By combining these tools, we can monitor ecdysteroid entry into the cells in real time, making it a reliable system to assess EcI-mediated steroid hormone incorporation into animal cells.

Highlights

Steroid hormones are cholesterol derivatives essential for development and homeostasis in a wide variety of animals
We developed a novel assay system for detecting cellular uptake of ecdysteroids in a heterologous system by combining the ecdysone receptor (EcR)-retinoid X receptor (RXR) NanoLuc Binary Technology (NanoBiT) assay and HEK293-Ecdysone Importer (EcI) cells
The EcR-RXR NanoBiT assay monitors formation of the EcR-RXR heterodimer complex upon 20E binding in real time, making it a reliable tool to assess the incorporation of steroid hormone into animal cells as mediated by EcI

Summary

Introduction

Steroid hormones are cholesterol derivatives essential for development and homeostasis in a wide variety of animals. They regulate physiological processes including immune response and sexual maturation (Sapolsky et al, 2000; Sisk and Foster, 2004; Rhen and Cidlowski, 2005; Wilson and Davies, 2007; Oakley and Cidlowski, 2011; Sakiani et al, 2013), and affect pathological processes such as cancer progression (Clemons and Goss, 2001; Rhen and Cidlowski, 2005; Attard et al, 2009). The EcR/USP heterodimer complex binds to the ecdysone

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