Modulation of hormone-dependent glucocorticoid receptor function using a tetracycline-regulated expression system

Abstract

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor capable of stimulating and inhibiting the expression of target genes. To better understand the biological action of glucocorticoids and the function of GR, we have utilized the tetracycline (Tc)-regulated mammalian expression system to develop a novel cell line, E8.2/GR3, derived from GR null mouse L929 fibroblasts, that exhibits conditional expression of rat GR. The intracellular concentration of rGR in E8.2/GR3 cells — from undetectable levels to levels more than 10-fold greater than that observed in wild-type L929 cells — could be manipulated by varying the Tc concentration in the culture media. Similarly, dexamethasone (DEX)-dependent transactivation of the mouse mammary tumor virus long terminal repeat and transrepression of the cadmium-induced activity of the mouse heme oxygenase-1 gene enhancer, SX2, were strictly dependent on the presence of rGR, and the levels of these activities could be modulated by Tc. Similar levels of Tc, and thus rGR, were required for half-maximal transactivation and transrepression whereas a 6-fold lower concentration of DEX was required for half-maximal transrepression than for transactivation. RU486 inhibited both DEX-dependent transactivation and transrepression. DEX decreased the steady-state level of rGR mRNA and protein in a Tc dependent manner. DEX also induced morphological changes in E8.2/GR3 cells that were dependent on rGR as no alterations were observed in the presence of Tc. These cells provide a powerful system for examining the various activities of GR, particularly as a function of different intracellular receptor concentrations.

Introduction

Glucocorticoids and other steroid hormones play important roles in regulating development, differentiation, and homeostasis1, 2. The glucocorticoid receptor (GR), which belongs to a superfamily of ligand-dependent transcription factors, regulates the expression of target genes in response to glucocorticoid hormones3, 4. In many cases, this regulation occurs at the level of transcription, where the expression of certain genes is stimulated while other genes are inhibited5, 6, 7. Furthermore, several studies have suggested that the GR level in the cells is the limiting factor in the process of gene regulation by hormone. Thus, the intracellular concentration of GR protein can control the magnitude of the cellular response8, 9, 10. The GR protein mediates significant physiological functions in cells and is capable of interfering with other important signal transduction pathways. Recent advances in our understanding of nuclear receptor structure and function have led to important insights into their molecular mechanism of action[11].

To better understand the function of GR protein in regulating its target genes, we employed a tetracycline-based eukaryotic expression system[12]to conditionally express GR proteins in the E8.2 cell, a mouse L929 fibroblast variant that does not contain any endogenous GR protein or mRNA transcripts[13]. In this study, we report the establishment and characterization of the E8.2/GR3 cell line, in which the GR level is tightly and quantitatively regulated by tetracycline. We observed that the endogenous GR protein levels directly determine the magnitude of the effect of glucocorticoids on the activity of target genes, and that GR protein is required for autoregulation of its own mRNA transcripts by the cognate ligand.