The Journal of Steroid Biochemistry and Molecular Biology
Modulation of hormone-dependent glucocorticoid receptor function using a tetracycline-regulated expression system
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.
Section snippets
Cell culture
Mouse fibroblast E8.2 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum. E8.2/GR3 cells were maintained in the presence of 1 μg/ml tetracycline (Tc, Sigma), 200 μg/ml of G418 (Geneticin, GIBCO) and 200 μg of Hygromycin B (Sigma). GR3.SM cells were maintained under the same condition as E8.2/GR3 cells, except that puromycin was added to a final concentration of 5 μg/ml. All the cells were grown at 37°C in a humidified incubator under 6% CO2.
Plasmid constructs
The tTA (tet
Construction of a tetracycline responsive GR expression plasmid
In the tetracycline dependent eukaryotic expression system, the Tc-controlled transactivator (tTA), a fusion protein consisting of the Tet repressor and the activation domain of the Herpes Simplex virus VP16 protein, can induce the expression of a gene inserted downstream of the tet operator, the binding site for the tet repressor. Tc prevents tTA from binding to the operator and, therefore, removal of tetracycline stimulates transcription of a gene under the control of this regulatory sequence
Discussion
The ability to reversibly turn genes off and on is a powerful tool for investigating the function of various gene products. Efforts have been made to manipulate gene expression by various inducible promoters including those responsive to heavy metal ions37, 38, hormones39, 40, 41, and isopropyl-β-d-thiogalactoside (IPTG)42, 43, 44. The recent development of the tetracycline-regulatory system for inducible gene expression has provided a powerful methodology for quantitative and tight control of
Acknowledgements
This research was supported by NIH Grants #DK47211 (to WVV), #DK43 135 (to JA) and #DK47951 (to PRH), and a Student Research Grant from the Cancer Association of Greater New Orleans (to PW).
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