Induction Properties of a Transiently Transfected Glucocorticoid-responsive Gene Vary with Glucocorticoid Receptor Concentration

Abstract

Transient transfections of steroid receptors have yielded much of the data used to construct the current models of steroid hormone action. These experiments invariably examine the ability of receptors to regulate transcription when occupied by saturating concentrations of steroid. We now report that other induction properties of a transiently transfected gene are not constant but vary with the concentration of transiently transfected glucocorticoid receptors. Thus, the percentage of maximal induction seen with subsaturating concentrations of glucocorticoid could be dramatically increased, and an antiglucocorticoid could be converted into a partial glucocorticoid, simply by increasing the concentration of glucocorticoid receptors. This behavior was observed in HeLa cells, containing endogenous receptors, or in CV-1 cells, containing almost no endogenous receptor, with either homologous or heterologous receptors. These increases were relatively insensitive to the concentration of reporter gene, suggesting the titration of some transcription factor(s) involved in regulating the position of the glucocorticoid dose-response curve and the agonist activity of an antiglucocorticoid. This property of transfected glucocorticoid receptors required a full-length, functionally active receptor but was retained, albeit reduced in magnitude, in the absence of binding to a glucocorticoid response element. Furthermore, this phenomenon was specific in that the A form of the human progesterone receptor had no effect under the same conditions. These variations in induction properties of antiglucocorticoids and of subsaturating concentrations of glucocorticoid, in a manner that was proportional to the amount of transfected receptor, reveal processes that are not operative with saturating concentrations of glucocorticoid. These variations also demonstrate that caution should be exercised in making mechanistic conclusions based solely on experiments conducted with saturating concentrations of glucocorticoid.The overriding experimental advantage of transient transfections is time. Thus, the biological consequences of altered nucleotide compositions in the cDNAs encoding active proteins, and in genomic sequences, can be examined in a fraction of the time required to establish cell lines with the same sequences stably integrated into the cellular genome. In the field of steroid receptors, most of the recent advances have emerged from transient transfection experiments, including the contributions of cis-acting elements (1, 2), of different nucleotides in receptor binding to the hormone-responsive element (3), of various regions of receptors in steroid binding and biological activity (reviewed in Ref. 4), of promoter structure and cell type as determinants for the activity of antisteroid activity (5), and of overlapping signaling systems such as dopamine (6), epidermal growth factor (7), and protein kinase A inducers (8, 9). The utility of transient transfections has been further enhanced by the development of the "two-hybrid" (10, 11) and...

Differential modulation of glucocorticoid and progesterone receptor transactivation

“…A recurrent theme with these modulatory proteins is that the V max can be altered independently of EC 50 and partial agonist activity Jr., 1993;Szapary et al, 1996;Szapary et al, 1999;Chen et al, 2000;Zeng et al, 2000;Giannoukos et al, 2001;Song et al, 2001;He et al, 2002;Kaul et al, 2002;Wang et al, 2004a;Wang et al, 2004b;Cho et al, 2005;Kim et al, 2006). This phenomenon is again observed here with GMEB-2 (Table 3A).…”

“…P values for statistical significance are in parentheses. Table 3 Summary of Cofactor Interactions with PR vs. GR Summary is a composite of results from (A) this study and (B) previous studies for PR and GR (Szapary et al, 1996;Szapary et al, 1999;Giannoukos et al, 2001), TIF2 Giannoukos et al, 2001), and NCoR and SMRT (Song et al, 2001). Definition of symbols describing effect on parameters following addition of various factors: --, much less than; -, less than; =, equal to; ≈, approximately equal to; ≥, greater than or equal to; +, more than.…”

“…Monolayer cultures of Cos-7, CV-1, and 1470.2 cells were grown at 37 °C as described previously (Szapary et al, 1996;Giannoukos et al, 2001). Cells are transfected for 18 h using Lipofectamine (Life Technologies, Inc., Gaithersburg, MD) or FuGene6 (Roche Diagnostics, Indianapolis, IN) as recommended by the supplier.…”

A theoretical framework for gene induction and experimental comparisons

“…Considerations of EC 50 are of major importance in intact organisms, as seen with ecdysone regulation of Drosophila embryo development (33), GR concentration in glucocorticoidinduced apoptosis of mouse thymocytes (34), the role of coactivators in the pituitary resistance to thyroid hormone syndrome (35), and gene-selective responses in human peripheral blood mononuclear cells (30). An FHDC implies that changes in factor concentration act like a rheostat to give a continuum of responses (2,4,36,37), which suggests that gene expression by steroid receptors during development, differentiation, and homeostasis employs differential control, as seen with other transcription factors and morphogens (38)(39)(40)(41). This rheostat model is fully compatible with the many observations that the binding of steroid receptors and transcription factors is rapid and readily reversible (12,13,42).…”

“…This is the dose response for the actual amount of protein product measured, not the receptor occupancy or an initial reaction rate. The addition of various cofactors can shift the EC 50 and A max yet preserve the shape of the dose-response curve (2)(3)(4). The fact that the multiple steps between ligand-receptor binding and translation to protein can yield a first-order Hill dose-response curve (FHDC) that can change position is remarkable.…”