Abstract
The dose-response curve of steroid hormones and the associated EC(50) value are critical parameters both in the development of new pharmacologically active compounds and in the endocrine therapy of various disease states. We have recently described three different variables that can reposition the dose-response curve of agonist-bound glucocorticoid receptors (GRs): a 21-base pair sequence of the rat tyrosine aminotransferase gene called a glucocorticoid modulatory element (GME), GR concentration, and coactivator concentration. At the same time, each of these three components was found to influence the partial agonist activity of antiglucocorticoids. In an effort to determine whether these three processes proceed via independent pathways or a common intermediate, we have examined several mechanistic details. The effects of increasing concentrations of both GR and the coactivator TIF2 are found to be saturable. Furthermore, saturating levels of either GR or TIF2 inhibit the ability of each protein, and the GME, to affect further changes in the dose-response curve or partial agonist activity of antisteroids. This competitive inhibition suggests that all three modulators proceed through a common step involving a titratable factor. Support for this hypothesis comes from the observation that a fragment of the coactivator TIF2 retaining intrinsic transactivation activity is a dominant negative inhibitor of each component (GME, GR, and coactivator). This inhibition was not due to nonspecific effects on the general transcription machinery as the VP16 transactivation domain was inactive. The viral protein E1A also prevented the action of each of the three components in a manner that was independent of E1A's ability to block the histone acetyltransferase activity of CBP. Collectively, these results suggest that three different inputs (GME, GR, and coactivator) for perturbing the dose-response curve, and partial agonist activity, of GR-steroid complexes act by converging at a single step that involves a limiting factor prior to transcription initiation.
Highlights
Quently studied situations, these rates are increased following the binding of agonist steroids to their cognate receptors
This abbreviated assay includes a single point with a saturating concentration of antisteroid so that we can simultaneously determine the effect of the specific reaction conditions on the partial agonist activity of an antiglucocorticoid
Two parameters of glucocorticoid receptors (GRs) induction of responsive genes that are important for the differential control of gene expression are the positioning of the steroid dose-response curve, or EC50, and the amount of partial agonist activity displayed by antiglucocorticoids
Summary
Quently studied situations, these rates are increased following the binding of agonist steroids to their cognate receptors. The recent discovery of histone acetyltransferase (HAT) activity in most coactivators (4 –7), and of histone deacetylase activity with corepressors (8 –10), has permitted models of histone acetylation-induced modifications of nucleosome structure [11,12,13] to be incorporated into those for steroid regulated gene expression (14 –16) Both the responses to endogenous steroids and the pharmacology of steroid hormone-based endocrine therapies depend critically on the dose-response curve, which relates steroid concentration to biological activity. A lower EC50, which is synonymous with a left-shifted doseresponse curve, is highly desirable in clinical situations as it leads to fewer adverse side effects This is because the responses of other regulated genes with higher EC50 values would be greatly reduced at the lower steroid concentrations. This coincidence of phenotypical properties suggested that each process might modify the GR induction properties via a common step that could involve a titratable factor
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