The experimental protocols used over the last 40+ years to unravel the mysteries of steroid hormone action almost invariably employed a single saturating, or pharmacological, concentration of agonist steroid. This approach yields only the maximum amount of steroid-regulated gene expression, as shown in for the induction of two genes A and B. For the purposes of this review, we call the maximum activity the Amax
. This value is also called the efficacy of the steroid but due to some confusion as to its precise meaning we will use Amax
here. More information is obtained if lower concentrations of agonist steroid are included to examine steroid potency (). Under these circumstances, the concentration of steroid required for half-maximal induction, or EC50
, can be determined. As was found many years ago, the concentration of circulating cortisol in humans (80–600 nM) is about the same as the EC50
for some cortisol-induced effects (19
). Importantly, changing the EC50
for steroid-regulated gene expression will obviously alter the response of that gene. Thus, Gene B, with a lower EC50
, will be maximally induced at a lower steroid concentration than gene A, which is important for the differential induction of gene B vs. A during development, differentiation, and homeostasis. This also means that the activity with 100 nM agonist should not be assumed to be maximal. A right shift in the dose-response curve can easily move the EC50
to values ≥100 nM (20
). It should also be noted that there is no observed, or theoretical, correlation between Amax
() and EC50
() so that studies organized as in are unable to give any information about the EC50
of the gene of interest.
Fig. 2 Data plots for experiments on steroid hormone action. (A) Induction of gene expression of transiently transfected luciferase reporter genes A and B with a saturating concentration of agonist steroid. Raw luciferase data are plotted. Background levels (more ...)
Additional information is obtained if pharmacological concentrations of an antagonist, or antisteroid, are included (). Antisteroids are used at these concentrations during endocrine therapies to block the actions of agonist steroids. However, the amount of residual agonist activity of each antisteroid is currently impossible to predict and must be experimentally determined. A useful parameter, when discussing the residual agonist activity of antisteroids, is the percent partial agonist activity.
While the precise values for the EC50, or potency, and percent partial agonist activity can be calculated from plots of the raw data, as in , it is much easier if the data are first replotted as percent of maximal induction for each gene, as in . In this case, the EC50 and percent partial agonist activity can be readily read off of the graph. Such plots also clearly demonstrate that the EC50 is independent of the Amax of gene induction. The importance of comparing the activities of antagonist and agonist under the same conditions cannot be overemphasized. The residual activity of the antisteroid is about the same for genes A and B () but that the percent partial agonist activity is much greater with gene B than with gene A (). Replotting the data as in will also immediately clarify whether or not reports of increased sensitivity (i.e, more activity at low concentrations of agonist), or more response with high levels of antisteroid, actually reflect a change in EC50, or percent partial agonist activity respectively.
The current model of steroid hormone action says little about the determinants of the EC50
. Early observations of a close correlation between the Kd
for steroid binding to receptors and the EC50
for steroid-regulated gene induction were viewed as evidence that the steroid was acting via binding to its cognate receptor. In fact, it has been known for many years that such a correlation is not general (22
and reviewed in 23
). It should also be noted that high steroid binding affinity does not assure high efficacy of biological response (Amax
). RU486 has a high affinity for GR (25
) but a negligible Amax
for gene induction under most conditions (26
). Under conditions where the affinity label Dexamethasone 21-mesylate (Dex-Mes) covalently binds to >90% of the receptors, it displays ≤7% of maximal activity seen with Dex (28
). Thus, detailed studies of the EC50
with sub-saturating concentrations of agonists, and the percent partial agonist activity with saturating concentrations of antagonists, are required to gain an understanding of the determinants of these relevant parameters. Finally, as is described below, such experiments have revealed that the EC50
, and percent partial agonist activity, are not constant even for a single gene but can be modulated by a variety of factors.