Initial Intracellular Events Of Steroid Hormone Action

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The focus of our research is the determinants of the steroid hormone dose-response curve and the partial agonist activity of antisteroids. The dose-response curve is closely related to the EC50, or steroid concentration at which half-maximal response is seen, and is a crucial but poorly understood component of steroid hormone pharmacology. The partial agonist activity of antisteroids is an important consideration for endocrine therapies. Previous studies from our laboratory have defined a role for various trans-acting factors (coactivators, corepressors, and the receptors themselves) in regulating the EC50 and partial agonist activity for glucocorticoid receptors (GRs). In an effort to determine the generality and scope of these observations, we investigated the ability of these factors to modulate the transcriptional properties of another member of the steroid receptor superfamily, i.e., progesterone receptors (PRs). At the same time, we have searched for additional modulatory factors. In order to comprehensively examine the modulation of PR transactivation properties, we needed an antiprogestin with partial agonist activity under a wide variety of conditions so that possible changes in activity could be monitored. No such compound existed at the start of this study. We now report that dexamethasone-mesylate (Dex-Mes) and dexamethasone-oxetanone (Dex-Ox), each a derivative of the glucocorticoid-selective steroid dexamethasone (Dex), are two new antiprogestins with significant amounts of agonist activity with both the A and B isoforms of PR, for different progesterone responsive elements, and in several cell lines. These compounds continue to display activity under conditions where another partial antiprogestin (RTI-020) is inactive. These new antiprogestins were used to determine that elevated PR or TIF2 concentrations increase the partial agonist activity of Dex-Mes and Dex-Ox, and lower the EC50 of agonists, independently of changes in total gene transactivation. Furthermore, the corepressors SMRT and NCoR each suppresses gene induction but NCoR acts opposite to SMRT and, like the coactivator TIF2, reduces the EC50 and increases the partial agonist activity of antiprogestins. These comparable responses of GR and PR suggest that variations in receptor and coregulator concentrations may be a general mechanism for altering the induction properties of other steroid receptors. Finally, the properties of Dex-Mes and Dex-Ox, plus the sensitivity of their activity to cellular differences in PR and coregulator concentrations, make these steroids potential new SPRMs (selective progesterone receptor modulators) that should prove useful as probes of PR induction properties. A major unanswered question of glucocorticoid and progesterone action is how different whole cell responses arise when both of the cognate receptors can bind to, and activate, the same hormone response elements (HREs). Having documented that the EC50 of agonist complexes, and the partial agonist activity of antagonist complexes, of both GRs and PRs are modulated by increased amounts of homologous receptor and of coregulators, we next asked whether these components differentially alter GR and PR transcriptional properties. To remove possible cell-specific differences, we have examined both receptors in the common environment of a line of mouse mammary adenocarcinoma (1470.2) cells. In order to segregate the responses that might be due to unequal nucleosome reorganization by the two receptors from those reflecting interactions with other components, we chose a transiently transfected reporter containing a simple glucocorticoid response element, or GRE (i.e., GREtkLUC). No significant differences are found with elevated levels of either receptor. However, major, qualitative differences are seen with the corepressors SMRT and NCoR, which afford opposite responses with GR and PR. Studies with chimeric GR/PR receptors indicate that no one segment of PR or GR is responsible for these properties and that the composite response likely involves interactions with both the N- and C-termini of receptors. We recently reported that the modulation of GR transactivation properties by the above factors is inhibited by the 13S form of the adenovirus protein E1A. Surprisingly, we now find that the C-terminal fragment of E1A-13S (E1A-133C) is another positive modulator of GR induction properties. However, while saturating levels of GR and added inhibitors can prevent the added GR or TIF2 from further modifying the activation properties of GR, they are ineffective with E1A-133C. These results imply that the modification of GR properties by E1A-133C proceeds via a step that is downstream of that we previously established is a common and rate limiting step for GR and TIF2. hSur2 binds to E1A-133C and similarly modifies GR transactivation properties in the presence of saturating concentrations of GR, thus suggesting that the activity of E1A-133C is mediated by hSur2. These combined studies suggest the existence of a second pathway that can reposition the GR dose-response curve and change the partial agonist activity of antisteroid complexes. This second pathway is proposed to involve other components of the transcriptional machinery than have been implicated in earlier studies. Collectively, the data suggest that GR and PR induction of responsive genes in a given cell can be modulated by the same factors. However, this modulation of receptor activities can be differentially controlled by the same non-receptor factors, in part, by unequal interactions with multiple receptor domains. New modulatory factors have also been uncovered, which suggests that these factors may be of general importance for the differential control of gene expression during development, differentiation, and homeostasis. These combined findings contribute to our long term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.