Initial Intracellular Events Of Steroid Hormone Action

Diabetes, Digestive, Kidney Diseases

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Abstract

The focus of our research is the determinants of two transcriptional properties of steroid receptors: the dose-response curve of agonists and the partial agonist activity of antisteroids. The dose-response curve defines the EC50, or steroid concentration at which half-maximal response is seen, and is a crucial but poorly understood component of steroid hormone endocrinology. Differences in the EC50s of regulated genes provide a mechanism for differential expression by the common concentration of circulating steroid hormone in an organism. The partial agonist activity of antisteroids is an important consideration for limiting unwanted side effects during endocrine therapies by potentially allowing partial expression of genes other than the one targeted for suppression. While the EC50 and partial agonist activity were long considered to be invariant, it is now clear that they are not fixed and can be modulated by equilibrium interactions with a variety of factors. However, our initial findings suggest that the effects of specific factors are not the same with different receptors. Thus, receptor-specific responses to cofactors may offer a mechanism whereby unequal responses can be elicited by different classes of receptors even though they can bind to the same DNA sequences of regulated genes. For classical steroid receptors, such as glucocorticoid receptors (GRs) and progesterone receptors (PRs), one important group of modulatory factors is the corepressors NCoR and SMRT. Corepressors are known to interact via their receptor interaction domains (RIDs) with the ligand binding domain in the carboxyl terminal half of steroid/nuclear receptors. We now report that a portion of the AF-1 domain of glucocorticoid (GRs) and progesterone receptors (PRs), which is the major transactivation sequence in the amino-terminal half of each receptor, is necessary but not sufficient for corepressor (NCoR and SMRT) RID binding to GRs and PRs in both mammalian two-hybrid and co-immunoprecipitation assays. Importantly, these two receptor sequences are functionally interchangeable in the context of GRs for transactivation, corepressor binding, and corepressor modulatory activity assays. This suggests that corepressors act in part by physically blocking portions of receptor AF-1 domains. However, differences do exist in corepressor binding to GRs and PRs. The C-terminal domain of PRs has a higher affinity for corepressor than that of GRs. The ability of some segments of the coactivator TIF2 to competitively inhibit corepressor binding to receptors is different for GRs and PRs. With each receptor, the cell-free binding of corepressors to ligand-free receptor is prevented by sodium molybdate, which is a well-known inhibitor of receptor activation to the DNA-binding state. This suggests that receptor activation precedes binding to corepressors. A similar phenomenon may be responsible for the ligand-free binding of steroid receptors to a variety of other transcriptional cofactors. Collectively, these results indicate that corepressor binding to GRs and PRs involve both N- and C-terminal sequences of activated receptors but differ in ways that may contribute to the unique biological responses of each receptor in intact cells. Our mechanistic studies to date repeatedly indicate that the pathway utilized for steroid receptor-mediated increases in gene expression is separate and distinct from the process(es) that dictates both the position of the dose-response curve (and the value of the EC50) for an agonist and the amount of partial agonist activity for an antisteroid. This presence of two independent reaction schemes therefore suggests that novel proteins may be involved in the expression of the EC50 and the amount of partial agonist activity. This conclusion was supported by our finding a fragment of the coactivator TIF2 that was not known to bind any protein but retained the ability of modulate th EC50 and partial agonist activity of GR complexes. A yeast two-hybrid screen was used to isolate the predicted factor as a new 1277 amino acid endogenous protein. This novel protein also binds to the small modulatory domain of another coactivator, SRC-1. For this reason, this new protein is called STAMP, or SRC-1 and TIF2 associated modulatory protein. STAMP selectively binds to a sub-set of the steroid/nuclear receptors, including GRs, in mammalian two-hybrid assays. Importantly, transfected STAMP increases the modulatory effects of TIF2 in GR- and androgen receptor (AR), but not thyroid receptor (TR), -mediated induction. Endogenous STAMP colocalizes with GR on the promoters of an endogenous GR-induced gene. The co-immunoprecipitation of GR, TIF2, and STAMP indicates that they all are present in a macromolecular complex and exists in a complex with endogenous GRs. Another clinically important property of GRs is their immunosuppressive activity that appears to result from GR repression of a variety of induced genes. It had been reported by others that the region of TIF2 that binds STAMP is also involved in augmenting the fold repression of GR-agonist complexes. We found that STAMP increases the repressive activity of GR-agonist complexes without or with exogenous TIF2. Endogenous STAMP colocalizes in the same complex with GR on the promoters of an endogenous GR-repressed gene. The level of repression seen with endogenous and exogenous STAMP is reduced by STAMP siRNAs. Collectively, these data suggest that STAMP is an important new, down-stream component of GR action in both gene activation and gene repression. Furthermore, the selectivity of STAMP in modulating the transcriptional properties of ARs but not TRs is unusual among the previously described cofactors and potentially offers a novel control point for therapeutical interventions. As a result of the above studies, we have gained new molecular information about the modulation of the dose-response curve of agonists and the partial agonist activity of antisteroids. These results suggest new avenues for controlling these parameters, which are of general and widespread importance for the differential control of gene expression during development, differentiation, and homeostasis and for more selective endocrine therapies. 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.