To learn more about the mechanism(s) by which the EC50 and percent partial agonist activity of GR complexes are modulated for glucocorticoid-regulated gene induction, we have been conducting our studies both with saturating and subsaturating concentrations of agonists and with saturating concentrations of antisteroids. Using a panel of GR ligands, we investigated the relationships between the EC50 and Amax for transactivation (of a glucocorticoid response element GRE) vs. transrepression (via both NFκB and AP-1 sites), the relative binding affinity of ligand for the GR, and the interaction with coactivators vs. corepressors. Ligand-selective differences in EC50 and Amax were observed that varied between the three promoters. The EC50 of agonists and partial agonists paralleled GR-affinity in transactivation but not transrepression. Conversely, Amax was unrelated to ligand affinity for GR in both transactivation and transrepression. A significant correlation between full-length coactivator binding in two-hybrid assays and Amax, as well as EC50, for different receptor-steroid complexes in both transactivation and transrepression suggests that coactivator recruitment plays a major role in the determination of ligand-selective transcriptional activity. Furthermore, ligand-selective GR binding to the coactivator GRIP-1, as determined by both two-hybrid and DNA pull-down assays, correlated positively with ligand-selective Amax with both a synthetic GRE reporter with expressed GR as well as of an endogenous gene with endogenous GR. The receptor interacting domain of the corepressor SMRT exhibited robust interaction with both agonists and partial agonists, similar to the results for coactivators, suggesting a possible role for SMRT in activation of transcription. These results provide strong support for the model in which GR-mediated ligand-selective differential interaction with coactivators and corepressors is a major determinant of ligand-selective and promoter-specific differences in potency and efficacy, for both transactivation and transrepression. In an effort to provide evidence that variations in the above induction parameters occur in physiologically relevant settings, we examined the effect of reducing the intracellular concentration of TIF2 (the human homolog of GRIP1) with transfected siRNA in human peripheral mononuclear cells (PBMCs) on GR-mediated induction of three endogenous genes (GILZ, CD163, and THBS1). As expected from our previous experiments in tissue culture cells, lower TIF2 levels reduced the percent partial agonist activity of the antiglucocorticoid Dex-Mes, and raised the EC50 of the agonist Dex, in a gene-selective manner. This is the first documentation that altering the level of a specific endogenous transcription factor can change some induction properties of endogenous genes by endogenous receptor in primary human cells. These results support the hypothesis that the modulation of GR induction properties is a relevant feature of human physiology and can provide a viable mechanism for the differential control of gene expression during development, differentiation, and homeostasis. As a result of the above studies, we have gained new molecular information both about the determinants of glucocorticoid steroid activity and about the modulation of the dose-response curve of agonists and the percent partial agonist activity of antisteroids by cofactors. These modulatory factors permit a continuum of responses and constitute new therapeutic targets for differential control of gene expression by steroid hormones during development, differentiation, homeostasis, and 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.
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