The nuclear corepressors NCoR and SMRT are key regulators of both ligand- and 8-bromo-cyclic AMP-dependent transcriptional activity of the human progesterone receptor

Abstract

Previously, we defined a novel class of ligands for the human progesterone receptor (PR) which function as mixed agonists. These compounds induce a conformational change upon binding the receptor that is different from those induced by agonists and antagonists. This establishes a correlation between the structure of a ligand-receptor complex and its transcriptional activity. In an attempt to define the cellular components which distinguish between different ligand-induced PR conformations, we have determined, by using a mammalian two-hybrid assay, that the nuclear receptor corepressor (NCoR) and the silencing mediator for retinoid and thyroid hormone receptor (SMRT) differentially associate with PR depending upon the class of ligand bound to the receptor. Specifically, we observed that the corepressors preferentially associate with antagonist-occupied PR and that overexpression of these corepressors suppresses the partial agonist activity of antagonist-occupied PR. Binding studies performed in vitro, however, reveal that recombinant SMRT can interact with PR in a manner which is not influenced by the nature of the bound ligand. Thus, the inability of SMRT or NCoR to interact with agonist-activated PR when assayed in vivo may relate more to the increased affinity of PR for coactivators, with a subsequent displacement of corepressors, than to an inherent low affinity for the corepressor proteins. Previous work from other groups has shown that 8-bromo-cyclic AMP (8-bromo-cAMP) can convert the PR antagonist RU486 into an agonist and, additionally, can potentiate the transcriptional activity of agonist-bound PR. In this study, we show that exogenous expression of NCoR or SMRT suppresses all 8-bromo-cAMP-mediated potentiation of PR transcriptional activity. Further analysis revealed that 8-bromo-cAMP addition decreases the association of NCoR and SMRT with PR. Thus, we propose that 8-bromo-cAMP-mediated potentiation of PR transcriptional activity is due, at least in part, to a disruption of the interaction between PR and the corepressors NCoR and SMRT. Cumulatively, these results suggest that NCoR and SMRT expression may play a pivotal role in PR pharmacology.

FIG. 1

Mammalian two-hybrid analysis reveals that PR and the corepressors NCoR and SMRT interact in a ligand-dependent manner. (A) Schematic indicating the regions of NCoR (ΔN4) and SMRT (C′SMRT) which were fused to the GAL4 DNA binding domain (GAL4-DBD) for use in mammalian two-hybrid analysis. The repressive domains (RD) and interaction domains (ID) previously identified for NCoR and SMRT are indicated. (B and C) HepG2 cells were transiently transfected as indicated in Materials and Methods with a luciferase reporter plasmid containing five GAL4-responsive elements (5xGAL4-TAT-LUC), the CMV-β-Gal expression vector to control for transfection efficiency, and ΔN4 (B) or C′SMRT (C) with either an empty expression vector containing the VP16 activation domain (ΔN4), the PR-VP16 expression vector, or the TR-VP16 expression vector. After transfection, the cells were incubated with either no hormone (NH) or the designated ligands (100 nM but 10 nM for T3) for 24 h and subsequently harvested for luciferase and β-galactosidase assays. The data are presented as a normalized response, representing the absolute luciferase activity corrected for transfection efficiency by normalizing against the β-galactosidase activity. The data from a representative experiment are shown. Each data point represents the average of triplicate determinations (+ standard error of the mean).

FIG. 2

In vitro binding analysis reveals that the interaction of PR with SMRT occurs in a ligand-independent manner. Recombinant GST-C′SMRT or GST alone was produced in bacteria and immobilized on gluthathione-Sepharose beads. Full-length PR was produced by in vitro translation and labeled with [³⁵S]methionine. The integrity of this protein was assessed by running an aliquot on a denaturing gel (lane 1, 10% of input labeled receptor). Labeled PR was then incubated either with equimolar amounts of GST alone (lane 2) or with GST-C′SMRT either in the absence of added ligand (lane 3), in the presence of progesterone (lane 4), or in the presence of RU486 (lane 5).

FIG. 3

Overexpression of NCoR or SMRT decreases PR transcriptional activity in a ligand-dependent manner. (A and B) HeLa cells were transiently transfected as indicated in Materials and Methods with the luciferase reporter plasmid PRE-TK-LUC, a CMV-β-Gal expression vector (CMV-β-Gal) to control for transfection efficiency, and a PR expression vector either alone or with an expression vector for either NCoR (A) or SMRT (B). After transfection, the cells were incubated with either no hormone (NH) or designated ligand R5020 (10 pM to 1 nM), RTI-020 (10 pM to 1 nM), or RU486 (1 to 100 nM) for 48 h and subsequently harvested for luciferase and β-galactosidase assays. Luciferase data was normalized to the β-galactosidase activity and the data are represented as the fold induction over the response in the absence of hormone. The data presented are representative of multiple independent experiments. (C) HeLa cells were transfected with an expression vector for either PR alone, PR and SMRT, or PR with increasing amounts of C′SMRT expression vector as indicated. After transfection, the cells were incubated with either no hormone (NH) or 10⁻⁷ M RU486. The C′SMRT protein contains the major receptor-interacting domains but not the repression domains.

FIG. 4

NCoR or SMRT overexpression prevents RU486 agonist activity on the PR-891 mutant. HeLa cells were transiently transfected with the PRE-TK-LUC reporter plasmid, the CMV-β-Gal expression vector, and the PR-891 expression plasmid either alone or with the NCoR or SMRT expression plasmid as described in Materials and Methods. After transfection, the cells were incubated as indicated with RU486 for 48 h and subsequently assayed for luciferase and β-galactosidase activities. The data from a representative experiment are shown.

FIG. 5

Corepressor overexpression prevents 8-bromo-cAMP-mediated potentiation of PR transcriptional activity. HeLa cells were transiently transfected as indicated previously with expression vectors for PR alone or for PR along with NCoR (A) or SMRT (B). After transfection, the cells were incubated in the absence or presence of 8-bromo-cAMP (8Br) (1 mM) with either no hormone (NH) or designated ligand R5020 (10 pM to 1 nM), RTI-020 (10 pM to 1 nM), or RU486 (1 to 100 nM) for 48 h and subsequently harvested for luciferase and β-galactosidase assays. Luciferase data were normalized to the β-galactosidase activity, and the data are represented as the fold induction over the response in the absence of hormone. The data presented are representative of multiple independent experiments.

FIG. 6

Mammalian two-hybrid analysis reveals that 8-bromo-cAMP reduces the interaction between PR and the corepressors. (A and B) HepG2 cells were transiently transfected as described previously with either ΔN4 (A) or C′SMRT (B) in the absence or presence of PR-VP16. (C and D) Cells were transfected with an expression vector for either ΔN4 (C) or C′SMRT (D) in the presence or absence of TR-VP16 as indicated. After transfection, the cells were incubated with either no hormone (NH) or the designated ligand (100 nM, but 10 nM for T3) in the presence (+) or absence (−) of 1 mM 8-bromo-cAMP for 48 h, and subsequently assayed for luciferase and β-galactosidase activities. The data from a representative experiment are shown.

FIG. 7

Possible mechanisms by which 8-bromo-cAMP decreases PR-corepressor association. The association of PR with both NCoR and SMRT is decreased upon the addition of 8-bromo-cAMP. This decreased association between PR and the corepressors is accompanied by an increase in receptor transcriptional activity. We propose three possible models by which 8-bromo-cAMP reduces the association between these proteins. In model 1, 8-bromo-cAMP activates a pathway which leads to the phosphorylation of the corepressor, causing it to dissociate. Simple loss of the corepressor would result in an increase in transcriptional activity. In model 2, phosphorylation of an unidentified factor X would result in association of that factor with PR, causing the dissociation of the corepressor. In this model, the increased transcriptional activity may result from both the loss of association of PR with the corepressor and the association of PR with factor X. In model 3, factor Y becomes phosphorylated by an 8-bromo-cAMP-induced signaling pathway. The corepressor has a higher affinity for the phosphorylated factor Y than for PR and is therefore titrated away from the receptor.