An estrogen receptor-selective coregulator that potentiates the effectiveness of antiestrogens and represses the activity of estrogens

Abstract

The action of nuclear hormone receptors is tripartite, involving the receptor, its ligands, and its coregulator proteins. The estrogen receptor (ER), a member of this superfamily, is a hormone-activated transcription factor that mediates the stimulatory effects of estrogens and the inhibitory effects of antiestrogens such as tamoxifen in breast cancer and other estrogen target cells. To understand how antiestrogens and dominant negative ERs suppress ER activity, we used a dominant negative ER as bait in two-hybrid screening assays from which we isolated a clone from breast cancer cells that potentiates the inhibitory activities of dominant negative ERs and antiestrogen-liganded ER. At higher concentrations, it also represses the transcriptional activity of the estradiol-liganded ER, while having no effect on other nuclear hormone receptors. This clone, denoted REA for "repressor of estrogen receptor activity," encodes a 37-kDa protein that is an ER-selective coregulator. Its competitive reversal of steroid receptor coactivator 1 enhancement of ER activity and its direct interaction with liganded ER suggest that it may play an important role in determining the sensitivity of estrogen target cells, including breast cancer cells, to antiestrogens and estrogens.

Figure 1

Amino acid and nucleotide sequence of human REA. Potential protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites and a nuclear receptor-interaction box (NR, LXXLL) are underlined, and a nuclear localization sequence (NLS) in REA is boxed.

Figure 2

REA enhances the potency of antiestrogens and dominant negative ER as suppressors of ER activity, but has no affect on the potency of antiprogestin as a progesterone receptor antagonist. In A– C, CHO cells were transfected with an expression vector for the wild-type ER (5 ng) and the reporter construct (ERE)₂-TATA-CAT in the absence or presence of an expression vector for REA (10 ng). In A, cells were cotransfected with increasing amounts of an expression vector for the dominant negative L540Q ER and were treated with 10⁻⁸ M E₂ for 24 h. In B and C, cells were treated with 10⁻⁸ M E₂ along with increasing concentrations of the antiestrogen TOT (B) or ICI182,780 (ICI) (C) for 24 h. (D) The effect of REA on the repressive action of antiprogestin on R5020-mediated activation of the PR also was tested. CHO cells were transfected with an expression vector for human PR-B (5 ng), the PR responsive reporter construct MMTV-CAT in the absence or presence of REA expression vector (10 ng), and were treated with 10⁻⁸ M progestin R5020 plus increasing concentrations of the antiprogestin RU486 for 24 h. All cells in A– D also were transfected with a β-galactosidase internal control reporter to correct for transfection efficiency. Cell extract CAT activity values, normalized for β-galactosidase activity, are the means ± SD from three separate experiments.

Figure 3

REA is an ER-selective coregulator, suppressing transcriptional activation of ERα and ERβ, but not that of PR, RAR, or Gal4-VP16. CHO cells (A and B and D–F) were transfected with 5 ng of the expression vector for various activators and 2 μg of the reporter construct indicated. (A) ERα/(ERE)₂-TATA-CAT. (B) ERβ/(ERE)₂-TATA-CAT. (D) PR/MMTV-CAT. (E) RAR/DR-5-CAT. (F) Gal4-VP-16/G5-E1-CAT. In C, MDA-MB-231 breast cancer cells were transfected with 100 ng of the expression vector for ERα and 6 μg of the (ERE)₂-pS2-CAT reporter. The cells were cotransfected with increasing concentrations of an expression vector for REA as indicated and with a β-galactosidase internal control reporter to correct for transfection efficiency. Cells then were treated for 24 h with 10⁻⁸ M ligands (ER, E₂; PR, R5020; RAR, all-trans retinoic acid). Cell extracts were prepared, and CAT activity, normalized for β-galactosidase activity, is shown. Values are the means ± SD from three separate experiments. Numbers at the top of the leftmost bar show the fold induction in CAT activity by hormone (receptor-transfected, plus vs. minus hormone) in the absence of added REA.

Figure 4

Mapping the regions of REA required for repression of ER activity. The regions of REA required for repression of ER activity were monitored by using the N- and C-terminal truncated REAs indicated. Repressive activity was monitored by the ability of the cotransfected REA to repress ER-stimulated transcription as measured by CAT assay from the estrogen-responsive reporter (ERE)₂-pS2-CAT. MDA-MB-231 human breast cancer cells were cotransfected with 100 ng pCMV-ERα, 500 ng of pCMV-REA construct, and internal control β-galactosidase plasmid. Cells were treated with 10⁻⁸ M E₂ for 24 h. The level of repression of ER activity by full-length REA is set at 100%. The effectiveness of the truncated forms of REA in repressing ER activity is listed as a percentage of full-length REA. Values are the means ± SD of three to eight determinations. REA (1–226) was found to suppress ER activity to the same level as full-length REA when higher amounts of REA (1–226) expression plasmid were utilized.

Figure 5

Direct interaction of REA with ER. (Upper) In vitro translated, [³⁵S]methionine-labeled REA was incubated with GST-L540Q-ER (lanes 1–3) or GST alone bound to Sepharose beads (lane 4) in the presence of 0.1% ethanol control vehicle (−), 10⁻⁶ M TOT (T), or 10⁻⁶ M estradiol (E). (Lower) In vitro translated, [³⁵S]methionine-labeled L540Q ER (lanes 1 and 2) or wild-type ER (lanes 3 and 4) were incubated with GST-REA (REA bound to GST-Sepharose beads) in the presence of control vehicle (−) or estradiol (E). Bound protein was eluted and analyzed by 12.5% SDS/PAGE. Typically, 12–15% of input-radiolabeled wild-type ER was bound to GST-REA in the presence of ligand. The numbers at the right indicate molecular size markers in kDa.

Figure 6

REA suppresses SRC-1-mediated enhancement of ER transcriptional activity. CHO cells were transfected with 5 ng of expression vector for ER and 2 μg of (ERE)₂-TATA-CAT reporter construct. The cells were cotransfected with increasing concentrations of an expression vector for SRC-1 in the presence or absence of REA as indicated. The cells also were transfected with a β-galactosidase internal control reporter to correct for transfection efficiency. Cells then were treated for 24 h with 10⁻⁸ M E₂. Cell extracts were prepared and analyzed for CAT activity and β-galactosidase activity. Values are the means ± SD from three separate experiments.

Figure 7

Model for REA potentiation of antiestrogen-inhibitory effectiveness. See text for description.