Transactivation Function-2 of Estrogen Receptor α Contains Transactivation Function-1-regulating Element

Abstract

ERα has a ligand-dependent transactivation function in the ligand binding domain of ERα C terminus (AF-2) and a ligand-independent activation function in the N terminus (AF-1). It is still not fully understood how AF-1 and AF-2 activities are regulated cooperatively by ligands. To evaluate the AF-1 involvement in the estrogenic activities of various compounds, we analyzed these transactivation functions using AF-1-truncated and AF-2-mutated ERα mutants. AF-2 is composed of two domains with flexible and static regions. We used an AF-2 flexible region mutant and an AF-2 static region mutant. Both mutants have been reported as non-E2 responsive due to disruption of E2-mediated coactivator recruitment to the AF-2. The AF-2 mutants were not activated by agonists, but surprisingly antagonists and selective estrogen receptor modulators (SERMs) activated the AF-2 mutants. This antagonist reversal activity was derived from AF-1. Furthermore, we demonstrated that the AF-2 contains an AF-1 suppression function using C-terminal-truncated ERα mutants. From these findings we hypothesized that the mutation of AF-2 disrupted its ability to suppress AF-1, causing the antagonist reversal. To assess the AF-2-mediated AF-1 suppression, we analyzed the transcription activity of physically separated AF-1 and AF-2 using a novel hybrid reporter assay. We observed that the AF-1 activity was not suppressed by the physically separated AF-2. Furthermore, SERMs did not induce the AF-1-mediated activity from the separated mutant AF-2, which differed from the intact protein. These results imply that SERM activity is dependent on a conformational change of the full-length ERα molecule, which allows for AF-1 activation.

Keywords: agonist; antagonist; estrogen; estrogen receptor; protein structure; selected estrogen receptor modulators; steroid hormone; steroid hormone receptor.

FIGURE 1.

Chemical structures. Chemical structures are illustrated. The chemicals are categorized in estrogen, mycotoxin, phytoestrogens, EDCs, SERMs, and antagonist. GSK1648229 and GSK1648230 are enantiomers with undetermined absolute configuration of trans-cyclopropanes.

FIGURE 2.

Schematic structure of mERα WT and mutants. A, ERα consists of six domains named A to F (FL-ERα). A/B-domain possesses AF-1 activity (AF1). E-domain possesses ligand-dependent transcription activation domain (AF-2). AF-2 is composed of static region (AF2-S) and flexible region (AF2-F). 121-ERα, the mutant with entire AF-1 domain deletion from FL-ERα; AF2ER, leucines 543 and 544 in AF2-F were mutated to alanines; mERα-I362D, isoleucine 362 in AF2-S was mutated to aspartic acid; AF2ER-I362D, the combined mutation of AF2ER and I362D; ERα339, the mutant with entire AF-2 truncation; ERα384, the mutant with AF2-F truncation but retains AF2-S; ERα384-I362D, ERα384 contains AF2-S mutation (I362D). B, whole cell lysates extracted from the plasmid-transfected HepG2 cells were analyzed by immunoblotting with anti-ERα antibody (MC-20 and H-184) to demonstrate expression levels of ERα WT and mutants. β-Actin was used as a loading control (Actin). * suggests nonspecific signals. A representative Western blot analysis is shown.

FIGURE 3.

Agonist activity of xenochemicals for 3xERE-TATA reporter. HepG2 cells were cotransfected with the reporter gene (3xERE-Luc), reference gene (pRL-TK), and expression vectors for full-length ERα (FL-ERα) (A) or N-terminal-truncated ERα (121-ERα) (B) and treated with either vehicle (EtOH) or 100 nm chemical. BPA, bisphenol A; BPAF, bisphenol AF. C, the cells were cotransfected with 3xERE-Luc, pRL-TK, and FL-ERα then treated with vehicle (0 μm; DMSO), 0.001–1.0 μm E2, or 0.1–10 μm indicated chemical. The cells were cotransfected with 3xERE-Luc, pRL-TK, and FL-ERα (D) or 121-ERα (E) and treated with either vehicle (DMSO) or 10 μm chemical (E2, 4OHT, and clomifene were 1 μm). The luciferase activities for each panel are represented as -fold change over vehicle. The activity is represented as the mean ± S.E. One-way ANOVA was performed to indicate significant differences against vehicle in each panel. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05.

FIGURE 4.

Agonist activity of xenochemicals for C3-T1 reporter. HepG2 cells were cotransfected with the reporter gene (C3-T1-Luc), reference gene (pRL-TK), and expression vectors for FL-ERα (A) or 121-ERα (B) and treated with either vehicle (EtOH) or 100 nm chemical. BPA, bisphenol A; BPAF, bisphenol AF. C, the cells were cotransfected with 3xERE-Luc, pRL-TK, and FL-ERα then treated with vehicle (0 μm; DMSO), 0.001–1.0 μm E2, or 0.1–10 μm indicated chemical. The cells were cotransfected with 3xERE-Luc, pRL-TK, and FL-ERα (D) or 121-ERα (E) and treated with either vehicle (DMSO) or 10 μm chemical (E2, 4OHT and clomifene were 1 μm). The luciferase activities for each panel are represented as -fold change over vehicle. The activity is represented as the mean ± S.E. One-way ANOVA was performed to indicate significant differences against vehicle in each panel. ****, p < 0.0001; ***, p < 0.001; **. p < 0.01; *, p < 0.05.

FIGURE 5.

Antagonist activity of xenochemicals. HepG2 cells were cotransfected with the reporter gene (3xERE-Luc), reference gene (pRL-TK), and expression vectors for FL-ERα (A) or 121-ERα (B) then treated with 10 nm E2 and 1 μm chemical (apigenin, 4-nonylphenol, p,p'-DDT, triclosan, 1-bromopropane, and GW5638 were 10 μm). The luciferase activities for the each panel are represented as relative luciferase activity compared with the level of 10 nm E2-dependent activity (white column; DMSO), which is indicated as 100. Dotted lines denote the E2-mediated luciferase activity level. The activity is represented as the mean ± S.E. One-way ANOVA was performed to indicate significant reductions against E2-dependent activity in each panel. ****, p < 0.0001. BPA, bisphenol A; BPAF, bisphenol AF.

FIGURE 6.

The profile of xenochemical activity through an ERα AF-2 flexible region mutant (AF2ER). HepG2 cells were cotransfected with the reporter gene (3xERE-Luc), reference gene (pRL-TK), and expression vectors for the L543A,L544A-mutated ERα (AF2ER) (A) or N-terminal-truncated AF2ER (121-AF2ER) (B) and treated with either vehicle (EtOH) or 100 nm chemical. BPA, bisphenol A; BPAF, bisphenol AF. C, the cells were cotransfected with 3xERE-Luc, pRL-TK, and AF2ER or 121-AF2ER and treated with either vehicle (DMSO) or 10 μm chemical (E2, 4OHT, and clomifene were 1 μm). The luciferase activities for each panel are represented as -fold change over vehicle. The activity is represented as the mean ± S.E. One-way ANOVA was performed to indicate significant differences against vehicle in each panel. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01.

FIGURE 7.

The profile of xenochemical activity through an ERα AF-2 static region mutant (mERα-I362D). HepG2 cells were cotransfected with the reporter gene (3xERE-Luc), reference gene (pRL-TK), and expression vectors for the I362D-mutated ERα (mERα-I362D) (A) or N-terminal-truncated mERα-I362D (121-mERα-I362D) (B) and treated with either vehicle (EtOH) or 100 nm chemical. BPA, bisphenol A; BPAF, bisphenol AF. C, the cells were cotransfected with 3xERE-Luc, pRL-TK, and mERα-I362D or 121-mERα-I362D and treated with either vehicle (DMSO) or 10 μm chemical (E2, 4OHT, and clomifene were 1 μm). The luciferase activities for each panel are represented as -fold change over vehicle. The activity is represented as the mean ± S.E. One-way ANOVA was performed to indicate significant differences against vehicle in each panel. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05.

FIGURE 8.

The profile of xenochemical activity through a compound AF-2 mutant (AF2ER-I362D). A, HepG2 cells were cotransfected with the reporter gene (3xERE-Luc), reference gene (pRL-TK), and the expression vector for I362D, L543A, and L544A mutated ERα (AF2ER-I362D) and treated with either vehicle (EtOH) or 100 nm chemical. BPA, bisphenol A; BPAF, bisphenol AF. B, the cells were cotransfected with 3xERE-Luc, pRL-TK, and AF2ER-I362D or 121-AF2ER-I362D and treated with either vehicle (DMSO) or 10 μm chemical (E2, 4OHT, and clomifene were 1 μm). The luciferase activity is represented as -fold change over vehicle. The activity is represented as the mean ± S.E. One-way ANOVA was performed to indicate significant differences against vehicle. ****, p < 0.0001; ***, p < 0.001. C, HepG2 cells were cotransfected with 3xERE-Luc, pRL-TK, and the expression vector for AF2ER-I362D and treated with either vehicle (0 nm; EtOH) or 0.1–100 nm indicated chemical. Luciferase activities are represented as -fold change over vehicle, and the activity is represented as the mean ± S.E. D, HepG2 cells were cotransfected with 3xERE-Luc, pRL-TK, and the expression plasmids for AF2ER-I362D (FL) or 121-AF2ER-I362D (ΔN) then treated with 100 nm chemical. Luciferase activities are represented as relative activity compared with the empty expression plasmid transfected cells for each chemical (pcDNA3). The activity is represented as the mean ± S.E.

FIGURE 9.

AF-2 static region harbors AF-1 repression activity. A, schematic diagram of C-terminal truncated ERα mutants. AF2-S and AF2-F denote the static and flexible regions of AF-2 constituent elements respectively. B, HepG2 cells were cotransfected with the 3xERE-Luc, pRL-TK, and the expression vector for ERα339, ERα384, ERα384-I362D, or FL-ERα treated with vehicle (EtOH) and 100 nm E2 for FL-ERα. The luciferase activities are represented as relative activity compared with the empty expression plasmid transfected cells (pcDNA3). The activity is represented as the mean ± S.E. One-way ANOVA was performed to indicate significant differences. ns, not significant. C, whole cell lysates extracted from the plasmid-transfected HepG2 cells were analyzed by immunoblotting with anti-ERα antibody (H-184) to demonstrate expression levels of ERα WT and mutants. β-Actin was used as a loading control (Actin). A representative Western blot analysis is shown.

FIGURE 10.

Conformational change of ERα molecule is necessary for SERM-mediated AF-1 activation. A, schematic diagram of a hybrid reporter assay. The reporter gene contains a Gal4-binding element (Gal4RE) juxtaposed to an ERE, coexpressed with ERα339 and pBIND-EF/WT or pBIND-EF/AF2ER-I362D (pBIND-EF/m3). The activities were normalized by the activity of 121-ERα339 and pBIND-cotransfected cells (white column). B, HepG2 cells were cotransfected with the hybrid reporter (2x(ERE-m17)-TATA-Luc), 121-ERα339, ERα339, pBIND, and pBIND-EF/WT as indicated in figure then treated with either vehicle (EtOH) or 100 nm chemical. The activity of pBIND-EF/AF2ER-I362D (same result as vehicle in C) is displayed in right column as a reference. C, HepG2 cells were cotransfected with the hybrid reporter, 121-ERα339, ERα339, pBIND, and pBIND-EF/AF2ER-I362D (pBIND-EF/m3) as indicated then treated with either vehicle (EtOH) or 100 nm chemical. The activity of pBIND-EF/WT (same result as vehicle in B) is displayed in the right column as a reference. Normalized activity is represented as the mean ± S.E. Two-way ANOVA was performed to indicate the significance of ligand-dependent activation of ERα339 and pBIND-EF/WT (green column in B) or ERα339 and pBIND-EF/AF2ER-I362D (green column in C) cotransfected cells comparing vehicle. $ suggests significant difference; ns denotes non significant difference. Zea, zearalenone; Tor, toremifene; Osp, ospemifene; Clo, clomifene; Idx, idoxifene; Ral, raloxifene; Baz, bazedoxifene; Las, lasofoxifene. DBAC, des-bis(acetoxy)cyclofenil.

FIGURE 11.

AF2ER-I362D LBD dimerization is associated with AF-1-mediated activation. A, schematic diagram of a mammalian two-hybrid assay. HepG2 cells were cotransfected with pG5-Luc and expression vector for Gal4 DBD-fused ERα LBD (pBIND-EF/WT or pBIND-EF/AF2ER-I362D) in the presence of expression vector for VP16AD (pACT) alone or VP16AD-fused ERα LBD (pACT-EF/WT or pACT-EF/AF2ER-I362D). B, left panel, the result of mammalian two-hybrid assay for pACT and pBIND-EF/WT-cotransfected samples (black column) and pACT-EF/WT and pBIND-EF/WT-cotransfected samples (white column). The cells were treated with 10 nm chemical. BPA, bisphenol A; BPAF, bisphenol AF. The luciferase activity is represented as -fold over vehicle (EtOH) in the pACT and pBIND-EF/WT-cotransfected cells. B, right panel, the result of mammalian two-hybrid assay for pACT and pBIND-EF/AF2ER-I362D cotransfected samples (black column) and pACT-EF/AF2ER-I362D and pBIND-EF/AF2ER-I362D-cotransfected samples (white column). The cells were treated with 100 nm chemical. The activity is represented as -fold over vehicle (EtOH) in the pACT and pBIND-EF/AF2ER-I362D-cotransfected cells. Luciferase activity is represented as the mean ± S.E. C, the normalized luciferase activities are represented. Left panel, the luciferase activity in the pACT-EF/WT and pBIND-EF/WT-cotransfected samples (white column in B) was normalized over the pACT and pBIND-EF/WT-cotransfected samples (black column in B) in each compound. Right panel, the luciferase activity in the pACT-EF/AF2ER-I362D and pBIND-EF/AF2ER-I362D-cotransfected samples (white column in B) was normalized over the pACT and pBIND-EF/AF2ER-I362D-cotransfected samples (black column in B) in each compound. Normalized activity is represented as the mean ± S.E. One-way ANOVA was performed to indicate significant differences against vehicle. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01. The activities of samples denoted as ♦ are <1.

FIGURE 12.

Group A agonists induce AF-1 and AF-2 cooperative activity. HepG2 cells were cotransfected with the hybrid reporter, 121-ERα339, ERα339, pBIND, and pBIND-EF/WT as displayed in the figure then treated with either vehicle (EtOH) or chemical (0.1 or 1.0 μm). The activities were normalized by the activity of 121-ERα339 and pBIND-cotransfected cells, which are represented as 1. Normalized activity is represented as the mean ± S.E. Two-way ANOVA was performed to indicate a significant difference of ligand-dependent enhancement of ERα339 and pBIND-EF/WT-cotransfected cells against vehicle. $ suggests significant difference; ns denotes non significant difference.