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. 2011 Sep 6;108(36):14986-91.
doi: 10.1073/pnas.1109180108. Epub 2011 Aug 22.

Estrogen receptor α AF-2 mutation results in antagonist reversal and reveals tissue selective function of estrogen receptor modulators

Yukitomo Arao  1 , Katherine J Hamilton, Manas K Ray, Gregory Scott, Yuji Mishina, Kenneth S Korach
Affiliations

Estrogen receptor α AF-2 mutation results in antagonist reversal and reveals tissue selective function of estrogen receptor modulators

Yukitomo Arao et al. Proc Natl Acad Sci U S A. .

Abstract

The estrogen receptor (ER) is a ligand-dependent transcription factor containing two transcriptional activation domains. AF-1 is in the N terminus of the receptor protein and AF-2 activity is dependent on helix 12 of the C-terminal ligand-binding domain. Two point mutations of leucines 543 and 544 to alanines (L543A, L544A) in helix 12 minimized estrogen-dependent transcriptional activation and reversed the activity of the estrogen antagonists ICI182780 (ICI) and tamoxifen (TAM) into agonists in a similar manner that TAM activated WT ERα through AF-1 activation. To evaluate the physiological role of AF-1 and AF-2 for the tissue-selective function of TAM, we generated an AF-2-mutated ERα knock-in (AF2ERKI) mouse model. AF2ERKI homozygote female mice have hypoplastic uterine tissue and rudimentary mammary glands similar to ERα-KO mice. Female mice were infertile as a result of anovulation from hemorrhagic cystic ovaries and elevated serum LH and E2 levels, although the mutant ERα protein is expressed in the AF2ERKI model. The AF2ERKI phenotype suggests that AF-1 is not activated independently, even with high serum E2 levels. ICI and TAM induced uterotropic and ER-mediated gene responses in ovariectomized AF2ERKI female mice in the same manner as in TAM- and E2-treated WT mice. In contrast, ICI and TAM did not act as agonists to regulate negative feedback of serum LH or stimulate pituitary prolactin gene expression in a different manner than TAM- or E2-treated WT mice. The functionality of the mutant ERα in the pituitary appears to be different from that in the uterus, indicating that ERα uses AF-1 differently in the uterus and the pituitary for TAM action.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Transcription function of AF2ER (L543A, L544A mutated ERα). (A) Schematic illustration of the ERα mutants. (B) HepG2 or HeLa cells were transfected with the reporter gene (3xERE-TATA-luc) and expression vectors for WT or mutated receptors were maintained with or without ligands. (C) HepG2 or HeLa cells were transfected with the reporter gene (7xAP1-TATA-luc) and expression vectors for WT or AF2ER. The cells were maintained with or without ligands. The luciferase activities for the each treatment were represented as fold change for the empty expression vector, pcDNA3 (no ERα). (D) Transrepression function of WT ERα and AF2ER. HeLa cells were transfected with the NF-κB reporter gene (3xMHC-luc), and expression vectors for p65/RelA and WT ERα or AF2ER were maintained with or without ligands. The luciferase activities were expressed relative to p65 activity in the absence of ligand (100%). (E) The effect of cofactors on ICI- or OHT-dependent AF2ER activation. HeLa cells were transfected with the 3xERE reporter gene and expression vectors for cofactors and AF2ER. The cells were maintained with or without ligands (100 nM E2, ICI, or OHT were used for treatments). Luciferase activity is represented as the mean ± SD of three independent experiments.
Fig. 2.
Fig. 2.
Targeting strategy and confirmation of L543A, L544A ERα knock-in mutation. (A) Schematic illustration of the targeting strategy used to introduce mutation. Diagrams show the WT ERα locus, targeting construct, targeted mutant allele in the ES cells/chimera mice, and F1 mutant allele after ACN cassette self-excision. The targeting construct contained ERα exon 9 (light gray boxes show coding sequence and dark gray box shows 3′ UTR), the L543A, L544A mutations (“mutation”), an extra XbaI site (Xb), and a 6xHis-tag epitope (6xHis). The ACN cassette was flanked at the 5′ and 3′ ends by loxP sites (closed arrowheads). Open box suggests the position of 5′ external probe for Southern blot (5′Ex), and pairs of open arrowheads suggest PCR primer sets for 3′ external PCR (3′Ex PCR), His-tag PCR, and PCR genotyping. D, DrdI; Xm, XmnI; B, BamHI; Xh, XhoI; H, HindIII. (B) Representative results of Western blot probed for the ERα, His-tagged ERα (His-Tag), and β-tubulin in the 8-wk-old individual mouse uterus are shown. β-Tubulin was used as a loading control. +/+, WT; KI/+, heterozygote; KI/KI, homozygote. (C) Uterine ERα immunohistochemistry of 8-wk-old representative mice. (D) Morphology of AF2ERKI female reproductive organs in the 8-wk-old representative mice. (E) Histology of 8-wk-old representative AF2ERKI female mice. Uterine (Top) and ovarian (Middle) tissue H&E staining from WT (Left) and AF2ERKI homozygote (Right) mice. (Scale bar: 100 μm.) Mammary gland (Bottom) whole-mount Carmine alum staining from 8-wk-old representative mice. (Scale bar: 1 cm.) (F) The mRNA level of Foxa2 was quantified by real-time PCR. The mRNA levels were represented as fold change for the WT. Values are presented as mean ± SD.
Fig. 3.
Fig. 3.
Assessment of AF2ER function in the AF2ERKI mouse uterus. (A) Uterine wet weight after vehicle (Veh), ICI (2 mg/kg), TAM (2 mg/kg), E2 10 μg/kg, or E2 2 mg/kg treatments for three consecutive days. Values are presented as mean ± SEM. (B) Uterine histology after vehicle, ICI, TAM, or E2 (10 μg/kg) treatments for three consecutive days in representative mice. (C) ICI and TAM induce the proliferation of endometrial epithelial cells in AF2ERKI uterus. Uterine EdU incorporation was analyzed. Hoechst was used as a counterstain to visualize tissue. (D) The mRNA levels of Ltf, Igf1, and Cox7a2l genes were quantified by real-time PCR. The mRNA levels were represented as fold change vs. vehicle treatment of WT. Values are shown as mean ± SD; *P < 0.05 vs. vehicle in each genotype. (E) Representative results of Western blots probed for ERα and β-tubulin from the vehicle- and ICI-treated individual mouse uteri are shown. The ERα level was normalized to the level of β-tubulin in each sample and presented as fold change vs. vehicle in each group.
Fig. 4.
Fig. 4.
Functional difference of AF2ER in the AF2ERKI mouse pituitary. (A) Serum LH levels were determined after vehicle (Veh), ICI (2 mg/kg), TAM (2 mg/kg), or E2 (10 μg/kg) treatments for three consecutive days. Values are presented as mean ± SEM; *P < 0.05 vs. vehicle in each genotype. The pooled serum from the non-OVX WT and αERKO female mice was used as reference. (B) The mRNA level of Lhb was quantified by real-time PCR. (C) The mRNA level of Prl was quantified by real-time PCR. The mRNA levels are presented as fold change for vehicle of WT. Values are presented as mean ± SD; *P < 0.05 vs. vehicle in each genotype. (D) The mRNA level of Pit1 was quantified by real-time PCR. The mRNA levels are presented as fold change vs. WT. Values are presented as mean ± SD. (E) Representative results of Western blots probed for the ERα, His-tagged ERα, and β-tubulin from WT and AF2ER homozygote individual mouse uteri and pituitaries are shown.
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References

    1. Brzozowski AM, et al. Molecular basis of agonism and antagonism in the oestrogen receptor. Nature. 1997;389:753–758. - PubMed
    1. Wu YL, et al. Structural basis for an unexpected mode of SERM-mediated ER antagonism. Mol Cell. 2005;18:413–424. - PubMed
    1. Danielian PS, White R, Lees JA, Parker MG. Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors. EMBO J. 1992;11:1025–1033. - PMC - PubMed
    1. Valentine JE, Kalkhoven E, White R, Hoare S, Parker MG. Mutations in the estrogen receptor ligand binding domain discriminate between hormone-dependent transactivation and transrepression. J Biol Chem. 2000;275:25322–25329. - PubMed
    1. Mahfoudi A, Roulet E, Dauvois S, Parker MG, Wahli W. Specific mutations in the estrogen receptor change the properties of antiestrogens to full agonists. Proc Natl Acad Sci USA. 1995;92:4206–4210. - PMC - PubMed

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