The uterine and vascular actions of estetrol delineate a distinctive profile of estrogen receptor α modulation, uncoupling nuclear and membrane activation

Abstract

Estetrol (E4) is a natural estrogen with a long half-life produced only by the human fetal liver during pregnancy. The crystal structures of the estrogen receptor α (ERα) ligand-binding domain bound to 17β-estradiol (E2) and E4 are very similar, as well as their capacity to activate the two activation functions AF-1 and AF-2 and to recruit the coactivator SRC3. In vivo administration of high doses of E4 stimulated uterine gene expression, epithelial proliferation, and prevented atheroma, three recognized nuclear ERα actions. However, E4 failed to promote endothelial NO synthase activation and acceleration of endothelial healing, two processes clearly dependent on membrane-initiated steroid signaling (MISS). Furthermore, E4 antagonized E2 MISS-dependent effects in endothelium but also in MCF-7 breast cancer cell line. This profile of ERα activation by E4, uncoupling nuclear and membrane activation, characterizes E4 as a selective ER modulator which could have medical applications that should now be considered further.

Keywords: endothelium; estetrol; estrogen receptor; uterus.

Figure 1. Structure of E₂, E₃ and E₄ and their respective complexed structure with ERα ligand binding domain

A Chemical structures of E₂, E₃, and E₄. B, C Structure of ERαLBD complexed with E₂ (blue), E₃ (red), or E₄ (green). Shown are ribbon diagrams of the ERαLBD monomer. Ligand-binding site (B), shown in ball-and-stick rendering of the ligands along with their interacting residues. Hydrogen bonds are shown as dotted lines. Ligand-binding domain (C) and peptide fragment of the GRIP1 coactivator protein in complex with E₃ or E₄ only (darker red and darker green). Ligand is represented as a space-filled model. Position of the helix 12 is indicated by an arrow.

Figure 2. E₄ induces ERE transcriptional activity in a cellular context-dependent manner in vitro in a manner similar to that of E₂

A, B HeLa (A, B) and HepG2 (B) cells were transiently transfected with the ERE-TK-Luc reporter constructs in the presence of pCR-ERα, pCR-ERαΔ79, pCR-ERαAF-1⁰, or empty pCR vector. Cells were treated with indicated dose of E₂ and E₄ or vehicle (Ctrl) for 24 h. Normalized luciferase activities were expressed as fold increase above values measured with empty pCR and vehicle. Data correspond to the mean values ± SEM of at least three separate transfection experiments.

Figure 3. Comparison of E₂ and E₄ on uterine gene regulation in ovariectomized mice

Seven-week-old ovariectomized C57Bl/6J mice were subcutaneously injected with vehicle (Ctrl, castor oil), E₂ (8 μg/kg), or E₄ (1 mg/kg) and were euthanized 6 h after treatment.

1. Data obtained from 96.96 Dynamic Arrays were used to generate a cluster diagram of the significant gene expression changes. Each vertical line represents a single gene. Each horizontal line represents an individual sample. Genes that were up-regulated at least twofold following E₂ administration relative to placebo are in red, whereas down-regulated genes are in green. The color intensity indicates the degree of variation in expression.

2. Clustering pattern of the gene whose expression is affected by E₂ and/or E₄.

Figure 4. Comparison of E₂ and E₄ on uterine histological parameters and epithelial proliferation

Seven-week-old ovariectomized C57Bl/6J mice were injected subcutaneously with vehicle (Ctrl, castor oil), E₂ (8 μg/kg), and/or E₄ (200 μg/kg or 1 mg/kg) and were euthanized 24 h after treatment. A, B Luminal epithelial height (LEH) (A) and stromal height (SH) (B) were measured. C, D Representative (C) and quantification (D) of Ki-67 detection in transverse uterus sections (scale bar = 50 μm). Data information: Results are expressed as mean ± SEM. To test the respective roles of each treatment, a one-way ANOVA was performed and a Bonferroni's multiple comparison test (n = 4–6 mice/group).

Figure 5. E₄ prevents aortic sinus lipid deposition in hypercholesterolemic mice

Four-week-old ovariectomized ERα^+/+LDL-r^−/− or ERα^−/−LDL-r^−/− mice were switched to atherogenic diet from the age of 6–18 weeks added with placebo (Ctrl) or E₄ (0.6 or 6 mg/kg/day). A, B Representative micrographs of Oil red-O (ORO) lipid-stained cryosections of the aortic sinus (A) and quantification of lipid deposition (B) are represented. C Gremlin 2 (Grem2) mRNA level from aorta of these mice was quantified by qPCR and normalized to Tpt1 mRNA levels. Result was expressed according to the level in aorta from placebo set as 1. Data information: Results are expressed as mean ± SEM. Significance of the observed effects was evaluated using one-way or two-way ANOVA followed by Bonferroni's post hoc test (n = 4–8 mice/group).

Figure 6. E₄ fails to accelerate reendothelialization and to increase NO production

Seven-week-old ovariectomized C57Bl/6J mice were given placebo (Ctrl), E₂ (80 μg/kg/day) or E₄ (0.3–6 mg/kg/day), or E₂ (80 μg/kg/day) + E₄ (6 mg/kg/day) for 2 weeks.

1. Electric injury was applied to the distal part (3 mm precisely) of the common carotid artery, and the endothelial regeneration process was evaluated 3 days postinjury. Quantification of the reendothelialized area evaluated by Evans blue staining, and results were expressed as mean ± SEM (n = 7–23 mice per group). Significance of the observed effects was evaluated using one-way ANOVA followed by Bonferroni's post hoc test.

2. Quantification expressed as mean ± SEM (n = 7 mice per group, upper panel) and representative Western blot (lower panel) of phospho-eNOS/eNOS abundance in isolated aortae treated by E₂ (10⁻⁸ M), E₄ (10⁻⁶ M), combination of both E₂ and E₄ or acetylcholine (Ach) used as a positive control during 30 min. Significance of the observed effects was evaluated using one-way ANOVA followed by Bonferroni's post hoc test (n = 8 mice/group).

3. Representative trace of ex vivo amperometric measurements of NO release of aortae from 10- to 12-week-old C57Bl/6J mice exposed to E₂ (10⁻⁸ M) or E₄ (10⁻⁶ M) during 5 min.

4. For cotreatment experiment, E₄ (10⁻⁶ M) or vehicle (DMSO) was pre-incubated during 10 min prior to E₂ (10⁻⁸ M) treatment. To test the respective roles of each treatment, a one-way ANOVA was performed followed by a Bonferroni's post hoc test.

Source data are available online for this figure.

Figure 7. E₄ promotes ERα-src interaction less efficiently than does E₂ but induces similar ERE-dependent transcriptional activity in MCF-7

1. MCF-7 cells were grown in medium containing 2.5% charcoal-stripped serum with vehicle or with E₂ (10⁻⁸ M), E₄ (10⁻⁶ M) or in combination for 5 min. After fixation, in situ PLA for ERα-Src dimers was performed with ERα- and Src-specific antibodies. The detected dimers are represented by red dots, and the nuclei were counterstained with DAPI (blue). Quantification of the number of signals per cell was performed by computer-assisted analysis as reported in the Materials and Methods section. Values correspond to the mean ± SEM of at least three separate experiments, and columns with different superscripts differ significantly using Student's t-test.

2. mRNA level of the indicated gene from MCF-7 cells treated with vehicle, E₂ (10⁻⁸ M), E₄ (10⁻⁶ M) or combined treatment and analyzed after 24 h by qPCR. Values correspond to the mean ± SD of at least three separate experiments. To test the respective roles of each treatment, a one-way ANOVA was performed and a Bonferroni's multiple comparison test.