The transactivating function 1 of estrogen receptor alpha is dispensable for the vasculoprotective actions of 17beta-estradiol

Abstract

Full-length 66-kDa estrogen receptor alpha (ERalpha) stimulates target gene transcription through two activation functions (AFs), AF-1 in the N-terminal domain and AF-2 in the ligand binding domain. Another physiologically expressed 46-kDa ERalpha isoform lacks the N-terminal A/B domains and is consequently devoid of AF-1. Previous studies in cultured endothelial cells showed that the N-terminal A/B domain might not be required for estradiol (E2)-elicited NO production. To evaluate the involvement of ERalpha AF-1 in the vasculoprotective actions of E2, we generated a targeted deletion of the ERalpha A/B domain in the mouse. In these ERalphaAF-1(0) mice, both basal endothelial NO production and reendothelialization process were increased by E2 administration to a similar extent than in control mice. Furthermore, exogenous E2 similarly decreased fatty streak deposits at the aortic root from both ovariectomized 18-week-old ERalphaAF-1(+/+) LDLr(-/-) (low-density lipoprotein receptor) and ERalphaAF-1(0) LDLr (-/-) mice fed with a hypercholesterolemic diet. In addition, quantification of lesion size on en face preparations of the aortic tree of 8-month-old ovariectomized or intact female mice revealed that ERalpha AF-1 is dispensable for the atheroprotective action of endogenous estrogens. We conclude that ERalpha AF-1 is not required for three major vasculoprotective actions of E2, whereas it is necessary for the effects of E2 on its reproductive targets. Thus, selective ER modulators stimulating ERalpha with minimal activation of ERalpha AF-1 could retain beneficial vascular actions, while minimizing the sexual effects.

Fig. 1.

Generation and validation of ERα AF-1⁰ mice. (A) Schematic representation of the wild-type ERα gene (Esr1) and the targeted AF1⁰ allele (the gray area in exon 1 shows the sequence deleted in ERαAF-1⁰ mice corresponding to amino acids 2–148). The two physiologically-expressed ERα isoforms of 66-kDa (full length) and 46 kDa (AF-1 deficient) and the 49-kDa ERα protein expressed in ERα AF-1⁰ mice are represented. (B) ERα protein level were assessed by Western blot analysis of 50 μg of protein from ERα^+/+, ERα^−/−, and ERα AF-1⁰ mice uteri. (C) Uterine weight from ERα^+/+, ERα^−/−, and ERαAF-1⁰⁻ mice treated or not with E2 (80 μg/kg per day for 2 weeks).

Fig. 2.

ERα AF-1 function is dispensable in the effect of E2 on basal NO production and endothelial healing. The effect of E2 was studied in ERα^+/+ and ERα AF-1⁰ ovariectomized mice. (A) The basal NO release of aortic rings from mice treated either with placebo (filled bars) or E2 (empty bars) was evaluated from the N^G-nitro-l-arginine (100 μM)-induced contraction in rings precontracted with U-46619 (7.5 nM). Data were analyzed by two-way ANOVA, and no significant interaction (P = 0.83) was observed. (B) (Upper) Representative en face confocal immunohistochemical analysis of the intima tunica from a E2-treated mouse, 72 h after surgery with designation of the reendothelialized area, retrograde proliferating zone (RetroP), and regenerative endothelial area. Nuclei, stained with propidium iodide, appear in dark blue, and proliferating BrdU-positive cells appear in light blue. (Scale bar: 50 μm.) (Lower) Quantification (mean ± SEM) of the length of the above-mentioned zones from an average of 4 mice per group. Data were analyzed by two-way ANOVA, and no significant interaction was observed for RetroP length (P = 0.91) or reendothelialized area (P = 0.51).

Fig. 3.

Exogenous E2 prevents fatty streak deposits in 18-week-old ERαAF-1⁰ mice. Four-week-old ovariectomized ERαAF-1^+/+LDLr^−/− and ERαAF-1⁰LDLr^−/− mice were given either placebo or E2 (80 μg/kg per day for 12 weeks) and switched to atherogenic diet from the age of 6–18 weeks. (A) Representative micrographs of Oil red-O lipid stained cryosections of the aortic sinus. (Scale bars: 200 μm.) (B) Quantification (mean ± SEM) of lesion area at the aortic sinus from an average of 7–8 mice per group. Data were analyzed by two-way ANOVA, and no significant interaction (P = 0.85) was observed.

Fig. 4.

The atheroprotective effect of endogenous estrogens persists in 8-month-old ERαAF-1⁰ mice. Four-week-old ERαAF-1^+/+ LDLr^−/− and ERαAF-1⁰ LDLr^−/− mice were ovariectomized (OVX) or not (SHAM) and switched to atherogenic diet from the age of 6 weeks until euthanization. (A) Representative en face aorta preparations (1.8× magnification) from groups of 5 mice. (B) Quantification of lesions (mean ± SEM) from the thoracic and the abdominal aorta expressed as percentage of total aorta area. Data were analyzed by two-way ANOVA, and no significant interaction was observed for either thoracic (P = 0.87) or abdominal (P = 0.76) aortae.