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. 2011 Aug 9;108(32):13311-6.
doi: 10.1073/pnas.1105632108. Epub 2011 Jul 25.

Activation function 2 (AF2) of estrogen receptor-alpha is required for the atheroprotective action of estradiol but not to accelerate endothelial healing

Audrey Billon-Galés  1 , Andrée Krust, Coralie Fontaine, Anne Abot, Gilles Flouriot, Céline Toutain, Hortense Berges, Alain-Pierre Gadeau, Françoise Lenfant, Pierre Gourdy, Pierre Chambon, Jean-François Arnal
Affiliations

Activation function 2 (AF2) of estrogen receptor-alpha is required for the atheroprotective action of estradiol but not to accelerate endothelial healing

Audrey Billon-Galés et al. Proc Natl Acad Sci U S A. .

Abstract

17β-Estradiol (E2) regulates estrogen receptor-α (ERα) target gene transcription through the two independent activation functions (AFs), AF1 and AF2, located in the N-terminal and ligand binding domain of ERα, respectively. We previously reported that ERα is required for the E2 atheroprotective action as well as for its accelerative action on endothelial healing, but its AF1 function is dispensable. Here, we investigated the role of ERαAF2 in these two major beneficial actions of E2 by electively targeting ERαAF2 (named ERαAF2(0)). Our results prove four points. (i) Compared with WT ERα, the ability of ERαAF2(0) to stimulate the C3 complement or the estrogen response element-thymidine kinase promoter in two cell lines was dramatically decreased, confirming the importance of AF2 in the E2-induced transcriptional activity of ERα. (ii) The uterotrophic action of E2 was totally absent in ERαAF2(0) mice, showing the crucial role of ERαAF2 in E2-induced uterus hyperplasia. (iii) ERαAF2 was dispensable for the accelerative action of E2 on endothelial healing, underlining the functionality of ERαAF2(0) in vivo. (iv) Finally, the atheroprotective effect of E2 was abrogated in ERαAF2(0) LDL-r(-/-) mice. Thus, whereas ERαAF1 and ERαAF2 are both required for the uterotrophic action of E2, we show that only ERαAF2 is necessary for its atheroprotective effect.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Generation of ERαAF20 null mutant mice. Schematic representation of the ERα gene: the transcripts and proteins expressed in WT mice (Upper) and mice with specific inactivation of the activation function AF2 in ERα (ERαAF20 mutant mice; Lower).
Fig. 2.
Fig. 2.
Validation of the excision of the ERαAF2 function. HeLa (A) and HepG2 (B) cells were transiently transfected with the ERE-TK LUC or C3-LUC reporter constructs in the presence of pCR-ERα, pCR- ERαAF20, or empty pCR vectors. Cells were treated with E2 (10 nM) or vehicle (control) 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.
Fig. 3.
Fig. 3.
ERαAF2 is necessary for the E2 uterotrophic action. (A) Representative Western blot of ERα and β-actin protein levels (Upper). Proteins from uteri of 10-wk-old ERαAF2+/+ and ERαAF20 nonovariectomized mice were loaded as described in Methods. Protein levels (n = 4 for each genotype) were quantified using ImageJ software (Lower). (B) Uterine weight from ERαAF2+/+ and ERαAF20 ovariectomized mice treated or not treated with E2 (80 μg/kg per d).
Fig. 4.
Fig. 4.
ERαAF2 is dispensable for the effect of E2 on endothelial healing. (A) Representative en face confocal immunohistochemical analysis of the intima tunica of the carotid artery from an E2-treated mouse 72 h after surgery with designation of the regenerative endothelial area. Nuclei, stained with propidium iodide, appear in dark blue. (B) The effect of E2 was studied in the ERαAF2+/+ and ERαAF20 ovariectomized mice. Quantification (mean ± SEM) of the regenerative endothelial area was from an average of four mice per group. Analysis by a two-way ANOVA: effect of exogenous E2, P = 0.01; effect of genotype, not significant (P = 0.78); interaction, not significant (P = 0.45).
Fig. 5.
Fig. 5.
ERαAF2 is necessary for the effect of exogenous E2 on fatty streak deposit prevention in 18-wk-old mice. Four-week-old ovariectomized ERαAF2+/+LDL-r −/− and ERαAF20LDL-r −/− mice were given either placebo or E2 (80 μg/kg per d during 12 wk) and switched to an atherogenic diet from the age of 6–18 wk. (A) Representative micrographs of oil red-O lipid-stained cryosections of the aortic sinus. (B) Quantification of lesion area at the aortic sinus. Analysis was performed with a two-way ANOVA; because an interaction was observed between the two factors, effect of E2 treatment was studied in each genotype using a Bonferroni posttest (***P < 0.001).
Fig. 6.
Fig. 6.
ERαAF2 is necessary for the effect of exogenous E2 on aorta gene regulation in vivo. Four-week-old ovariectomized ERαAF2+/+LDL-r−/− and ERαAF20LDL-r−/− mice were given either placebo or E2 (80 μg/kg per d during 12 wk) and switched to an atherogenic diet from the age of 6–18 wk. VCAM-1 (A), ICAM-1 (B), UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase-like 2 (Galntl2) (C), and Gremlin 2 (D) mRNA levels from aortas were quantified by quantitative PCR and normalized to HPRT mRNA levels. Results are expressed according to the level in aortas from the ERαAF2+/+LDL-r−/− set as one. Analysis was with a two-way ANOVA; because an interaction was observed between the two factors, effect of E2 treatment was studied in each genotype using a Bonferroni posttest (***P < 0.001).
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