Mutation of the palmitoylation site of estrogen receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions

Abstract

Estrogen receptor alpha (ERα) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERα is targeted to plasma membrane and elicits membrane-initiated steroid signaling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss of function of ERα. The abrogation of membrane localization of ERα in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERα mice and endometrial epithelial proliferation was similar to wild type. However, E2 vascular actions such as rapid dilatation, acceleration of endothelial repair, and endothelial NO synthase phosphorylation were abrogated in C451A-ERα mice. A complementary mutant mouse lacking the transactivation function AF-2 of ERα (ERα-AF2(0)) provided selective loss of function of nuclear ERα actions. In ERα-AF2(0), the acceleration of endothelial repair in response to estrogen-dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERα-AF2(0), whereas in C451A-ERα it was nearly fully preserved, indicating that membrane-to-nuclear receptor cross-talk in vivo is modest in the uterus. Thus, this work genetically segregated membrane versus nuclear actions of a steroid hormone receptor and demonstrated their in vivo tissue-specific roles.

Keywords: fertility; genomic actions; nongenomic effects; vascular effects.

Fig. 1.

Validation of the C451A-ERα mouse model. (A) ERα protein levels in uterus, aorta, and hepatocytes homogenates from ovariectomized WT-C451 and C451A-ERα mice. (B) Representative Western blot of plasma membrane-associated ERα isolated from WT-C451 or C451A-ERα hepatocytes by sucrose gradient. Quantification of plasma membrane-associated ERα in WT-C451 or C451A-ERα hepatocytes, n = 3. Results are normalized to annexin II expression. (C) Phospho-MAPK/MAPK abundance in hepatocytes treated or not with E2 (10⁻⁷ M, 15 min), from WT-C451 and C451A-ERα mice, n = 3. Representative Western blot is shown. AU, arbitrary units.

Fig. 2.

Adult ovarian phenotype is disturbed in the C451A-ERα mice. (A) Representative images of WT-C451 and C451A-ERα ovaries (×25). CL, corpus luteum; HCF, hemorrhagic cystic follicles. (B) Percentages of corpora lutea and hemorrhagic/cystic follicles were determined in ovaries from 3-mo-old WT-C451 and C451A-ERα mice, n = 4. (C) Serum E2, progesterone (PG), FSH, and LH concentrations in 3-mo-old WT-C451 and C451A-ERα mice, n = 9.

Fig. 3.

Uterine proliferative response is maintained in C451A-ERα mice. (A) Representative images of intact uterus. (B) Uterine wet weight in ovariectomized WT-C451 and C451A-ERα mice treated or not with chronic (28 d) E2 treatment (8 µg/kg per day), n = 4–6. (C) Representative Ki-67 immunodetection in uterus sections from WT-C451A and C451A-ERα mice treated or not with E2 for 24 h. (Scale bar, 100 µm.) (D) Percentages of Ki-67–positive cells in epithelium and (E) luminal epithelial height (LEH) were measured, n = 4–8. For B, D, and E, statistical results of two-way ANOVA are shown, explaining the effect of treatment (Treat.), genotype (Gen.), and the interaction (Inter.) between these two variables.

Fig. 4.

Large-scale gene analysis on response to E2 on C451A-ERα and ERα-AF2⁰ mice. (A) Gene expression profiles were obtained by microarray analysis (Agilent SurePrint G3 Mouse GE, 8 × 60K) of uterine samples from C451A-ERα, ERα-AF2⁰ mice or their littermate WT controls treated with E2 (8 µg/kg, 6 h) or vehicle (Veh), n = 3–4. The heat map represents the data obtained on all samples for the differentially expressed probes exhibiting interaction between genotype and treatment (BH adjusted P < 0.001). (B) The Venn diagram illustrates the overlaps between the genes significantly (P < 0.05) up- or down-regulated in the uterus following E2 treatment in ERα-AF2⁰ and C451A-ERα mice. (C) The Venn diagram illustrates the overlaps between the genes significantly (BH adjusted P < 0.05) up- or down-regulated in the uterus following interaction between E2 treatment and genotypes, respectively, ERα-AF2⁰ and C451A-ERα mice.

Fig. 5.

Membrane-initiated endothelial effects of E2 are abrogated in C451A-ERα mice. (A) Percentage of relaxation of mesenteric arteries from WT-C451, ERα ^−/−, and C451A-ERα mice following addition of E2 (1 µM) or EDC (ethinyl-estradiol dendrimer conjugate, 1 µM), n = 8–9. (B) Phospho-eNOS/eNOS abundance in carotid arteries from WT-C451, ERα ^−/−, and C451A-ERα mice treated or not with E2 (10⁻⁸ M, 30 min), n = 6. Representative Western blot is shown. AU, arbitrary units. (C) Percentage of migration of primary endothelial cells treated or not with E2 (10⁻⁹ M, 16 h), n = 3–4. (D) Carotid artery reendothelialization in ovariectomized WT-C451 and C451A-ERα mice treated or not with E2 (80 µg⋅kg^–1⋅d^–1, 2 wk), n = 8–10. For C and D, statistical results of two-way ANOVA are shown, reporting the statistical interaction between treatment and genotype.

Fig. 6.

Membrane effects of ERα are preserved in ERα-AF2⁰ mutant mice. Carotid artery reendothelialization in ovariectomized WT-AF2 and AF2-ERα mice treated with E2 (80 µg⋅kg^–1⋅d^–1, 2 wk) or vehicle (Veh) and EDC (240 µg⋅kg^–1⋅d^–1, 2 wk) or control dendrimer, n = 8–10. Statistical results of two-way ANOVA are shown, explaining the effect of treatment, genotype, and interaction between these two variables.