Non-nuclear estrogen receptor alpha signaling promotes cardiovascular protection but not uterine or breast cancer growth in mice

Abstract

Steroid hormone receptors function classically in the nucleus as transcription factors. However, recent data indicate that there are also non-nuclear subpopulations of steroid hormone receptors, including estrogen receptors (ERs), that mediate membrane-initiated signaling of unclear basis and significance. Here we have shown that an estrogen-dendrimer conjugate (EDC) that is excluded from the nucleus stimulates endothelial cell proliferation and migration via ERalpha, direct ERalpha-Galphai interaction, and endothelial NOS (eNOS) activation. Analysis of mice carrying an estrogen response element luciferase reporter, ER-regulated genes in the mouse uterus, and eNOS enzyme activation further indicated that EDC specifically targets non-nuclear processes in vivo. In mice, estradiol and EDC equally stimulated carotid artery reendothelialization in an ERalpha- and G protein-dependent manner, and both agents attenuated the development of neointimal hyperplasia following endothelial injury. In contrast, endometrial carcinoma cell growth in vitro and uterine enlargement and MCF-7 cell breast cancer xenograft growth in vivo were stimulated by estradiol but not EDC. Thus, EDC is a non-nuclear selective ER modulator (SERM) in vivo, and in mice, non-nuclear ER signaling promotes cardiovascular protection. These processes potentially could be harnessed to provide vascular benefit without increasing the risk of uterine or breast cancer.

Figure 1. EDC does not activate nuclear ER function in cultured cells.

(A) HEK293 cells were transfected with either an ERα-based or an ERβ-based Gal4-ER-LBD chimera and the Gal4-responsive luciferase reporter plasmid TKMH100X4-Luc, and reporter activity was determined after 24 hours exposure to vehicle (control [Con]), 10^–8 M E₂, dendrimer (Dend) alone at a concentration equivalent to EDC, or EDC at a concentration equivalent to 10^–8 M E₂. (B) BAECs were cotransfected with 3ERE-Luc luciferase reporter plasmid and either empty vector or ERα construct, and reporter activity was determined after exposure to vehicle, E₂ or EDC as in A for 48 hours. In A and B, values are mean ± SEM, n = 3–4. *P < 0.05 versus Con; ^†P < 0.05 versus vector. (C–E) EDC does not enter the endothelial cell nucleus. BAECs were treated with Cy5-labeled EDC (red) or fluorescein-labeled estrogen (yellow) for 24 hours, and subcellular localization was evaluated by laser scanning confocal microscopy (C) or by epifluorescence microscopy (D and E). Nuclei were imaged with DAPI (blue). Scale bars in C–E: 20 μm.

Figure 2. Non-nuclear ERα activation stimulates eNOS via Gαi.

(A) eNOS activation was assessed in intact BAECs by measuring ¹⁴C-l-arginine to ¹⁴C-l-citrulline conversion during 15-minute incubations with vehicle (control), 10^–8 M E₂, EDC at a concentration equivalent to 10^–8 M E₂, or dendrimer alone at a concentration equivalent to EDC at 10^–8 M. EDC at 10^–10 M estrogen equivalents did not activate eNOS (data not shown). (B) Parallel experiments were performed in cells incubated with EDC (10^–8 M estrogen equivalents) in the absence or presence of 10^–5 M ICI 182,780 or 100 ng/ml pertussis toxin (PTX). In A and B, values are mean ± SEM, n = 4. *P < 0.05 versus control. (C) eNOS activation by EDC and VEGF (100 ng/ml) above basal activity (B) was evaluated following siRNA knockdown of ERα. Inset shows ERα abundance in control siRNA cells (lane 1) and ERα siRNA cells (lane 2). Values in C are mean ± SEM, n = 4. *P < 0.05 versus basal; ^†P < 0.05 versus control.

Figure 3. Non-nuclear ER activation stimulates endothelial cell proliferation and migration via ER, Gαi, Src, and eNOS and through direct interaction of the receptor with Gαi.

(A) Cell number was determined after 48 hours treatment with vehicle or dendrimer controls, or 10^–8 M E₂ or EDC (10^–8 M E₂ equivalents), in the absence or presence of 10^–5 M ICI 182,780, 100 ng/ml pertussis toxin, 10 μM PP2, or 2 mM L-NAME. (B and C) To evaluate migration, cells were wounded and treated as in A for 24 hours, and the number of cells that had migrated past the wound edge per high-power field was quantified. Images in B are representative optical fields. In A and C, values are expressed as percent of control treatment, mean ± SEM, n = 6. *P < 0.05 versus control; ^†P < 0.05 versus E₂ or EDC alone. (D) Migration was assessed in BAECs transfected with sham plasmid (control), plasmid encoding the dominant-negative mutant receptor ERαΔ250–260, or plasmid that expressed a peptide consisting of amino acids 251–260 of the receptor (ERα251–260). Treatments were vehicle (basal), 10^–8 M E₂, EDC (10^–8 M E₂ equivalents), 100 ng/ml VEGF, or 10% serum for 24 hours. Summary data are provided in E. White bars, cells transfected with sham plasmid (control); gray bars, cells transfected with the mutant receptor ERαΔ250–260; black bars, cells expressing ERα251–260 peptide. The approximately 50% attenuation of the migration responses to E₂ and EDC caused by the interventions is consistent with the transfection efficiency attained under these conditions. Values are mean ± SEM, n = 6. *P < 0.05 versus basal; ^†P < 0.05 versus control.

Figure 4. EDC distinguishes non-nuclear from nuclear ER function in vivo.

(A) Quantitative RT-PCR was performed to assess ERβ expression in the uteri of mice treated 24 days with vehicle, E₂, dendrimer, or EDC. iNOS (B) and C3 expression (C) were similarly evaluated. In A–C, values are mean ± SEM, n = 4. *P < 0.05 versus vehicle. (D) Using HEK293 cells cotransfected with 3ERE-Luc luciferase reporter plasmid and ERα cDNA, ER-dependent gene transcription was evaluated over 48 hours in response to serum from mice treated 24 days with vehicle, E₂, dendrimer, or EDC. Values are mean ± SEM, n = 5–8. *P < 0.05 versus vehicle. (E) ER-dependent, acute NOS activation was assessed over 15 minutes in BAECs by measuring ¹⁴C-l-arginine to ¹⁴C-l-citrulline conversion in the absence versus presence of 10^–5 M ICI 182,780. Responses to 10% serum from vehicle-, E₂-, dendrimer-, or EDC-treated animals were compared. Values are mean ± SEM, n = 8. *P < 0.05 versus vehicle or dendrimer.

Figure 5. Non-nuclear ER signaling via Gαi promotes endothelial monolayer integrity in vivo.

(A) Carotid artery reendothelialization was evaluated following perivascular electric injury in ovariectomized female mice. The intimal surface of Evans blue–stained arteries from vehicle- versus E₂-treated mice 3 days after injury is shown (upper panels). Area of denudation that incorporated dye was quantified and expressed in arbitrary units (lower panel). (B) Parallel experiments were performed in dendrimer- (Dend) versus EDC-treated mice. In A and B, values are mean ± SEM, n = 10–12. *P < 0.05 versus vehicle or dendrimer. In addition, the impact of ER antagonism with ICI 182,780 on reendothelialization was determined in E₂- (C) or EDC-treated mice (D). In C and D, values are mean ± SEM, n = 8–9. *P < 0.05 versus vehicle or dendrimer; ^†P < 0.05 versus no ICI 182,780. Furthermore, the requirement for G proteins was assessed in E₂- (E) or EDC-treated mice (F) also treated with B-oligomer (control) or pertussis toxin (PTX). In E and F, values are mean ± SEM, n = 8–9. *P < 0.05 versus vehicle or dendrimer; ^†P < 0.05 versus no PTX.

Figure 6. Non-nuclear ER signaling prevents neointima formation.

Neointima formation in the carotid artery was evaluated in ovariectomized female Apoe^–/– mice 2 weeks following endothelial denudation. Representative arteries are shown for mice treated with (A) vehicle, (B) E₂, (C) empty dendrimer, or (D) an estrogen equivalent amount of EDC. Scale bars in A–D: 100 μm. Summary data for intimal area are shown in E. Values are mean ± SEM, n = 14–17. *P < 0.05 versus vehicle or dendrimer; ^†P < 0.05 versus E₂.

Figure 7. Non-nuclear ER signaling does not induce a uterotrophic response or promote breast cancer tumor growth in vivo.

(A) Uteri from mice treated 24 days with vehicle, E₂, dendrimer, or EDC. (B) Uterine wet weight/body weight ratio. In additional experiments, uteri were obtained from mice that were also treated with either ICI 182,780 (C) or pertussis toxin (D). In B–D, values are mean ± SEM, n = 8–9. *P < 0.05 versus vehicle; ^†P < 0.05 versus no ICI 182,780. (E) MCF-7 cell tumor xenografts were established with E₂ treatment for 28 days in SCID mice, and treatment was then randomized to continue with E₂ for 21 days or switched to vehicle, dendrimer, or EDC for 21 days. Representative tumors are shown in E. (F) Tumor weight at end of study. In F, values are mean ± SEM, n = 11–18. *P < 0.05 versus vehicle.