Estradiol-17beta and its cytochrome P450- and catechol-O-methyltransferase-derived metabolites stimulate proliferation in uterine artery endothelial cells: role of estrogen receptor-alpha versus estrogen receptor-beta

Abstract

Estradiol-17beta (E(2)beta) and its metabolites, which are sequentially synthesized by cytochrome P450s and catechol-O-methyltransferase to form 2 and 4-hydroxyestradiol (OHE(2)) and 2- and 4-methoxestradiol (ME(2)), are elevated during pregnancy. We investigated whether cytochrome P450s and catechol-O-methyltransferase are expressed in uterine artery endothelial cells (UAECs) and whether E(2)beta and its metabolites modulate cell proliferation via ER-alpha and/or ER-beta and play roles in physiological uterine angiogenesis during pregnancy. Cultured ovine UAECs from pregnant and nonpregnant ewes were treated with 0.1 to 100.0 nmol/L of E(2)beta, 2-OHE(2), 4-OHE(2), 2-ME(2), and 4-ME(2). ER-alpha or ER-beta specificity was tested using ICI 182 780, ER-alpha-specific 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinyleth oxy)phenol]-1H-pyrazole dihydrochloride, ER-beta-specific 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo [1,5-a]pyrim idin-3-yl]phenol antagonists and their respective agonists ER-alpha-specific 4,4',4"-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol and ER-beta-specific 2,3-bis(4-Hydroxyphenyl)-propionitrile. Angiogenesis was evaluated using 5-bromodeoxyuridine proliferation assay. Using confocal microscopy and Western analyses to determine enzyme location and levels, we observed CYP1A1, CYP1A2, CYP1B1, CYP3A4, and catechol-O-methyltransferase expression in UAECs; however, expressions were similar between nonpregnant UAECs and pregnant UAECs. E(2)beta, 2-OHE(2), 4-OHE(2), and 4-ME(2) treatments concentration-dependently stimulated proliferation in pregnant UAECs but not in nonpregnant UAECs; 2-ME(2) did not stimulate proliferation in either cell type. Proliferative responses of pregnant UAECs to E(2)beta were solely mediated by ER-beta, whereas responses to E(2)beta metabolites were neither ER-alpha nor ER-beta mediated. We demonstrate an important vascular role for E(2)beta, its cytochrome P450- and catechol-O-methyltransferase-derived metabolites, and ER-beta in uterine angiogenesis regulation during pregnancy that may be dysfunctional in preeclampsia and other cardiovascular disorders.

Figure 1

(A) Immunoblots showing expression of CYP1A1, CYP1A2, CYP1B1, CYP3A4, COMT, and GAPDH in NP-UAECs and P-UAECs. (B) Densitometric analyses (Relative protein expression = enzyme expression OD /GAPDH OD) showed no difference between NP-UAECs (n=6) and P-UAECs (n=6); (P=0.949, One-Way ANOVA).

Figure 2

Immunofluorescence microscopy showing intracellular localization of (A) CYP1A1, (B) CYP1A2, (C) CYP1B1, (D) CYP3A4, (E) COMT and (F) Negative Control in P-UAECs. Positive staining is green fluorescence with nuclei depicted in blue (DAPI). Pictures are representative of three experiments.

Figure 3

Concentration-dependent cell proliferation responses of NP-UAECs and P-UAECs to (A) E₂β, (B) 2-OHE₂, (C) 4-OHE₂, (D) 2-ME₂ and (E) 4-ME₂. A biphasic proliferative response was observed in P-UAECs in response to E₂β, 2-OHE₂, and 4-OHE₂ but not 4-ME₂ compared to control with maximum responses at a physiologic concentration of 0.1 nmol/L (Two-Way ANOVA; Pregnancy × Concentration effect; E₂β, F_4,40=8.16, P<0.0001; 2-OHE₂, F_4,40=4.07, P=0.0073;_, 4-OHE₂, F_4,40=3.69, P=0.0119; and 4-ME₂, F_4,40=5.05, P=0.002). NP-UAECs did not respond to E₂β or its metabolites. No proliferation effect was observed with 2ME₂. *Indicates an increase (P<0.05; n=6) in P-UAEC proliferation compared with both the respective NP-UAEC (n=7) group and untreated control.

Figure 4

The effects of 1 μmol/L ICI on P-UAEC proliferative responses to 0.1 nmol/L of E₂β, 2-OHE₂, 4-OHE₂, 2-ME₂, and 4-ME₂. ICI abrogated the response of P-UAECs to E₂β but not in response to 2-OHE₂, 4-OHE₂ and 4-ME₂ respectively (Two-Way ANOVA; Antagonist × Group effect; F_5,60=25.272, P<0.001.*Indicates an increase (P<0.05, n=6) in P-UAEC proliferation compared to untreated control. τ Indicates inhibition (P<0.05) of P-UAEC proliferation with ICI; λ indicates lower P-UAEC proliferation (P<0.05) compared to E₂β responses alone.

Figure 5

The effects of the ER-α antagonist MPP (1μmol/L) on P-UAEC proliferation responses to 0.1 nmol/L of E₂β, 2-OHE₂, 4-OHE₂, 2-ME₂ and 4-ME₂. MPP had no effect on the proliferation responses of P-UAECs to 0.1 nmol/L of E₂β, 2-OHE₂, 4-OHE₂, 2-ME₂ and 4-ME₂ (Two-Way ANOVA; Group effect, F_5,60=14.315, P<0.001). Neither a main effect of MPP nor an interaction was noted. *Indicates an increase (P<0.05; n=6) in P-UAEC proliferation compared to untreated control; λ indicates lower P-UAEC proliferation (P<0.05) compared to E₂β responses alone.

Figure 6

The effects of the ER-β antagonist PHTPP (1μmol/L) on P-UAEC proliferative responses to 0.1 nmol/L of E₂β, 2-OHE₂, 4-OHE₂, 2-ME₂, and 4-ME₂ (Two-Way ANOVA; Antagonist × Group effect; F_5,60=17.517, P<0.001. *Indicates an increase (P<0.05; n=6) in P-UAEC proliferation compared to untreated control. τ Indicates inhibition (P<0.05) of P-UAEC proliferation with PHTPP. λ Indicates lower P-UAEC proliferation (P<0.05) compared to E₂β responses alone.

Figure 7

Concentration-dependent effects of (A) ER-α agonist PPT (B) ER-β agonist DPN and (C) their combination on cell proliferation responses of P-UAECs. Blockade of ER-β with PHTPP (1μmol/L) prior to treatment with ER-β agonist DNP is shown in (C). *Indicates an increase (P<0.05; n=7) in P-UAEC proliferation compared to untreated controls. λ Indicates a difference (P< 0.05) in P-UAEC proliferation in response to DPN or the combination of DNP and PPT compared to E₂β only responses. τ Indicates inhibition (P<0.05) of P-UAEC proliferation with PHTPP.