Vascular cell signaling by membrane estrogen receptors

Abstract

The definition of estrogen's actions has expanded from transcriptional regulation to the rapid, membrane-initiated activation of numerous signal transduction cascades. Multiple biological effects of estrogen have been shown in numerous animals, cellular and molecular studies, which support the favorable effects of estrogen on vascular structure, function, and cell signaling. Work from several laboratories has shown that these effects are mediated by distinct forms of estrogen receptor (ER) alpha. This includes estrogen-stimulated rapid activation of endothelial nitric oxide synthase (eNOS), resulting in the elaboration of the athero-protective, angiogenesis-promoting product nitric oxide (NO). We have described the expression of ER46, an N-terminus truncated isoform of the ERalpha, in human endothelial cells (EC), and its critical role in membrane-initiated, rapid responses to 17beta-estradiol (E2). We have proposed an ER46-centered, eNOS activating molecular complex in human EC caveolar membranes, containing c-Src, phosphatidylinositol 3-kinase (PI3K), Akt and eNOS. Our previous studies support estrogen-induced rapid eNOS activation via a sequential c-Src/PI3K/Akt cascade in EC. In this review, we describe estrogen-induced, rapid, non-genomic actions in endothelium, driven by c-Src-ER46-caveolin-1 interactions, with consequent activation of eNOS. Amidst ongoing controversies in hormone replacement therapy, these molecular and cellular data, defining favorable estrogenic effects on the endothelium, provide a strong impetus to resolve these clinical questions.

Fig. 1. c-Src-dependent basal and E2-induced vasorelaxation

(A) Impaired E2 induced vasodilation in PE-preconstricted c-Src−/− aortas. Control and Src−/−thoracic aorta rings were isolated, preconstricted with 10⁻⁶ M PE, treated with E2, and the vascular tone was recorded by vessel myograph system. (B) Partially NO and receptor-dependent vasorelaxation (10⁻⁸ M E2, 10⁻⁵ M ICI 182,780, 10⁻⁴ M L-NAME). (C) Suppression of basal NO-dependent vasomotion in c-Src−/− aortas. Rings were pretreated with L-NAME (10⁻⁴ M) 20 min before PE preconstriction and remained in the bath throughout the experiment. *, p<0.05 (two-tailed ANOVA). Reprinted with permission [14].

Fig. 2. Interdependent incorporation of c-Src and ER46 in plasma membranes

COS-7 cells were transfected with ER46, avian Src or avian SrcG2A, treated with 30 nM E2 for 10 min, fractionated and immunoblotted. Reprinted with permission [14].

Fig. 3. Requirement of membrane c-Src kinase activity for eNOS activation in ECs

EA.hy926 cells electroporated with Src or SrcG2A were treated with 30 nM E2 or E2BSA for 10 min, fractionated, immunoblotted (A), and subjected to eNOS activity assay (n=3) (B). Reprinted with permission [14].

Fig. 4. Requirement of c-Src SH2 or SH3 domain in ER46-c-Src interaction

COS-7 cells were transfected with ER46, eNOS and the indicated avian c-Srcs, E2-deprived for 48 h, serum-starved for 12 h, and stimulated with 33 nM E2 for 10 min. Cell lysates were used for immunoprecipitation with F-10 (anti-ERα C-terminal antibody), and the co-immunoprecipitated avian c-Src was examined by immunoblotting. SrcW118K: c-Src SH3 mutant; SrcR175L: c-Src SH2 mutant. Reprinted with permission [14].

Fig. 5. Caveolin-1-ER46 colocalization in endothelial cells

Confocal images of cell-surface caveolin-1 and ER46 with anti-cav-1 and anti-ERα (F10) antibodies. Arrows indicate caveolin-1 and ER46 colocalization in digitonin-permeabilized, fixed EA.hy926 cells. Reprinted with permission [13].