Analysis of Estrogen Receptors Alpha Versus Beta in the Uterine Endothelial Cell Plasma Membrane and Their Interaction with Cav-1 for eNOS Activation.

Abstract

Uterine endothelial nitric oxide (NO) production is partly responsible for the maintenance of vasodilatation during physiologic states of high circulating estrogen levels such as pregnancy. Estrogen receptors (ER-a and/or ER-β) are classically thought of as nuclear transcription factor receptors; however, a small pool (3-5%) of ERs is localized to the plasma membrane of endothelial cells that are responsible for nongenomic vasodilatory responses. We and others have previously shown that ERs induce very rapid activation of ERK signaling and eNOS to increase NO production. In addition, these rapid changes were observed with estradiol-17β bound to plasma membrane impermeable BSA and the NO responses were ER-mediated since they were blocked by ICI, 182,780. Various estrogenic effects have been shown to be mediated by one or both ERs and may be dependent on interactions with Cav-1, the main caveolae scaffolding protein. It is unclear whether rapid NO productions are mediated by plasma membrane Cav-1 associated ER-a and/or ER-β. We hypothesize that ER-a and ER-β maintain similar spatial partitioning between the plasma membrane and nucleus of Uterine Artery Endothelial Cell (UAECs), similar interactions with Cav-1 protein at the plasma membrane, and are capable of changing the eNOS phosphorylation patterns indicative of temporal and special NO activation state. UAECs lysates were subjected to Immunoisolation column chromatography using Cav-1 antibody to identify protein-protein interactions between Cav-1 and ER-a or ER-β. Transmission Electron Microscopy (TEM) was employ to visualized caveolae structures at the plasma membrane of UAECs under control and estrogen treatment. TEM ImmunoGold label visualization was employed to localize ER-a and ER-β at the UAEC plasma membrane and within the caveolae structures. Lastly, UAECs were treated with control or increasing concentrations of estradiol-17β (0.1-100nM) and changes in eNOS stimulatory phosphorylation sites, Ser 1177, Ser 635 vs. inhibitory site Thr 495 were evaluated via Western blot analyzes. UAEC protein Cav-1 immunoisolation columns showed that ER-a was tightly bound to Cav-1. In contrast, Cav-1 did not appear to interact with ER-β with high affinity. TEM revealed that UAECs have substantial numbers of caveolae structures on the plasma membrane. Immunogold labeling revealed that ER-a has relatively low expression bet very discrete focal caveolar allocations within the UAEC plasma membrane. However, ER-β showed a much higher expression patterns throughout the cell but similar focal patterns as ER-a at the plasma membrane. Increasing estradiol-17β concentrations directly increased stimulatory Ser 1177, Ser 635 phosphorylation with a concurrent decrease in inhibitory Thr 495 phosphorylation in eNOS, indicating elevations in eNOS activation. These data support the hypothesis that plasma membrane ERs mediate NO production through the activation of signaling cascades that alter eNOS multi-site phosphorylation state, protein-protein interactions with Cav-1, and its temporal spatial distribution. NIH GM083252, HL49210, HD38843, HL87144, AA19446.