Abstract 877: Estrogen Dendrimer Conjugate Reveals that Estrogen-induced Endothelial Cell Migration and Proliferation are Mediated by Non-nuclear Estrogen Receptors that Interact Directly with Gαi
Estrogen-related cardiovascular protection is primarily due to direct effects on vascular cells including endothelium. We have previously shown that estradiol (E2) stimulates endothelial NO synthase (eNOS) enzymatic activity via the activation of plasma membrane-associated estrogen receptor α (ERα) that are G protein-coupled. However, due to prior limitations in the characteristics of conjugated forms of E2, the direct contribution of signaling by non-nuclear ERα to the regulation of endothelial cell phenotype and the most proximal mechanisms underlying non-nuclear ERα action have not been well-defined. We used a new estrogen dendrimer conjugate (EDC) to exclusively investigate the role of non-nuclear ERα in the regulation of bovine aortic endothelial cell (BAEC) migration and proliferation. EDC contains a large, nondegradable poly(amido)amine dendrimer conjugated to multiple estrogen molecules through hydrolytically stable linkages. In contrast to fluorescently-labeled E2 which localized to the nucleus, labeled EDC was excluded from the nucleus and localized to plasma membrane and cytoplasmic vesicles. Whereas E2 activated the promoter-reporter construct ERE- Luciferase in BAEC, EDC did not. However, E2 and EDC caused comparable stimulation of eNOS activity and cell migration and proliferation, and responses to both E2 and EDC were blocked by ICI 182,780, pertussis toxin, the Src antagonist PP2, and N-nitro-L-arginine methyl ester. In our recent studies determining how ERα is coupled to G proteins, pulldown experiments with recombinant proteins demonstrated dynamic, direct protein-protein interaction between Gαi and ERα mediated by a 10 amino acid domain within nuclear localization signal 3 of the receptor. Transfection of BAEC with an ERα mutant lacking the newly-identified Gαi binding domain or a peptide representing the domain prevented E2 and EDC-stimulated migration. These cumulative results indicate that estrogen-induced endothelial cell migration and proliferation are mediated by signaling that is initiated exclusively by non-nuclear ERα through their direct interaction with Gαi. Thus, novel, non-nuclear processes enable ERα to regulate endothelial cell phenotypes of importance to vascular health.