First Author: Seda Babroudi
Additional authors: Qing Lu, Richard Karas
Heart disease is the leading cause of death in both men and women in the United States despite disproportionately affecting men. In younger post-menopausal women, estrogen supplementation may decrease the risk of heart disease through its interaction with ER-alpha and ER-beta in the vasculature. Upon binding ER, estrogen initiates two pathways: the rapid signaling pathway and the genomic signaling pathway, traditionally associated with dilatation of vessels and proliferation of endothelial cells, respectively. Estrogen supplementation, however, has been associated with an increased risk of breast and uterine cancer. Here we hypothesize that isolating the rapid pathway alone, using the novel compound membrane-tethered E2 (MT E2), is sufficient to mediate the beneficial vascular effects of estrogen without inducing the potential adverse effects.
Human endothelial somatic hybrid cells (EA.hy926) were treated with 100 nM MT E2 with the appropriate ligand controls. Activation of the rapid signaling pathway was measured by phosphorylation of specific downstream proteins (eNOS, AKT, and ERK). Activation of the genomic signaling pathway was measured by an ERE- luciferase assay. ER-alpha was localized by immunocytochemistry, and proliferation and migration were measured by a cell viability and scratch-wound assay, respectively.
Cells treated with MT E2 exhibited increased phosphorylation of eNOS, AKT, and ERK, suggesting that MT E2 activates the rapid signaling pathway. Cells treated with MT E2 did not, however, upregulate luciferase via an estrogen response element (ERE), suggesting that MT E2 is incapable of activating the genomic pathway. Unlike treatment with E2, MT E2 did not drive ER-alpha to the nucleus, though it did induce proliferation and migration of vascular cells to an equal or greater extent than E2, suggesting that the rapid pathway is sufficient to induce vascular cell function.
In conclusion, we confirmed our hypothesis that the novel compound, MT E2, activates the ER rapid pathway without activating the genomic pathway, and by doing so, is sufficient to induce proliferation and migration of human endothelial cells to an equal or greater extent than conventional estrogen, supporting its potential vascular benefit in vivo.