Selected Phytoestrogens Distinguish Roles of ERα Transactivation and Ligand Binding for Anti-Inflammatory Activity

Estrogen receptor alpha (ER alpha) is a ligand-activated transcriptional activator that is also involved vascular inflammation and atherosclerosis. Whether different ligands may affect this activity has not been explored. We screened a panel of phytoestrogens for their role in ERa binding and transcriptional transcription, and correlated the findings to anti-inflammatory activities in vascular endothelial cells stably expressing either a wild-type or mutant form of ER alpha deficient in its membrane association. Taxifolin and silymarin were high binders for ER alpha ligand binding; quercetin and curcumin were high activators for ER alpha transactivation. Using these phytoestrogens as functional probes, we found, in endothelial cells expressing wild-type ER alpha, the ER alpha high activator, but not the ER alpha high binder, promoted ER alpha nuclear translocation, estrogen response element (ERE) reporter activity, and the downstream gene expression. In endothelial cells expressing membrane association-deficient mutant ER alpha, the ER alpha nuclear translocation was significantly enhanced by taxifolin and silymarin, which still failed to activate ER alpha. Inflammation response was examined using the systemic or vascular inflammation inducers lipopolysaccharide or oxidized low-density lipoprotein. In both cases, only the ER alpha high activator inhibited nuclear translocation of nuclear factor kappa B, JNK, and p38, and the production of inflammatory cytokines IL-1 beta and TNF alpha. We confirm a threshold nuclear accumulation of ER alpha is necessary for its transactivation. The anti-inflammatory activity of phytoestrogens is highly dependent on ER alpha transactivation, less so on the ligand binding, and independent of its membrane association. A pre-examination of phytoestrogens for their mode of ER alpha interaction could facilitate their development as better targeted receptor modifiers.