The sex hormone estradiol (E<sub>2</sub>) appears to mediate both anti-atherogenic and pro-inflammatory effects in premenopausal women, suggesting a complex immunomodulatory role. Tumor necrosis factor (TNF) is a key pro-inflammatory cytokine involved in the pathogenesis of atherosclerosis and other inflammatory diseases. Alterations at the TNF receptors (TNFRs) and their downstream signaling/transcriptional pathways can affect inflammatory responses. Given this background, we hypothesized that chronic E<sub>2</sub> exposure would alter endothelial inflammatory response involving modulation at the levels of TNFRs and signaling pathways. HUVECs were used as the model system. Pre-treatment with E<sub>2</sub> did not significantly alter TNF-induced upregulation of pro-inflammatory molecules ICAM-1 (3–6 times) and VCAM-1 (5–7 times). However, pharmacological inhibition of transcriptional pathways suggested a partial shift from NF-ĸB (from 97 to 64%) towards the JNK/AP-1 pathway in ICAM-1 upregulation on E<sub>2</sub> treatment. In contrast, VCAM-1 expression remained NF-ĸB dependent in both control (∼96%) and E<sub>2</sub> treated (∼85%) cells. The pro-inflammatory TNF effects were mediated by TNFR1. Interestingly, E<sub>2</sub> pre-treatment increased TNFR2 levels in these cells. Concomitant TNFR2 activation (but not TNFR1 activation alone) led to the shift towards JNK/AP-1-mediated ICAM-1 upregulation in E<sub>2</sub>-treated cells, suggesting the effects of chronic E<sub>2</sub> to be dependent on TNFR2 signaling
