Orail-dependent Ca2+ entry plays an essential role in inflammatory response through regulating T cell and macrophage activation and neutrophil infiltration. However, whether Orail Ca2+ entry contributes to endothelial activation, one of the early steps of vascular inflammation, remains elusive. In the present study, we observed that knockdown of Orail reduced, whereas overexpression of Orail potentiated, TNF-alpha-induced expression of adhesion molecules such as ICAM-1 and VCAM-1 in HUVECs, and subsequently blocked adhesion of monocyte to HUVEC5. In vivo, Orail downregulation attenuated TNFa.induced ICAM-1 and VCAM-1 expression in mouse aorta and the levels of pro-inflammatory cytokines in the serum. In addition, Orail knockdown also dramatically decreased the expression of pro inflammatory cytokines and neutrophil infiltration in the lung after TNFa treatment, and thus protected lung tissue injury. Notably, among all isoforms of nuclear factor of activated T cells (NFAT5), TNF alpha only triggered NFATc4 nuclear accumulation in HUVECs. Knockdown of Orail or inhibition of calcineurin prevented TNF-alpha-induced NFATc4 nuclear translocation and reduced ICAM-1 and VCAM-1 expression in HUVEC5. Overexpression of NFATc4 further enhanced ICAM-1 and VCAM-1 expression induced by TNF alpha. Our study demonstrates that Orai1-Ca2-calcineurin-NFATc4 signaling is an essential inflammatory pathway required for TNF alpha-induced endothelial cell activation and vascular inflammation. Therefore, Orail may be a potential therapeutic target for treatment of inflammatory diseases. (C) 2017 Elsevier Inc. All rights reserved.