EphA2 stimulates VCAM-1 expression through calcium-dependent NFAT1 activity

Endothelial cell activation by proinflammatory stimuli drives leukocyte recruitment through enhanced expression of counter-receptors such as vascular cell adhesion molecule-1 (VCAM-1). We previously demonstrated that activation of the receptor tyrosine kinase EphA2 with its ligand ephrin-Al induces VCAM-1 expression. Here, we sought to characterize the proinflammatory signaling pathways involved. Analysis of over-represented transcription factors in ephrin-A1-induced genes identified multiple potential transcriptional regulators, including the Rel family members nuclear factor-kappa B (NF-kappa B/p65) and nuclear factor of activated T-cells (NFAT). While ephrin-Al failed to induce endothelial NF-kappa B activation, NF-kappa B inhibitors prevented ephrin-Al-induced VCAM-1 expression, suggesting basal NF-kappa B activity is required. In contrast, ephrin-A1 induced a robust EphA2-dependent increase in NFAT activation, and mutation of the NF-kappa B/NFAT-binding sites in the VCAM-1 promoter blunted ephrin-A1-induced promoter activity. NFAT activation classically occurs through calcium-dependent calcineurin activation, and inhibiting NFAT signaling with calcineurin inhibitors (cyclosporine A, FK506) or direct NFAT inhibitors (A 285222) was sufficient to block ephrin-Al-induced VCAM-1 expression. Consistent with robust NFAT activation, ephrin-Al-induced an EphA2-dependent calcium influx in endothelial cells that was required for ephrin-A1-induced NFAT activation and VCAM-1 expression. This work provides the first data showing EphA2-dependent calcium influx and NFAT activation and identifies NFAT as a novel EphA2-dependent proinflammatory pathway in endothelial activation.