Department of Cell Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, LA 71130, United States; Department of Internal Medicine, Renal Division, Washington University, St. Louis, MO 63110, United States.
Department of Cell Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, LA 71130, United States.
Cell Signal. 2018 Sep;49:30-38. doi: 10.1016/j.cellsig.2018.05.008. Epub 2018 May 21.
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-A1 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-κB (NF-κB/p65) and nuclear factor of activated T-cells (NFAT). While ephrin-A1 failed to induce endothelial NF-κB activation, NF-κB inhibitors prevented ephrin-A1-induced VCAM-1 expression, suggesting basal NF-κB activity is required. In contrast, ephrin-A1 induced a robust EphA2-dependent increase in NFAT activation, and mutation of the NF-κB/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-A1-induced VCAM-1 expression. Consistent with robust NFAT activation, ephrin-A1-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.
促炎刺激物激活内皮细胞会通过增强细胞间黏附分子-1（VCAM-1）等反受体的表达来驱动白细胞募集。我们之前的研究表明，受体酪氨酸激酶 EphA2 与其配体 ephrin-A1 的激活会诱导 VCAM-1 的表达。在这里，我们试图描述涉及的促炎信号通路。对 ephrin-A1 诱导基因中过表达的转录因子进行分析，确定了多个潜在的转录调节剂，包括核因子-κB（NF-κB/p65）和激活 T 细胞的核因子（NFAT）等 Rel 家族成员。虽然 ephrin-A1 未能诱导内皮 NF-κB 激活，但 NF-κB 抑制剂可阻止 ephrin-A1 诱导的 VCAM-1 表达，表明基础 NF-κB 活性是必需的。相反，ephrin-A1 诱导 EphA2 依赖性 NFAT 激活的显著增加，并且 VCAM-1 启动子中 NF-κB/NFAT 结合位点的突变削弱了 ephrin-A1 诱导的启动子活性。NFAT 激活经典地通过钙依赖性钙调神经磷酸酶激活发生，并且用钙调神经磷酸酶抑制剂（环孢菌素 A、FK506）或直接 NFAT 抑制剂（A-285222）抑制 NFAT 信号足以阻断 ephrin-A1 诱导的 VCAM-1 表达。与 EphA2 依赖性钙内流的强烈激活一致，ephrin-A1 诱导内皮细胞中 EphA2 依赖性钙内流，这是 ephrin-A1 诱导的 NFAT 激活和 VCAM-1 表达所必需的。这项工作首次提供了 EphA2 依赖性钙内流和 NFAT 激活的数据，并确定 NFAT 是内皮细胞激活中 EphA2 依赖性新的促炎途径。
