Department of Pediatrics (Neonatology), Lung Biology and Disease Program, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, United States.
Department of Pediatrics (Neonatology), Lung Biology and Disease Program, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, United States.
Cell Signal. 2018 Apr;44:103-117. doi: 10.1016/j.cellsig.2018.01.011. Epub 2018 Jan 11.
Nucleocytoplasmic shuttling via importins is central to the function of eukaryotic cells and an integral part of the processes that lead to many human diseases. In this study, we addressed the role of α and β importins in the mechanism of endothelial cell (EC) inflammation and permeability, important pathogenic features of many inflammatory diseases such as acute lung injury and atherosclerosis. RNAi-mediated knockdown of importin α4 or α3 each inhibited NF-κB activation, proinflammatory gene (ICAM-1, VCAM-1, and IL-6) expression, and thereby endothelial adhesivity towards HL-60 cells, upon thrombin challenge. The inhibitory effect of α4 and α3 knockdown was associated with impaired nuclear import and consequently, DNA binding of RelA/p65 subunit of NF-κB and occurred independently of IκBα degradation. Intriguingly, knockdown of importins α4 and α3 also inhibited thrombin-induced RelA/p65 phosphorylation at Ser, showing a novel role of α importins in regulating transcriptional activity of RelA/p65. Similarly, knockdown of importin β1, but not β2, blocked thrombin-induced activation of RelA/p65 and its target genes. In parallel studies, TNFα-mediated inflammatory responses in EC were refractory to knockdown of importins α4, α3 or β1, indicating a stimulus-specific regulation of RelA/p65 and EC inflammation by these importins. Importantly, α4, α3, or β1 knockdown also protected against thrombin-induced EC barrier disruption by inhibiting the loss of VE-cadherin at adherens junctions and by regulating actin cytoskeletal rearrangement. These results identify α4, α3 and β1 as critical mediators of EC inflammation and permeability associated with intravascular coagulation.
核质穿梭通过输入蛋白是真核细胞功能的核心，也是导致许多人类疾病的过程的一个组成部分。在这项研究中，我们研究了α和β输入蛋白在血管内皮细胞（EC）炎症和通透性机制中的作用，这是许多炎症性疾病（如急性肺损伤和动脉粥样硬化）的重要致病特征。RNAi 介导的α4 或 α3 输入蛋白敲低均抑制 NF-κB 激活、促炎基因（ICAM-1、VCAM-1 和 IL-6）表达，从而抑制凝血酶刺激后的 HL-60 细胞与内皮细胞的黏附性。α4 和 α3 敲低的抑制作用与核输入受损有关，进而 NF-κB 的 RelA/p65 亚基的 DNA 结合受到影响，并且与 IκBα 降解无关。有趣的是，输入蛋白 α4 和 α3 的敲低也抑制了凝血酶诱导的 RelA/p65 丝氨酸磷酸化，表明 α 输入蛋白在调节 RelA/p65 的转录活性方面具有新的作用。同样，β1 输入蛋白的敲低，但不是 β2，阻断了凝血酶诱导的 RelA/p65 及其靶基因的激活。在平行研究中，TNFα 介导的 EC 炎症反应对输入蛋白 α4、α3 或 β1 的敲低具有抗性，表明这些输入蛋白对 RelA/p65 和 EC 炎症具有刺激特异性调节。重要的是，α4、α3 或 β1 的敲低也通过抑制黏着连接处 VE-钙黏蛋白的丢失和调节肌动蛋白细胞骨架重排来防止凝血酶诱导的 EC 屏障破坏。这些结果表明，α4、α3 和 β1 是与血管内凝血相关的 EC 炎症和通透性的关键介质。
