Differential regulation of human lung epithelial and endothelial barrier function by thrombin
Differential regulation of human lung epithelial and endothelial barrier function by thrombin
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DOI：
10.1165/rcmb.2003-0432oc
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发表时间：
2004-11-01
影响因子：
6.4
通讯作者：
Garcia, JGN
中科院分区：
文献类型：
--
作者：
Kawkitinarong, K;Linz-McGillem, L;Garcia, JGN
肺上皮和内皮屏障功能障碍是急性肺损伤所观察到的生理性紊乱的关键，但仍知之甚少。我们利用人肺泡上皮细胞(A549)和内皮细胞(EC)研究了细胞骨架重塑、肌球蛋白轻链(MLC)磷酸化和凝血酶诱导的屏障调节。凝血酶刺激的人EC单层表现为MLC磷酸化增加，肌动蛋白应激纤维形成，屏障完整性丧失，反映为跨单层电阻(TER)降低。相反，凝血酶使肌动蛋白纤维在周向明显定位，增加了MLC的磷酸化，并增加了跨上皮单层的TER，这与屏障保护一致。在两种类型的肺细胞中，用MLC激酶(ML-7)和Rho激酶(Y-27632)的药物抑制剂预处理诱导的MLC磷酸化减少显著地减弱凝血酶介导的TER变化和MLC磷酸化。在上皮和EC中，凝血酶产生的Rho GTP酶具有时间依赖性的激活，而Rac GTP酶的激活则是。仅在A549细胞中观察到。通过腺病毒转导显性-阴性RAC突变体的分子抑制RAC活性，可消除凝血酶诱导的肺泡上皮细胞TER增加。最后，A549细胞，而不是内皮，显示凝血酶后细胞-细胞界面区域紧密连接蛋白(ZO-1和occludin)水平增加，与凝血酶诱导的屏障保护有关。这些结果表明，通过独特的肌动球蛋白重塑和与紧密连接复合体的细胞骨架相互作用，对水肿性刺激产生选择性屏障反应，对肺内皮细胞和肺泡上皮屏障具有不同的调节作用。
Lung epithelial and endothelial barrier dysfunction is critical to the physiologic derangement observed in acute lung injury, but remains poorly understood. We utilized human alveolar epithelial (A549) and endothelial cells (EC) to study cytoskeletal remodeling, myosin light chain (MLC) phosphorylation and barrier regulation evoked by the eclemagenic agent, thrombin. Thrombin-challenged human EC monolayers demonstrated increased MLC phosphorylation, actin stress fiber formation and loss of barrier integrity reflected by decreased transmonolayer electrical resistance (TER). In contrast, thrombin produced prominent circumferential localization of actin fibers, increased MLC phosphorylation and increased TER across epithelial monolayers, consistent with barrier protection. Reductions in MLC phosphorylation induced by cell pretreatment with pharmacological inhibitors of MLC kinase (ML-7) and Rho kinase (Y-27632) significantly attenuated thrombin-mediated TER changes and MLC phosphorylation in both lung cell types. Thrombin-produced, time-dependent activation of Rho GTPase in both epithelial and EC, whereas Rac GTPase activation was. observed only in A549 cells. Molecular inhibition of Rac activity by adenoviral transfer of dominant-negative Rac mutant abolished thrombin-induced TER increases in alveolar epithelial cells. Finally, A549 cells, but not endothelium, demonstrated increased levels of tight junction proteins (ZO-1 and occludin) after thrombin at the cell-cell interface areas linked to thrombin-elicited barrier protection. These results demonstrate differential pulmonary endothelial and alveolar epithelial barrier regulation via unique actomyosin remodeling and cytoskeletal interactions with tight junction complexes, which confer selective barrier responses to edemagenic stimuli.