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Differential Regulation of Human Lung Epithelial and Endothelial Barrier Function by Thrombin

Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; and Department of Medicine, Chulalongkorn University, Bangkok, Thailand

Address correspondence to: Joe G. N. Garcia, M.D., Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle 4B-77, Baltimore, MD 21224-6801. E-mail: drgarcia{at}jhmi.edu

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 edemagenic 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.

Abbreviations: acute respiratory distress syndrome, ARDS • bovine serum albumin, BSA • dominant-negative, DN • endothelial cells, EC • fetal bovine serum, FBS • G-protein–binding domain, GBD • human bronchial epithelial cell, HBEC • hepatocyte growth factor, HGF • human pulmonary artery EC, HPAEC • horseradish peroxidase, HRP • immunoglobulin, Ig • myosin light chain, MLC • MLC kinase, MLCK • protease-activated receptor, PAR • phosphate-buffered saline, PBS • polyvinylidene fluoride, PVDF • diphosphorylated MLC, ppMLC • Rho-associated kinase, RhoK • sphingosine 1-phosphate, S1P • Tris-buffered saline, TBS • TBS–Tween 20, TBS-T • transmonolayer electrical resistance, TER • thrombin receptor–activating peptide, TRAP

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