Differential regulation of human lung epithelial and endothelial barrier function by thrombin.
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.
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Kamon
K
Kawkitinarong
Laura
L
Linz-McGillem
Konstantin G
KG
Birukov
Joe G N
JG
Garcia
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics; D000577:Amides / Q000494:pharmacology; D001381:Azepines / Q000378:metabolism; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004594:Electrophysiology; D004727:Endothelium / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology*; D004848:Epithelium / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008168:Lung / Q000166:cytology*; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D018994:Myosin Light Chains / Q000737:chemistry; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011725:Pyridines / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism* / Q000502:physiology*; D019108:Tight Junctions / Q000378:metabolism; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D062826:Zonula Occludens-1 Protein; D051176:beta Catenin; D054460:rho-Associated Kinases
D000199:Actins; D000577:Amides; D001381:Azepines; C495270:CTNNB1 protein, human; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D018994:Myosin Light Chains; D009281:Naphthalenes; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; D011725:Pyridines; C569042:TJP1 protein, human; D015534:Trans-Activators; D062826:Zonula Occludens-1 Protein; D051176:beta Catenin; C070571:ML 7; C108830:Y 27632; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase; D013917:Thrombin