Differential regulation of human lung epithelial and endothelial barrier function by thrombin

Kamon Kawkitinarong, Laura Linz-McGillem, Konstantin G. Birukov, Joe GN Garcia

Research output: Contribution to journalArticle

81 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)517-527
Number of pages11
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Volume31
Issue number5
DOIs
StatePublished - Nov 2004
Externally publishedYes

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Thrombin
Myosin Light Chains
Phosphorylation
Lung
Acoustic impedance
Electric Impedance
Endothelial cells
Alveolar Epithelial Cells
Endothelial Cells
Actins
Zonula Occludens-1 Protein
Monolayers
Occludin
Thrombin Time
Myosin-Light-Chain Kinase
Chemical activation
Actomyosin
rho-Associated Kinases
Stress Fibers
rho GTP-Binding Proteins

ASJC Scopus subject areas

  • Cell Biology
  • Pulmonary and Respiratory Medicine
  • Molecular Biology

Cite this

Differential regulation of human lung epithelial and endothelial barrier function by thrombin. / Kawkitinarong, Kamon; Linz-McGillem, Laura; Birukov, Konstantin G.; Garcia, Joe GN.

In: American Journal of Respiratory Cell and Molecular Biology, Vol. 31, No. 5, 11.2004, p. 517-527.

Research output: Contribution to journalArticle

Kawkitinarong, Kamon ; Linz-McGillem, Laura ; Birukov, Konstantin G. ; Garcia, Joe GN. / Differential regulation of human lung epithelial and endothelial barrier function by thrombin. In: American Journal of Respiratory Cell and Molecular Biology. 2004 ; Vol. 31, No. 5. pp. 517-527.
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