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 language | English (US) |
|---|---|
| Pages (from-to) | 517-527 |
| Number of pages | 11 |
| Journal | American Journal of Respiratory Cell and Molecular Biology |
| Volume | 31 |
| Issue number | 5 |
| DOIs | |
| State | Published - Nov 2004 |
| Externally published | Yes |
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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 journal › Article
}
TY - JOUR
T1 - Differential regulation of human lung epithelial and endothelial barrier function by thrombin
AU - Kawkitinarong, Kamon
AU - Linz-McGillem, Laura
AU - Birukov, Konstantin G.
AU - Garcia, Joe GN
PY - 2004/11
Y1 - 2004/11
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=7744223152&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=7744223152&partnerID=8YFLogxK
U2 - 10.1165/rcmb.2003-0432OC
DO - 10.1165/rcmb.2003-0432OC
M3 - Article
C2 - 15284075
AN - SCOPUS:7744223152
VL - 31
SP - 517
EP - 527
JO - American Journal of Respiratory Cell and Molecular Biology
JF - American Journal of Respiratory Cell and Molecular Biology
SN - 1044-1549
IS - 5
ER -