Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction

Anna A. Birukova, Ksenya Smurova, Konstantin G. Birukov, Kozo Kaibuchi, Joe G.N. Garcia, Alexander Dmitriyevich Verin

Research output: Contribution to journalArticle

213 Citations (Scopus)

Abstract

Thrombin-induced barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contraction, and gap formation. Phosphorylation of regulatory myosin light chains (MLC) is a key mechanism of endothelial cell (EC) contraction and barrier dysfunction, which is triggered by Ca2+/calmodulin-dependent MLC kinase (MLCK) and Rho-associated kinase (Rho-kinase). The role of MLCK in EC barrier regulation has been previously described; however, Rho-mediated pathway in thrombin-induced pulmonary EC dysfunction is not yet precisely characterized. Here, we demonstrate that thrombin-induced decreases in transendothelial electrical resistance (TER) indicating EC barrier dysfunction are universal for human and bovine pulmonary endothelium, and involve membrane translocation and direct activation of small GTPase Rho and its downstream target Rho-kinase. Transient Rho membrane translocation coincided with translocation of upstream Rho activator, guanosine nucleotide exchange factor p115-RhoGEF. Rho mediated activation of downstream target, Rho-kinase induced phosphorylation of the EC MLC phosphatase (MYPT1) at Thr686 and Thr850, resulting in MYPT1 inactivation, accumulation of diphospho-MLC, actin remodeling, and cell contraction. The specific Rho-kinase inhibitor, Y27632, abolished MYPT1 phosphorylation, MLC phosphorylation, significantly attenuated stress fiber formation and thrombin-induced TER decrease. Furthermore, expression of dominant-negative Rho and Rho-kinase abolished thrombin-induced stress fiber formation and MLC phosphorylation. Our data, which provide comprehensive analysis of Rho-mediated signal transduction in pulmonary EC, demonstrate involvement of guanosine nucleotide exchange factor, p115-RhoGEF, in thrombin-mediated Rho regulation, and suggest Rho, Rho-kinase, and MYPT1 as potential pharmacological and gene therapy targets critical for prevention of thrombin-induced EC barrier disruption and pulmonary edema associated with acute lung injury.

Original languageEnglish (US)
Pages (from-to)64-77
Number of pages14
JournalMicrovascular Research
Volume67
Issue number1
DOIs
StatePublished - Jan 1 2004
Externally publishedYes

Fingerprint

rho GTP-Binding Proteins
Endothelial cells
rho-Associated Kinases
Thrombin
Phosphorylation
Endothelial Cells
Myosin Light Chains
Lung
Rho Guanine Nucleotide Exchange Factors
Stress Fibers
Acoustic impedance
Guanosine
Chemical activation
Electric Impedance
Nucleotides
Myosin-Light-Chain Phosphatase
Membranes
Myosin-Light-Chain Kinase
Gene therapy
Actomyosin

Keywords

  • Actin
  • Myosin
  • Myosin phosphatase
  • Phosphorylation
  • Pulmonary endothelium
  • Rho-kinase
  • p115-RhoGEF

ASJC Scopus subject areas

  • Biochemistry
  • Cardiology and Cardiovascular Medicine
  • Cell Biology

Cite this

Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction. / Birukova, Anna A.; Smurova, Ksenya; Birukov, Konstantin G.; Kaibuchi, Kozo; Garcia, Joe G.N.; Verin, Alexander Dmitriyevich.

In: Microvascular Research, Vol. 67, No. 1, 01.01.2004, p. 64-77.

Research output: Contribution to journalArticle

Birukova, Anna A. ; Smurova, Ksenya ; Birukov, Konstantin G. ; Kaibuchi, Kozo ; Garcia, Joe G.N. ; Verin, Alexander Dmitriyevich. / Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction. In: Microvascular Research. 2004 ; Vol. 67, No. 1. pp. 64-77.
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abstract = "Thrombin-induced barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contraction, and gap formation. Phosphorylation of regulatory myosin light chains (MLC) is a key mechanism of endothelial cell (EC) contraction and barrier dysfunction, which is triggered by Ca2+/calmodulin-dependent MLC kinase (MLCK) and Rho-associated kinase (Rho-kinase). The role of MLCK in EC barrier regulation has been previously described; however, Rho-mediated pathway in thrombin-induced pulmonary EC dysfunction is not yet precisely characterized. Here, we demonstrate that thrombin-induced decreases in transendothelial electrical resistance (TER) indicating EC barrier dysfunction are universal for human and bovine pulmonary endothelium, and involve membrane translocation and direct activation of small GTPase Rho and its downstream target Rho-kinase. Transient Rho membrane translocation coincided with translocation of upstream Rho activator, guanosine nucleotide exchange factor p115-RhoGEF. Rho mediated activation of downstream target, Rho-kinase induced phosphorylation of the EC MLC phosphatase (MYPT1) at Thr686 and Thr850, resulting in MYPT1 inactivation, accumulation of diphospho-MLC, actin remodeling, and cell contraction. The specific Rho-kinase inhibitor, Y27632, abolished MYPT1 phosphorylation, MLC phosphorylation, significantly attenuated stress fiber formation and thrombin-induced TER decrease. Furthermore, expression of dominant-negative Rho and Rho-kinase abolished thrombin-induced stress fiber formation and MLC phosphorylation. Our data, which provide comprehensive analysis of Rho-mediated signal transduction in pulmonary EC, demonstrate involvement of guanosine nucleotide exchange factor, p115-RhoGEF, in thrombin-mediated Rho regulation, and suggest Rho, Rho-kinase, and MYPT1 as potential pharmacological and gene therapy targets critical for prevention of thrombin-induced EC barrier disruption and pulmonary edema associated with acute lung injury.",
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AU - Garcia, Joe G.N.

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AB - Thrombin-induced barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contraction, and gap formation. Phosphorylation of regulatory myosin light chains (MLC) is a key mechanism of endothelial cell (EC) contraction and barrier dysfunction, which is triggered by Ca2+/calmodulin-dependent MLC kinase (MLCK) and Rho-associated kinase (Rho-kinase). The role of MLCK in EC barrier regulation has been previously described; however, Rho-mediated pathway in thrombin-induced pulmonary EC dysfunction is not yet precisely characterized. Here, we demonstrate that thrombin-induced decreases in transendothelial electrical resistance (TER) indicating EC barrier dysfunction are universal for human and bovine pulmonary endothelium, and involve membrane translocation and direct activation of small GTPase Rho and its downstream target Rho-kinase. Transient Rho membrane translocation coincided with translocation of upstream Rho activator, guanosine nucleotide exchange factor p115-RhoGEF. Rho mediated activation of downstream target, Rho-kinase induced phosphorylation of the EC MLC phosphatase (MYPT1) at Thr686 and Thr850, resulting in MYPT1 inactivation, accumulation of diphospho-MLC, actin remodeling, and cell contraction. The specific Rho-kinase inhibitor, Y27632, abolished MYPT1 phosphorylation, MLC phosphorylation, significantly attenuated stress fiber formation and thrombin-induced TER decrease. Furthermore, expression of dominant-negative Rho and Rho-kinase abolished thrombin-induced stress fiber formation and MLC phosphorylation. Our data, which provide comprehensive analysis of Rho-mediated signal transduction in pulmonary EC, demonstrate involvement of guanosine nucleotide exchange factor, p115-RhoGEF, in thrombin-mediated Rho regulation, and suggest Rho, Rho-kinase, and MYPT1 as potential pharmacological and gene therapy targets critical for prevention of thrombin-induced EC barrier disruption and pulmonary edema associated with acute lung injury.

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