Novel role of microtubules in thrombin-induced endothelial barrier dysfunction.
Disturbances in endothelial cell (EC) barrier regulation are critically dependent upon rearrangements of EC actin cytoskeleton. However, the role of microtubule (MT) network in the regulation of EC permeability is not well understood. We examined involvement of MT remodeling in thrombin-induced EC permeability and explored MT regulation by heterotrimeric G12/13 proteins and by small GTPase Rho. Thrombin induced phosphorylation of MT regulatory protein tau at Ser409 and Ser262 and peripheral MT disassembly, which was linked to increased EC permeability. MT stabilization by taxol attenuated thrombin-induced permeability, actin remodeling, and paracellular gap formation and diminished thrombin-induced activation of Rho and Rho-kinase. Expression of activated Galpha12/13 subunits involved in thrombin-mediated signaling or their effector p115RhoGEF involved in Rho activation caused MT disassembly, whereas p115RhoGEF-specific negative regulator RGS preserved MT from thrombin-induced disassembly. Consistent with these results, expression of activated RhoA and Rho-kinase induced MT disassembly. Conversely, thrombin-induced disassembly of peripheral MT network was attenuated by expression of dominant negative RhoA and Rho-kinase mutants or by pharmacological inhibition of Rho-kinase. Collectively, our data demonstrate for the first time a critical involvement of MT disassembly in thrombin-induced EC barrier dysfunction and indicate G-protein-dependent mechanisms of thrombin-induced MT alteration.
Birukova
Anna A
AA
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, 5200 Eastern Ave., MFL Center Tower 660, Baltimore, MD 21224, USA. abirukov@jhmi.edu
Birukov
Konstantin G
KG
Smurova
Ksenya
K
Adyshev
Djanybek
D
Kaibuchi
Kozo
K
Alieva
Irina
I
Garcia
Joe G N
JG
Verin
Alexander D
AD
D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure*; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008168:Lung / Q000098:blood supply*; D008870:Microtubules / Q000502:physiology* / Q000648:ultrastructure; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D064067:Rho Guanine Nucleotide Exchange Factors; D013917:Thrombin / Q000494:pharmacology*; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000502:physiology; D016875:tau Proteins / Q000378:metabolism
D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D016875:tau Proteins; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D013917:Thrombin; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020962:Heterotrimeric GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein