Rationale Endothelial cell (EC) permeability is precisely controlled by cytoskeletal elements (actin filaments, microtubules, intermediate filaments). We have recently shown that the edemagenic agonist thrombin caused partial microtubule (MT) disassembly, which was linked to activation of small GTPase Rho, Rho-mediated actin remodeling, cell contraction, and EC barrier dysfunction. In this study we tested the hypothesis that MT-associated Rho-specific GEF-H1 may mediate Rho-dependent EC barrier dysfunction associated with partial MT disassembly.
Methods Endogenous GEF-H1 was depleted using siRNA technique. Functional GEF-H1 mutants were introduced to EC using transient transfection and nucleofection techniques. EC permeability changes were assessed by measurements of transendothelial electrical resistance. EC monolayer integrity and cytoskeletal remodeling were evaluated by immunofluorescent analysis of MLC phosphorylation and actin rearrangement. MLC and MYPT1 phosphorylation induced by thrombin (0.02 U/mL) or MT-depolymerizing agent nocodazole (0.2 μM) was detected by immunobloting with phosphospecific antibodies.
Results Depletion of GEF-H1 or expression of dominant negative GEF-H1 mutant significantly attenuated thrombin- and nocodazole-induced permeability increase and actin stress fiber formation associated with increased phosphorylation of Rho-Rho-kinase targets MLC and MYPT1. In contrast, expression of wild-type or activated GEF-H1 mutants dramatically enhanced thrombin and nocodazole effects on stress fiber formation and cell retraction.
Conclusions These results demonstrate for the first time a role for the GEF-H1 in the Rho activation induced by MT disassembly and suggest GEF-H1 as a key molecule involved in crosstalk between MT and actin cytoskeleton in agonist-induced Rho-dependent EC barrier regulation.
AHA SDG (A.A.B.) HL80675, HL058064 (A.D.V.), HL76259, HL75349 (K.G.B.).
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