title: GEF-H1 is involved in agonist-induced human pulmonary endothelial barrier dysfunction.
abstract: Endothelial cell (EC) permeability is precisely controlled by cytoskeletal elements [actin filaments, microtubules (MT), intermediate filaments] and cell contact protein complexes (focal adhesions, adherens junctions, tight junctions). We have recently shown that the edemagenic agonist thrombin caused partial MT disassembly, which was linked to activation of small GTPase Rho, Rho-mediated actin remodeling, cell contraction, and dysfunction of lung EC barrier. GEF-H1 is an MT-associated Rho-specific guanosine nucleotide (GDP/GTP) exchange factor, which in MT-unbound state stimulates Rho activity. In this study we tested hypothesis that GEF-H1 may be a key molecule involved in Rho activation, myosin light chain phosphorylation, actin remodeling, and EC barrier dysfunction associated with partial MT disassembly. Our results show that depletion of GEF-H1 or expression of dominant negative GEF-H1 mutant significantly attenuated permeability increase, actin stress fiber formation, and increased MLC and MYPT1 phosphorylation induced by thrombin or MT-depolymerizing agent nocodazole. In contrast, expression of wild-type or activated GEF-H1 mutants dramatically enhanced thrombin and nocodazole effects on stress fiber formation and cell retraction. These results show a critical role for the GEF-H1 in the Rho activation caused by MT disassembly and suggest GEF-H1 as a key molecule involved in cross talk between MT and actin cytoskeleton in agonist-induced Rho-dependent EC barrier regulation.
authors.affiliation: Sect. of Pulmonary and Critical Care, Dept. of Medicine, Biological Sciences Div., Univ. of Chicago, 929 E. 57th St., W410, Chicago, IL 60637, USA. abirukov@medicine.bsd.uchicago.edu
authors.forename: Anna A
authors.initials: AA
authors.lastname: Birukova
authors.forename: Djanybek
authors.initials: D
authors.lastname: Adyshev
authors.forename: Boris
authors.initials: B
authors.lastname: Gorshkov
authors.forename: Gary M
authors.initials: GM
authors.lastname: Bokoch
authors.forename: Konstantin G
authors.initials: KG
authors.lastname: Birukov
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Verin
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D002199:Capillary Permeability; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005799:Genes, Dominant; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology*; D006801:Humans; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011651:Pulmonary Artery / Q000166:cytology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D064067:Rho Guanine Nucleotide Exchange Factors; D013917:Thrombin / Q000494:pharmacology; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
chemical_list: C577356:ARHGEF2 protein, human; D000199:Actins; D000970:Antineoplastic Agents; D020662:Guanine Nucleotide Exchange Factors; D018994:Myosin Light Chains; D034741:RNA, Small Interfering; D064067:Rho Guanine Nucleotide Exchange Factors; D043263:Myosin-Light-Chain Phosphatase; C490372:PPP1R12A protein, human; D013917:Thrombin; D020741:rho GTP-Binding Proteins; D015739:Nocodazole