title: Isoprenylcysteine carboxyl methyltransferase modulates endothelial monolayer permeability: involvement of RhoA carboxyl methylation.
abstract: RhoA and Rac1 regulate formation of stress fibers and intercellular junctions, thus modulating endothelial monolayer permeability. Posttranslational modifications of RhoA and Rac1 regulate enzyme activity and subcellular localization, resulting in altered cellular function. The role of RhoA and Rac1 carboxyl methylation in modulating endothelial monolayer permeability is not known. In this study, we found that inhibition of isoprenylcysteine-O-carboxyl methyltransferase (ICMT) with adenosine plus homocysteine or N-acetyl-S-geranylgeranyl-l-cysteine decreased RhoA carboxyl methylation, RhoA activity, and endothelial monolayer permeability, suggesting that RhoA carboxyl methylation may play a role in the ICMT-modulated monolayer permeability. Similar studies showed no effect of ICMT inhibition on Rac1 carboxyl methylation or localization. Bovine pulmonary artery endothelial cells (PAECs) stably overexpressing ICMT-GFP cDNA were established to determine if increased ICMT expression could alter RhoA or Rac1 carboxyl methylation, activation, and endothelial monolayer permeability. PAECs stably overexpressing ICMT demonstrated increased RhoA carboxyl methylation, membrane-bound RhoA, and RhoA activity. Additionally, PAECs stably overexpressing ICMT had diminished VE-cadherin and beta-catenin at intercellular junctions, with resultant intercellular gap formation, as well as enhanced monolayer permeability. These effects were blunted by adenosine plus homocysteine and by inhibition of RhoA, but not by inhibition of Rac1. These results indicate that ICMT modulates endothelial monolayer permeability by altering RhoA carboxyl methylation and activation, thus changing the organization of intercellular junctions. Therefore, carboxyl methylation of RhoA may modulate endothelial barrier function.
authors.affiliation: Pulmonary Vascular Biology Laboratory, Providence VA Medical Center, Providence, R 02908, USA.
authors.forename: Qing
authors.initials: Q
authors.lastname: Lu
authors.forename: Elizabeth O
authors.initials: EO
authors.lastname: Harrington
authors.forename: Chi-Ming
authors.initials: CM
authors.lastname: Hai
authors.forename: Julie
authors.initials: J
authors.lastname: Newton
authors.forename: Megan
authors.initials: M
authors.lastname: Garber
authors.forename: Tetsuaki
authors.initials: T
authors.lastname: Hirase
authors.forename: Sharon
authors.initials: S
authors.lastname: Rounds
mesh_terms: D000241:Adenosine / Q000494:pharmacology; D022005:Adherens Junctions / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D003545:Cysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004224:Diterpenes / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology*; D049452:Green Fluorescent Proteins; D006710:Homocysteine / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D011496:Protein Methyltransferases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D004224:Diterpenes; D008164:Luminescent Proteins; C095716:N-acetyl-S-geranylgeranyl-cysteine; D011993:Recombinant Fusion Proteins; D006710:Homocysteine; D049452:Green Fluorescent Proteins; D011496:Protein Methyltransferases; C056501:protein-S-isoprenylcysteine O-methyltransferase; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; D000241:Adenosine; D003545:Cysteine