title: RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins.
abstract: Disruption of the endothelial barrier in response to Gram positive (G+) bacterial toxins is a major complication of acute lung injury (ALI) and can be further aggravated by antibiotics which stimulate toxin release. The integrity of the pulmonary endothelial barrier is mediated by the balance of disruptive forces such as the small GTPase RhoA, and protective forces including endothelium-derived nitric oxide (NO). How NO protects against the barrier dysfunction is incompletely understood and our goal was to determine whether NO and S-nitrosylation can modulate RhoA activity and whether this mechanism is important for G+toxin-induced microvascular permeability. We found that the G+toxin listeriolysin-O (LLO) increased RhoA activity and that NO and S-NO donors inhibit RhoA activity. RhoA was robustly S-nitrosylated as determined by biotin-switch and mercury column analysis. MS revealed that three primary cysteine residues are S-nitrosylated including cys16, cys20 and cys159. Mutation of these residues to serine diminished S-nitrosylation to endogenous NO and mutant RhoA was less sensitive to inhibition by S-NO. G+-toxins stimulated the denitrosylation of RhoA which was not mediated by S-nitrosoglutathione reductase (GSNOR), thioredoxin (TRX) or thiol-dependent enzyme activity but was instead stimulated directly by elevated calcium levels. Calcium-promoted the direct denitrosylation of WT but not mutant RhoA and mutant RhoA adenovirus was more effective than WT in disrupting the barrier integrity of human lung microvascular endothelial cells. In conclusion, we reveal a novel mechanism by which NO and S-nitrosylation reduces RhoA activity which may be of significance in the management of pulmonary endothelial permeability induced by G+-toxins.
authors.affiliation: Department of Forensic Medicine, Nanjing Medical University, Nanjing, Jiangsu, China; Vascular Biology Center, Augusta University, Augusta, Georgia 30912, USA. Electronic address: fchen@njmu.edu.cn.
authors.forename: F
authors.initials: F
authors.lastname: Chen
authors.affiliation: Vascular Biology Center, Augusta University, Augusta, Georgia 30912, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Vascular Biology Center, Augusta University, Augusta, Georgia 30912, USA.
authors.forename: R
authors.initials: R
authors.lastname: Rafikov
authors.affiliation: Vascular Biology Center, Augusta University, Augusta, Georgia 30912, USA.
authors.forename: S
authors.initials: S
authors.lastname: Haigh
authors.affiliation: Center for Biotechnology and Genomic Medicine, Augusta University, Augusta, Georgia 30912, USA.
authors.forename: W B
authors.initials: WB
authors.lastname: Zhi
authors.affiliation: Vascular Biology Center, Augusta University, Augusta, Georgia 30912, USA.
authors.forename: S
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Department of Pediatrics, Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA.
authors.forename: P T
authors.initials: PT
authors.lastname: Doulias
authors.affiliation: Vascular Biology Center, Augusta University, Augusta, Georgia 30912, USA.
authors.forename: O
authors.initials: O
authors.lastname: Rafikova
authors.affiliation: Institute for Medical Microbiology, Justus Liebig University, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Pillich
authors.affiliation: Institute for Medical Microbiology, Justus Liebig University, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Chakraborty
authors.affiliation: Vascular Biology Center, Augusta University, Augusta, Georgia 30912, USA; Department of Pharmacology, Augusta University, Augusta, Georgia 30912, USA.
authors.forename: R
authors.initials: R
authors.lastname: Lucas
authors.affiliation: Vascular Biology Center, Augusta University, Augusta, Georgia 30912, USA.
authors.forename: A D
authors.initials: AD
authors.lastname: Verin
authors.affiliation: Old Dominion University, Norfolk, VA, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Catravas
authors.affiliation: Center for Biotechnology and Genomic Medicine, Augusta University, Augusta, Georgia 30912, USA.
authors.forename: J X
authors.initials: JX
authors.lastname: She
authors.affiliation: Vascular Biology Center, Augusta University, Augusta, Georgia 30912, USA.
authors.forename: S M
authors.initials: SM
authors.lastname: Black
authors.affiliation: Vascular Biology Center, Augusta University, Augusta, Georgia 30912, USA; Department of Pharmacology, Augusta University, Augusta, Georgia 30912, USA. Electronic address: dfulton@augusta.edu.
authors.forename: D J R
authors.initials: DJR
authors.lastname: Fulton
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology*; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002522:Chlorocebus aethiops; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D057809:HEK293 Cells; D006360:Heat-Shock Proteins / Q000494:pharmacology*; D006460:Hemolysin Proteins / Q000494:pharmacology*; D006801:Humans; D008168:Lung / Q000098:blood supply; D055806:Microvessels / Q000378:metabolism; D009154:Mutation; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009603:Nitroso Compounds / Q000378:metabolism*; D010539:Permeability; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D009569:Nitric Oxide; D020742:rhoA GTP-Binding Protein; C038705:hlyA protein, Listeria monocytogenes; D002118:Calcium
keywords: Endothelial; G(+)-toxins; Nitric oxide; Permeability; RhoA; S-nitrosylation