title: PKC-dependent phosphorylation of eNOS at T495 regulates eNOS coupling and endothelial barrier function in response to G+ -toxins.
abstract: Gram positive (G+) infections make up ∼50% of all acute lung injury cases which are characterized by extensive permeability edema secondary to disruption of endothelial cell (EC) barrier integrity. A primary cause of increased permeability are cholesterol-dependent cytolysins (CDCs) of G+-bacteria, such as pneumolysin (PLY) and listeriolysin-O (LLO) which create plasma membrane pores, promoting Ca2+-influx and activation of PKCα. In human lung microvascular endothelial cells (HLMVEC), pretreatment with the nitric oxide synthase (NOS) inhibitor, ETU reduced the ability of LLO to increase microvascular cell permeability suggesting an endothelial nitric oxide synthase (eNOS)-dependent mechanism. LLO stimulated superoxide production from HLMVEC and this was prevented by silencing PKCα or NOS inhibition suggesting a link between these pathways. Both LLO and PLY stimulated eNOS T495 phosphorylation in a PKC-dependent manner. Expression of a phosphomimetic T495D eNOS (human isoform) resulted in increased superoxide and diminished nitric oxide (NO) production. Transduction of HLMVEC with an active form of PKCα resulted in the robust phosphorylation of T495 and increased peroxynitrite production, indicative of eNOS uncoupling. To determine the mechanisms underlying eNOS uncoupling, HLMVEC were stimulated with LLO and the amount of hsp90 and caveolin-1 bound to eNOS determined. LLO stimulated the dissociation of hsp90, and in particular, caveolin-1 from eNOS. Both hsp90 and caveolin-1 have been shown to influence eNOS uncoupling and a peptide mimicking the scaffolding domain of caveolin-1 blocked the ability of PKCα to stimulate eNOS-derived superoxide. Collectively, these results suggest that the G+ pore-forming toxins promote increased EC permeability via activation of PKCα, phosphorylation of eNOS-T495, loss of hsp90 and caveolin-1 binding which collectively promote eNOS uncoupling and the production of barrier disruptive superoxide.
authors.affiliation: Department of Forensic Medicine, Nanjing Medical University, Nanjing, Jiangsu, China; Vascular Biology Center Medical College of Georgia at Georgia Regents University, Augusta, Georgia, United States of America.
authors.forename: Feng
authors.initials: F
authors.lastname: Chen
authors.affiliation: Vascular Biology Center Medical College of Georgia at Georgia Regents University, Augusta, Georgia, United States of America.
authors.forename: Sanjiv
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Vascular Biology Center Medical College of Georgia at Georgia Regents University, Augusta, Georgia, United States of America.
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Vascular Biology Center Medical College of Georgia at Georgia Regents University, Augusta, Georgia, United States of America.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Aggarwal
authors.affiliation: Vascular Biology Center Medical College of Georgia at Georgia Regents University, Augusta, Georgia, United States of America.
authors.forename: Christine
authors.initials: C
authors.lastname: Gross
authors.affiliation: Vascular Biology Center Medical College of Georgia at Georgia Regents University, Augusta, Georgia, United States of America.
authors.forename: Yusi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Institute for Medical Microbiology, Justus Liebig University, Giessen, Germany.
authors.forename: Trinad
authors.initials: T
authors.lastname: Chakraborty
authors.affiliation: Vascular Biology Center Medical College of Georgia at Georgia Regents University, Augusta, Georgia, United States of America.
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Verin
authors.affiliation: Old Dominion University, Norfolk, Virginia, United States of America.
authors.forename: John D
authors.initials: JD
authors.lastname: Catravas
authors.affiliation: Vascular Biology Center Medical College of Georgia at Georgia Regents University, Augusta, Georgia, United States of America; Department of Pharmacology, Medical College of Georgia at Georgia Regents University, Augusta, Georgia, United States of America.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Lucas
authors.affiliation: Vascular Biology Center Medical College of Georgia at Georgia Regents University, Augusta, Georgia, United States of America.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Black
authors.affiliation: Vascular Biology Center Medical College of Georgia at Georgia Regents University, Augusta, Georgia, United States of America; Department of Pharmacology, Medical College of Georgia at Georgia Regents University, Augusta, Georgia, United States of America.
authors.forename: David J R
authors.initials: DJ
authors.lastname: Fulton
mesh_terms: D001427:Bacterial Toxins / Q000633:toxicity*; D051242:Caveolin 1 / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D006094:Gram-Positive Bacteria; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry / Q000378:metabolism*; D010539:Permeability / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism; D013912:Threonine
chemical_list: D001427:Bacterial Toxins; D051242:Caveolin 1; D018841:HSP90 Heat-Shock Proteins; D013481:Superoxides; D013912:Threonine; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D051571:Protein Kinase C-alpha