title: Obligatory role for endothelial heparan sulphate proteoglycans and caveolae internalization in catestatin-dependent eNOS activation.
abstract: The chromogranin-A peptide catestatin modulates a wide range of processes, such as cardiovascular functions, innate immunity, inflammation, and metabolism. We recently found that the cardiac antiadrenergic action of catestatin requires a PI3K-dependent NO release from endothelial cells, although the receptor involved is yet to be identified. In the present work, based on the cationic properties of catestatin, we tested the hypothesis of its interaction with membrane heparan sulphate proteoglycans, resulting in the activation of a caveolae-dependent endocytosis. Experiments were performed on bovine aortic endothelial cells. Endocytotic vesicles trafficking was quantified by confocal microscopy using a water-soluble membrane dye; catestatin colocalization with heparan sulphate proteoglycans and caveolin 1 internalization were studied by fluorimetric measurements in live cells. Modulation of the catestatin-dependent eNOS activation was assessed by immunofluorescence and immunoblot analysis. Our results demonstrate that catestatin (5 nM) colocalizes with heparan sulphate proteoglycans and induces a remarkable increase in the caveolae-dependent endocytosis and caveolin 1 internalization, which were significantly reduced by both heparinase and wortmannin. Moreover, catestatin was unable to induce Ser(1179) eNOS phosphorylation after pretreatments with heparinase and methyl-β-cyclodextrin. Taken together, these results highlight the obligatory role for proteoglycans and caveolae internalization in the catestatin-dependent eNOS activation in endothelial cells.
authors.affiliation: Department of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123 Turin, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Fornero
authors.affiliation: Department of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123 Turin, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Bassino
authors.affiliation: Department of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123 Turin, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Ramella
authors.affiliation: Department of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123 Turin, Italy.
authors.forename: Clara
authors.identifier: 0000-0002-0320-4282
authors.initials: C
authors.lastname: Gallina
authors.affiliation: Veterans Affairs San Diego Healthcare System, 3350 La Jolla Village Drive, San Diego, CA 92161, USA ; Department of Medicine, University of California, 9500-0838 Gilman Drive, La Jolla, San Diego, CA 92093, USA.
authors.forename: Sushil K
authors.initials: SK
authors.lastname: Mahata
authors.affiliation: DIBEST Department, University of Calabria, 87030 Arcavacata di Rende, Italy ; National Institute for Cardiovascular Research, Via Irnerio 48, 40126 Bologna, Italy.
authors.forename: Bruno
authors.initials: B
authors.lastname: Tota
authors.affiliation: Department of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123 Turin, Italy.
authors.forename: Renzo
authors.identifier: 0000-0003-0530-8496
authors.initials: R
authors.lastname: Levi
authors.affiliation: Department of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123 Turin, Italy ; National Institute for Cardiovascular Research, Via Irnerio 48, 40126 Bologna, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Alloatti
authors.affiliation: Department of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123 Turin, Italy.
authors.forename: Maria Pia
authors.identifier: 0000-0002-0382-7607
authors.initials: MP
authors.lastname: Gallo
mesh_terms: D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000378:metabolism; D002417:Cattle; D021941:Caveolae / Q000187:drug effects / Q000648:ultrastructure; D051242:Caveolin 1 / Q000378:metabolism; D053379:Chromogranin A / Q000008:administration & dosage* / Q000378:metabolism / Q000648:ultrastructure; D004705:Endocytosis / Q000187:drug effects*; D042783:Endothelial Cells / Q000187:drug effects / Q000648:ultrastructure; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010446:Peptide Fragments / Q000008:administration & dosage* / Q000378:metabolism / Q000648:ultrastructure; D022161:Transport Vesicles / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D051242:Caveolin 1; D053379:Chromogranin A; D019812:Heparan Sulfate Proteoglycans; D010446:Peptide Fragments; C109084:chromogranin A (344-364); D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III