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Obligatory role for endothelial heparan sulphate proteoglycans and caveolae internalization in catestatin-dependent eNOS activation.

Fornero S, Bassino E, Ramella R, Gallina C, Mahata SK, Tota B, Levi R, Alloatti G, Gallo MP - Biomed Res Int (2014)

Bottom Line: 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.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.Taken together, these results highlight the obligatory role for proteoglycans and caveolae internalization in the catestatin-dependent eNOS activation in endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123 Turin, Italy.

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.

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CST induces proteoglycan-dependent Cav1 internalization in GFP-Cav1 transfected BAE-1 cells. (a) Representative time course of the fluorescence intensity in single BAE-1 cells transfected with Cav-1-GFP and stimulated with H:ase + CST or with CST alone. Subpanels 1 and 2: confocal pseudocolor images of CST-treated BAE-1 cells from the correspondent time points indicated by the arrows, showing membrane (1) and cytosolic (2) localizations of GFP-Cav1 after CST treatment. (b) Bar graphs representing the percent variation with respect to control of the fluorescent GFP-Cav1 signal in the different experimental conditions (CST = 86.94 ± 25.51%; H:ase + CST = −0.07 ± 22.20%; n = 3 sets of experiments, 3 fields/sample, about 40 cells/fields; P < 0.05). (c) Bar graph representing the percent variation with respect to control of the fluorescent GFP-Cav1 signal during CST 5 nM treatment, respectively, in plasma membrane and in cytosol (membrane = −19.34 ± 1.22%; cytosol = 122.6 ± 3.49%; n = 17 cells; P < 0.05).
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fig4: CST induces proteoglycan-dependent Cav1 internalization in GFP-Cav1 transfected BAE-1 cells. (a) Representative time course of the fluorescence intensity in single BAE-1 cells transfected with Cav-1-GFP and stimulated with H:ase + CST or with CST alone. Subpanels 1 and 2: confocal pseudocolor images of CST-treated BAE-1 cells from the correspondent time points indicated by the arrows, showing membrane (1) and cytosolic (2) localizations of GFP-Cav1 after CST treatment. (b) Bar graphs representing the percent variation with respect to control of the fluorescent GFP-Cav1 signal in the different experimental conditions (CST = 86.94 ± 25.51%; H:ase + CST = −0.07 ± 22.20%; n = 3 sets of experiments, 3 fields/sample, about 40 cells/fields; P < 0.05). (c) Bar graph representing the percent variation with respect to control of the fluorescent GFP-Cav1 signal during CST 5 nM treatment, respectively, in plasma membrane and in cytosol (membrane = −19.34 ± 1.22%; cytosol = 122.6 ± 3.49%; n = 17 cells; P < 0.05).

Mentions: In transfected live cells GFP-Cav1 signal was confined in plasma membranes, while in the presence of CST 5 nM green fluorescence appeared clearly diffused in the cytosol, as a consequence of Cav1 internalization, thus producing a substantial increase of the overall fluorescent signal; pretreatment with H:ase strongly reduced CST ability to stimulate this process (Figures 4(a) and 4(b); percentage of fluorescence intensity variation above control: CST = 86.94 ± 25.51%; H:ase + CST = −0.07 ± 22.20%; n = 3 sets of experiments, 3 fields/sample, about 40 cells/fields; P < 0.05).


Obligatory role for endothelial heparan sulphate proteoglycans and caveolae internalization in catestatin-dependent eNOS activation.

Fornero S, Bassino E, Ramella R, Gallina C, Mahata SK, Tota B, Levi R, Alloatti G, Gallo MP - Biomed Res Int (2014)

CST induces proteoglycan-dependent Cav1 internalization in GFP-Cav1 transfected BAE-1 cells. (a) Representative time course of the fluorescence intensity in single BAE-1 cells transfected with Cav-1-GFP and stimulated with H:ase + CST or with CST alone. Subpanels 1 and 2: confocal pseudocolor images of CST-treated BAE-1 cells from the correspondent time points indicated by the arrows, showing membrane (1) and cytosolic (2) localizations of GFP-Cav1 after CST treatment. (b) Bar graphs representing the percent variation with respect to control of the fluorescent GFP-Cav1 signal in the different experimental conditions (CST = 86.94 ± 25.51%; H:ase + CST = −0.07 ± 22.20%; n = 3 sets of experiments, 3 fields/sample, about 40 cells/fields; P < 0.05). (c) Bar graph representing the percent variation with respect to control of the fluorescent GFP-Cav1 signal during CST 5 nM treatment, respectively, in plasma membrane and in cytosol (membrane = −19.34 ± 1.22%; cytosol = 122.6 ± 3.49%; n = 17 cells; P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4127283&req=5

fig4: CST induces proteoglycan-dependent Cav1 internalization in GFP-Cav1 transfected BAE-1 cells. (a) Representative time course of the fluorescence intensity in single BAE-1 cells transfected with Cav-1-GFP and stimulated with H:ase + CST or with CST alone. Subpanels 1 and 2: confocal pseudocolor images of CST-treated BAE-1 cells from the correspondent time points indicated by the arrows, showing membrane (1) and cytosolic (2) localizations of GFP-Cav1 after CST treatment. (b) Bar graphs representing the percent variation with respect to control of the fluorescent GFP-Cav1 signal in the different experimental conditions (CST = 86.94 ± 25.51%; H:ase + CST = −0.07 ± 22.20%; n = 3 sets of experiments, 3 fields/sample, about 40 cells/fields; P < 0.05). (c) Bar graph representing the percent variation with respect to control of the fluorescent GFP-Cav1 signal during CST 5 nM treatment, respectively, in plasma membrane and in cytosol (membrane = −19.34 ± 1.22%; cytosol = 122.6 ± 3.49%; n = 17 cells; P < 0.05).
Mentions: In transfected live cells GFP-Cav1 signal was confined in plasma membranes, while in the presence of CST 5 nM green fluorescence appeared clearly diffused in the cytosol, as a consequence of Cav1 internalization, thus producing a substantial increase of the overall fluorescent signal; pretreatment with H:ase strongly reduced CST ability to stimulate this process (Figures 4(a) and 4(b); percentage of fluorescence intensity variation above control: CST = 86.94 ± 25.51%; H:ase + CST = −0.07 ± 22.20%; n = 3 sets of experiments, 3 fields/sample, about 40 cells/fields; P < 0.05).

Bottom Line: 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.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.Taken together, these results highlight the obligatory role for proteoglycans and caveolae internalization in the catestatin-dependent eNOS activation in endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123 Turin, Italy.

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.

Show MeSH
Related in: MedlinePlus