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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 prevents Cav1/eNOS colocalization in a PI3K-dependent manner. (a) Confocal immunofluorescence images of BAE-1 cells showing the typical membrane localization of Cav1 (red staining) and eNOS (green staining). After CST administration, Cav1 dissociated from eNOS and staining diffused in the cytosol. This effect was reversed by Wm pretreatment. Immunofluorescence detection was carried out using Alexa Fluor 488 anti-mouse for total eNOS and Cy3 anti-rabbit for Cav1. Scale bar: 20 μm. (b) Bar graph representing Pearson correlation values relative to Cav1 and eNOS staining in each experimental condition (control = 0.84 ± 0.09; CST = −0.07 ± 0.05; Wm + CST = 0.69 ± 0.18; n = 8 sets of experiments, 3 fields/sample, about 30 cells/fields; P < 0.05).
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fig5: CST prevents Cav1/eNOS colocalization in a PI3K-dependent manner. (a) Confocal immunofluorescence images of BAE-1 cells showing the typical membrane localization of Cav1 (red staining) and eNOS (green staining). After CST administration, Cav1 dissociated from eNOS and staining diffused in the cytosol. This effect was reversed by Wm pretreatment. Immunofluorescence detection was carried out using Alexa Fluor 488 anti-mouse for total eNOS and Cy3 anti-rabbit for Cav1. Scale bar: 20 μm. (b) Bar graph representing Pearson correlation values relative to Cav1 and eNOS staining in each experimental condition (control = 0.84 ± 0.09; CST = −0.07 ± 0.05; Wm + CST = 0.69 ± 0.18; n = 8 sets of experiments, 3 fields/sample, about 30 cells/fields; P < 0.05).

Mentions: To verify this hypothesis we followed cellular colocalization of Cav1 and eNOS by immunofluorescence experiments (Figure 5). We observed that CST strongly reduced eNOS/Cav1 colocalization at plasma membrane detected in control condition. Moreover, Wm was able to restore this colocalization, confirming the role of PI3K in mediating CST intracellular signaling.


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 prevents Cav1/eNOS colocalization in a PI3K-dependent manner. (a) Confocal immunofluorescence images of BAE-1 cells showing the typical membrane localization of Cav1 (red staining) and eNOS (green staining). After CST administration, Cav1 dissociated from eNOS and staining diffused in the cytosol. This effect was reversed by Wm pretreatment. Immunofluorescence detection was carried out using Alexa Fluor 488 anti-mouse for total eNOS and Cy3 anti-rabbit for Cav1. Scale bar: 20 μm. (b) Bar graph representing Pearson correlation values relative to Cav1 and eNOS staining in each experimental condition (control = 0.84 ± 0.09; CST = −0.07 ± 0.05; Wm + CST = 0.69 ± 0.18; n = 8 sets of experiments, 3 fields/sample, about 30 cells/fields; P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

fig5: CST prevents Cav1/eNOS colocalization in a PI3K-dependent manner. (a) Confocal immunofluorescence images of BAE-1 cells showing the typical membrane localization of Cav1 (red staining) and eNOS (green staining). After CST administration, Cav1 dissociated from eNOS and staining diffused in the cytosol. This effect was reversed by Wm pretreatment. Immunofluorescence detection was carried out using Alexa Fluor 488 anti-mouse for total eNOS and Cy3 anti-rabbit for Cav1. Scale bar: 20 μm. (b) Bar graph representing Pearson correlation values relative to Cav1 and eNOS staining in each experimental condition (control = 0.84 ± 0.09; CST = −0.07 ± 0.05; Wm + CST = 0.69 ± 0.18; n = 8 sets of experiments, 3 fields/sample, about 30 cells/fields; P < 0.05).
Mentions: To verify this hypothesis we followed cellular colocalization of Cav1 and eNOS by immunofluorescence experiments (Figure 5). We observed that CST strongly reduced eNOS/Cav1 colocalization at plasma membrane detected in control condition. Moreover, Wm was able to restore this colocalization, confirming the role of PI3K in mediating CST intracellular signaling.

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