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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 stimulates a PI3K-proteoglycan dependent endocytotic machinery in BAE-1 cells. BAE-1 cells incubated with the water-soluble styryl pyridinium membrane dye (FM 1-43). Pseudocolor images better show the fluorescence intensity increase correlated with the rise in vesicles formation consequent to stimulation with CST 5 nM. In CST + H:ase and CST + Wm samples fluorescence intensity was comparable to control levels. Surface plots close to each image display 3D graphs of pixels intensities in a pseudocolor image. The height and the color represent the pixel intensity.
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fig1: CST stimulates a PI3K-proteoglycan dependent endocytotic machinery in BAE-1 cells. BAE-1 cells incubated with the water-soluble styryl pyridinium membrane dye (FM 1-43). Pseudocolor images better show the fluorescence intensity increase correlated with the rise in vesicles formation consequent to stimulation with CST 5 nM. In CST + H:ase and CST + Wm samples fluorescence intensity was comparable to control levels. Surface plots close to each image display 3D graphs of pixels intensities in a pseudocolor image. The height and the color represent the pixel intensity.

Mentions: We observed that CST (5 nM) induced a significant increase in the FM 1-43 fluorescence (Figure 1), indicating a stimulation of the endocytotic process.


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 stimulates a PI3K-proteoglycan dependent endocytotic machinery in BAE-1 cells. BAE-1 cells incubated with the water-soluble styryl pyridinium membrane dye (FM 1-43). Pseudocolor images better show the fluorescence intensity increase correlated with the rise in vesicles formation consequent to stimulation with CST 5 nM. In CST + H:ase and CST + Wm samples fluorescence intensity was comparable to control levels. Surface plots close to each image display 3D graphs of pixels intensities in a pseudocolor image. The height and the color represent the pixel intensity.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: CST stimulates a PI3K-proteoglycan dependent endocytotic machinery in BAE-1 cells. BAE-1 cells incubated with the water-soluble styryl pyridinium membrane dye (FM 1-43). Pseudocolor images better show the fluorescence intensity increase correlated with the rise in vesicles formation consequent to stimulation with CST 5 nM. In CST + H:ase and CST + Wm samples fluorescence intensity was comparable to control levels. Surface plots close to each image display 3D graphs of pixels intensities in a pseudocolor image. The height and the color represent the pixel intensity.
Mentions: We observed that CST (5 nM) induced a significant increase in the FM 1-43 fluorescence (Figure 1), indicating a stimulation of the endocytotic process.

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