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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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Both proteoglycans and caveolae are required to allow CST-dependent eNOS phosphorylation. (a) Typical Western blot experiment showing that CST-induced PSer1179eNOS was reduced by both MβCD (5 mM, 30 min) and H:ase (2 U/mL). (b) PSer1179eNOS/β-actin ratio of densitometric values from Western blots (%PSer1179eNOS/β-actin: control = 3.59 ± 1; CST = 18.81 ± 1.57; MβCD + CST = 3.48 ± 1.5, H:ase + CST = 1.63 ± 0.41; n = 3; P < 0.05).
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fig6: Both proteoglycans and caveolae are required to allow CST-dependent eNOS phosphorylation. (a) Typical Western blot experiment showing that CST-induced PSer1179eNOS was reduced by both MβCD (5 mM, 30 min) and H:ase (2 U/mL). (b) PSer1179eNOS/β-actin ratio of densitometric values from Western blots (%PSer1179eNOS/β-actin: control = 3.59 ± 1; CST = 18.81 ± 1.57; MβCD + CST = 3.48 ± 1.5, H:ase + CST = 1.63 ± 0.41; n = 3; P < 0.05).

Mentions: To strongly demonstrate the proposed pathway of a CST-dependent caveolae internalization and eNOS activation triggered by HSPGs, we evaluated the level of CST-induced PSer1179eNOS after both caveolae disruption by methyl-β-cyclodextrin (MBCD, 5 mM) and HSPGs removal (by H:ase, 3 h). Our results from Western blot experiments (Figure 6) showed a significant reduction of the CST-dependent eNOS phosphorylation in both conditions (%PSer1179eNOS/β-actin: control = 3.59 ± 1; CST = 18.81 ± 1.57; MβCD + CST = 3.48 ± 1.5, H:ase + CST = 1.63 ± 0.41; n = 3; P < 0.05), confirming the obligatory requirement of proteoglycans and caveolae integrity to allow the CST-dependent intracellular pathway activation.


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)

Both proteoglycans and caveolae are required to allow CST-dependent eNOS phosphorylation. (a) Typical Western blot experiment showing that CST-induced PSer1179eNOS was reduced by both MβCD (5 mM, 30 min) and H:ase (2 U/mL). (b) PSer1179eNOS/β-actin ratio of densitometric values from Western blots (%PSer1179eNOS/β-actin: control = 3.59 ± 1; CST = 18.81 ± 1.57; MβCD + CST = 3.48 ± 1.5, H:ase + CST = 1.63 ± 0.41; n = 3; P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Both proteoglycans and caveolae are required to allow CST-dependent eNOS phosphorylation. (a) Typical Western blot experiment showing that CST-induced PSer1179eNOS was reduced by both MβCD (5 mM, 30 min) and H:ase (2 U/mL). (b) PSer1179eNOS/β-actin ratio of densitometric values from Western blots (%PSer1179eNOS/β-actin: control = 3.59 ± 1; CST = 18.81 ± 1.57; MβCD + CST = 3.48 ± 1.5, H:ase + CST = 1.63 ± 0.41; n = 3; P < 0.05).
Mentions: To strongly demonstrate the proposed pathway of a CST-dependent caveolae internalization and eNOS activation triggered by HSPGs, we evaluated the level of CST-induced PSer1179eNOS after both caveolae disruption by methyl-β-cyclodextrin (MBCD, 5 mM) and HSPGs removal (by H:ase, 3 h). Our results from Western blot experiments (Figure 6) showed a significant reduction of the CST-dependent eNOS phosphorylation in both conditions (%PSer1179eNOS/β-actin: control = 3.59 ± 1; CST = 18.81 ± 1.57; MβCD + CST = 3.48 ± 1.5, H:ase + CST = 1.63 ± 0.41; n = 3; P < 0.05), confirming the obligatory requirement of proteoglycans and caveolae integrity to allow the CST-dependent intracellular pathway activation.

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