Regulation of Weibel-Palade body exocytosis by alpha-synuclein in endothelial cells - PubMed (original) (raw)
Regulation of Weibel-Palade body exocytosis by alpha-synuclein in endothelial cells
Kwang Soo Kim et al. J Biol Chem. 2010.
Abstract
alpha-Synuclein is a small presynaptic protein implicated in the pathogenesis of Parkinson disease. Nevertheless, its physiological roles and mechanisms remain incompletely understood. alpha-Synuclein is not only expressed in neurons but also in the vascular endothelium, which contains intracellular granules called Weibel-Palade bodies (WPBs) that contain a number of chemokines, adhesive molecules, and inflammatory cytokines. This study explored whether the exocytosis of WPB is regulated by alpha-synuclein. Phorbol 12-myristate 13-acetate-, thrombin-, or forskolin-induced von Willebrand factor release or translocation of P-selectin from endothelial cells were inhibited by alpha- and beta-synuclein but not gamma-synuclein. Three point mutants (A30P, A53T, and E46K) found in familial Parkinson disease also inhibited WPB exocytosis similar to that of wild-type alpha-synuclein. Furthermore, the negative regulation of WPB exocytosis required the N terminus or the nonamyloid beta-component of Alzheimer disease amyloid region of alpha-synuclein, but not the C-terminal acidic tail, and alpha-synuclein affected WPB exocytosis through interference with RalA activation by enhancing the interaction of RalGDS-beta-arrestin complexes. Immuno-EM analysis revealed that alpha-synuclein was localized close to WPBs. These findings imply that alpha-synuclein plays as a negative regulator in WPB exocytosis in endothelial cells.
Figures
FIGURE 1.
WPB exocytosis is inhibited by α-syn overexpression. HUVECs overexpressing by α-syn were stimulated with 100 n
m
PMA, 2 units/ml thrombin (Thr), or 20 μ
m
forskolin (For) together with 100 μ
m
IBMX for 1 h. A, cell-free supernatants were then analyzed for vWF by ELISA. Con, control. B, cells were stained with anti-vWF antibody (red) and observed under a confocal microscope. Blue indicates 4′,6-diamidino-2-phenylindole staining. C, after transfection for 24 h, vWF in total lysates was detected by Western blot. D and E, leukocyte adhesion assay was performed as described under “Experimental Procedures.” Green dots indicate CFDA-prelabeled THP-1 cells adhered to HUVECs. Scale bar indicates 20 μm. **, p < 0.01 against control.
FIGURE 2.
Effects of α-syn siRNA on WPB exocytosis. A, after transfection of HUVECs with 1 n
m
control (con) and α-syn siRNA, respectively, their expression levels were detected by Western blot. B, after stimulation with 100 n
m
PMA for 1 h, the cell-free supernatants were then analyzed by ELISA for vWF. C, leukocyte adhesion assay was performed as described under “Experimental Procedures.” **, p < 0.01 against control siRNA.
FIGURE 3.
Effects of α-, β-, γ-syn, and α-syn point mutants. A, after transfection of HUVECs with α-, β-, or γ-syn, respectively, their expression levels were detected by Western blot. B, after stimulation with 100 n
m
PMA for 1 h, the cell-free supernatants were then analyzed by ELISA for vWF. C, leukocyte adhesion assay was performed as described under “Experimental Procedures.” D, after transfection of HUVECs with point mutants of α-syn, they were detected by Western blot. con, control; WT, wild type. E, after stimulation with 100 n
m
PMA for 1 h, the cell-free supernatants were then analyzed for vWF by ELISA. **, p < 0.01 against control.
FIGURE 4.
Effects of deletion mutants of α-syn. A, after transfection of HUVECs with α-syn and GFP-α-syn, respectively, α-syn or GFP levels were detected by Western blot. con, control. B, after stimulation with 100 n
m
PMA for 1 h, the cell-free supernatants were then analyzed for vWF by ELISA. C, schematic representation of GFP-α-syn deletion mutants. D, after transfection of HUVECs with GFP-α-syn deletion mutants for 24 h, their levels were detected by Western blot against GFP. E, after stimulation with 100 n
m
PMA for 1 h, the cell-free supernatants were then analyzed for vWF by ELISA. F, leukocyte adhesion assay was performed as described under “Experimental Procedures.” *, p < 0.05; **, p < 0.01 against control.
FIGURE 5.
α-Syn overexpression inhibits small GTPase RalA activity in HUVECs. After transfection of HUVECs with GFP-α-syn (A) and GFP-α-syn deletion mutants (B), the cells were treated with 100 n
m
PMA, 2 units/ml thrombin, or 20 μ
m
forskolin together with 100 μ
m
IBMX for 1 h. GTP-bound form of RalA was then isolated from cell lysates incubated with the GST-RalBP and analyzed by Western blot with anti-RalA monoclonal antibody. The data were analyzed by the band intensities of Western blot. C, after cotransfection of HUVECs with α-syn and active RalA mutant (Q72L) and treatment with 100 n
m
PMA for 1 h, the expression level of α-syn and RalA was detected by Western blot. con, control. D, after stimulation with 100 n
m
PMA for 1 h, the cell-free supernatants were then analyzed for vWF by ELISA. *, p < 0.05; **, p < 0.01.
FIGURE 6.
α-Syn binds to both β-arrestin and RalGDS, and the interaction of β-arrestin and RalGDS is enhanced by α-syn overexpression. A, after transfection of HUVECs with GFP or GFP-α-syn and treatment with 100 n
m
PMA for 1 h, the cells were lysed and immunoprecipitated with anti-GFP and anti-β-arrestin antibodies. B, anti-GFP and anti-RalGDS antibodies; D, anti-RalGDS and anti-β-arrestin antibodies. con, control. After transfection of HUVECs with GFP, GFP-syn(1–95), or GFP-syn(96–140) and treatment with 100 n
m
PMA for 1 h, the cells were lysed and immunoprecipitated with anti-GFP (C) or anti-β-arrestin antibodies (E), and the immunoprecipitated proteins were analyzed by Western blot for the indicated antibodies.
FIGURE 7.
α-Syn is localized close to WPB in HUVECs. HUVECs were prepared as described under “Experimental Procedures,” and the sections were incubated overnight in the presence (A–C) or absence (D and E) of α-syn antibody at 4 °C and then with 10-nm gold conjugated goat anti-mouse IgG antibody. The sections were observed under an electron microscope. The regions of A and D in the boxes were magnified in the images of B, C and E, F, respectively. Note that α-syn was close to WPB in HUVECs. Arrow indicates the cluster of gold particles. Scale bar indicates 0.6 μm.
FIGURE 8.
Exogenously added recombinant α-syn also inhibits vWF release in HUVECs. A, after treatment of HUVECs with the indicated dose of recombinant α-syn for 1 h, the cells were stimulated with 100 n
m
PMA, 2 units/ml thrombin, or 20 μ
m
forskolin together with 100 μ
m
IBMX for 1 h. The cell-free supernatants were then analyzed for vWF by ELISA. B, after treatment of HUVECs with 1 μ
m
recombinant α-syn for 1 h, the cells were stimulated with 100 n
m
PMA. Leukocyte adhesion assay was then performed. C, after treatment of HUVECs with 1 μ
m
recombinant α-, β-, or γ-syn for 1 h, the cells were stimulated with 100 n
m
PMA. The cell-free supernatants were then analyzed for vWF by ELISA. **, p < 0.01 against control (con).
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