Role of MSK1 in the signaling pathway leading to VEGF-mediated PAF synthesis in endothelial cells
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Transcript of Role of MSK1 in the signaling pathway leading to VEGF-mediated PAF synthesis in endothelial cells
release of bFGF within 10 min into the pericellular matrix.Activation of PAR-1, -3 and -4, but not of PAR-2, by specificpeptides caused bFGF release, indicating a receptor-triggeredmechanism. Pretreatment with the highly specific Rho kinaseinhibitor Y-27632 or the protein kinase C (PKC) deltainhibitor rottlerin inhibited thrombin-induced release of bFGF,FGFR-1 phosphorylation and mitogenesis. Furthermore,incubation of SMC with cyclodextrin–cholesterol for 24 h,resulting in cellular enrichment with cholesterol as demon-strated by oil red staining, caused a strong increase in bFGFrelease. In contrast, incubation with cyclodextrin aloneresulted in decreased membrane fluidity and thrombin-induced release of bFGF. Cholesterol-enrichment resulted inincreased DNA synthesis after stimulation with thrombin,which was inhibited by a bFGF-neutralizing antibody.Thesedata suggest that thrombin causes release of bFGF fromhuman SMC via the Rho kinase–PKC delta pathway.Accumulated cellular cholesterol may increase bFGF release,resulting in an increased mitogenic response towardsthrombin.
doi:10.1016/j.vph.2006.08.344
B17.03
Role of MSK1 in the signaling pathway leading toVEGF-mediated PAF synthesis in endothelial cells
Catherine Marchand, Judith Favier, Martin G. Sirois
Montreal Heart Institute, Montreal, QC, Canada
Vascular endothelial growth factor (VEGF) inflammatoryeffects require acute platelet-activating factor (PAF) synthesisby endothelial cells (EC). We previously reported that VEGF-mediated PAF synthesis involves the activation of VEGFreceptor-2/neuropilin-1 complex, which is leading to theactivation of p38 and p42/44 mitogen-activated proteinkinases (MAPKs) and group V secretory phospholipase A2(sPLA2-V). As the mechanisms regulating sPLA2-V remainunknown, we addressed the role of the mitogen- and stress-activated protein kinase-1 (MSK1), which can be rapidly andtransiently activated by p38 or p42/44 MAPKs. In addition, ithas been reported that MSK1 is activated upon stimulation ofEC with VEGF. Together these different properties of MSK1led us to suggest that it might be involved in VEGF-mediatedPAF synthesis. Its implication was first assessed bycoimmuprecipitation and Western blot analyses. In nativebovine aortic endothelial cells (BAEC), we observed aconstitutive protein interaction between MSK1 with p38,p42/44 MAPKs and sPLA2-V. To assess the role of MSK1 N-and C-terminal kinase domains into these interactions, BAECwere transfected with the empty vector pCDNA3.1, wild-typeMSK1 (MSK1-WT), N-terminal (MSK1-D195A) or C-terminal (MSK1-D565A) dead kinase MSK1 mutants. Theprotein interactions were maintained in transfected BAEC
expressing pCDNA3.1, MSK1-WT or MSK1-D195A. How-ever, in BAEC expressing MSK1-D565A, the interactionbetween MSK1 and sPLA2-V was reduced by 82 and 90%under basal and VEGF-treated conditions as compared tonative BAEC. Treatment with VEGF for 15 min increasedbasal PAF synthesis in native BAEC, pCDNA3.1, MSK1-WTand MSK1-D195A by 166, 139, 125 and 82% respectively. Incontrast, PAF synthesis was prevented in cells expressingMSK1-D565A mutant. These results demonstrate the essentialrole of the C-terminal domain of MSK1 for its constitutiveinteraction with sPLA2-V, which appears essential to supportVEGF-mediated PAF synthesis. Supported by grants from theCanadian Institutes of Health Research and the Heart andStroke Foundation of Québec.
doi:10.1016/j.vph.2006.08.345
B17.04
The guanine exchange factor RalGDS is involved inregulated exocytosis of Weibel–Palade bodies fromendothelial cells
R. Bierings, A. Kragt, M.G. Rondaij, K.A. Gijzen, E. Sellink,J.A. van Mourik, M. Fernandez-Borja, J. Voorberg
Sanquin Research and Landsteiner Laboratory, Amsterdam,The Netherlands
The activation of the small GTPase RalA is directlyinvolved in exocytosis in many different cell types via itsassociation with the exocyst complex. Exocytosis of Weibel–Palade bodies, which are endothelial cell-specific secretoryorganelles that store von Willebrand Factor and P-selectin,was recently found to coincide with the activation of the smallGTPase RalA in response to Ca2+- as well as cAMP-raisingstimuli such as respectively thrombin and epinephrine. So far,little is known about the upstream events that lead toactivation of RalA and WPB exocytosis in response to G-protein coupled receptor activation. In this study we show thatsiRNA-mediated knock-down of the RalGEF RalGDS resultedin inhibition of thrombin — as well as epinephrine-inducedWeibel–Palade body exocytosis from HUVEC. In addition,overexpression of RalGDS induced RalA activation andstimulated the release of Weibel–Palade bodies in restingHUVEC. Moreover, a RalGDS mutant lacking its catalyticexchange domain was found to behave in a dominant negativemanner by blocking stimulus-induced exocytosis of theseorganelles. Together, these findings implicate RalGDS inagonist-induced RalA activation and the exocytosis ofWeibel–Palade bodies from endothelial cells.
doi:10.1016/j.vph.2006.08.346
e130 14th IVBM Abstracts