O204	FrzA/sFRP-1 activates non canonical Wnt signaling pathway in endothelial cells through interaction with Frizzled 4, 6 and 7.
1J.Ezan, 2P.Dufourcq, 1C.Moreau, 3T.Couffinhal, 1C.Duplaa
1Inserm U441, Pessac, FR; 2Universite de Bordeaux 2; Victor Segalen, Bordeaux, FR; 3Department of Cardiology, Hopital Haut Leveque, Pessac, FR.

sFRP-1/FrzA is a secreted frizzled related protein thought to interfere with Wnt/frizzled signaling pathway. sFRP-1/FrzA induced endothelial cell (EC) migration and differentiation in vitro and was shown to be a strong angiogenic factor in vivo. We showed that FrzA modified endothelial cell-extracellular matrix interactions increasing significantly endothelial cell spreading on gelatin, collagen type I and laminin. Blocking antibody experiment demonstrates that FrzA effects on endothelial cell spreading was alpha2beta1 integrin dependent. We demonstrated that FrzA-induced cell spreading involved a Ser9-Phospho GSK-3beta dependent pathway: infection of EC with adenoviral vectors encoding catalytically-inactive form of GSK-3beta blocked FrzA induced EC spreading. Whether FrzA/sFRP-1 activates canonical (beta-catenin) or non canonical pathway in these processes remain unclear as well as which Frizzled receptor (Fzd) could be involved. Here, we demonstrate by cell transfection studies that FrzA/sFRP-1 do not activate the canonical Wnt pathway in endothelial cells but activates a non canonical pathway involving activation of GSK-3beta (phosphorylation at serine-9 residue) and Rac/cdc42 GTPases activation in a cell specific manner. Using deleted β-catenin endothelial cell line, FrzA/sFRP-1 treatment can induce GSK-3beta activation regulating GSK-3beta phosphorylation at ser9 residue. Expression analysis of Fzd showed that Fzd 1, 2, 4, 5, 6, 7, 8 are expressed on endothelial cells in vitro and in mouse artery. Gene expression knockdown of distinct Fzd using siRNA demonstrated that Fzd4, Fzd6, Fzd7 are necessary to FrzA/sFRP-1 induced-endothelial cell spreading. Only Fzd4 suppression altered FrzA effect on GSK-3beta phosphorylation decrease at Ser-9. Biochemical analysis demonstrated that FrzA/sFRP-1 can bind to Fzd4 on the cell surface. Together, these data demonstrated that FrzA/sFRP-1 signals through Fzd4, 6 and7 and implicated theses Wnt coreceptors in regulation of endothelial properties during vessel formation.

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