145. WINGLESS (WNT) 3A INDUCES TROPHOBLAST MIGRATION AND MATRIX METALLOPROTEINASE-2 SECRETION THROUGH CANONICAL WNT SIGNALLING AND PROTEIN KINASE B/AKT ACTIVATION

Abstract

Human placenta and trophoblasts express WNT ligands and WNT receptors suggesting a role for WNT-signalling in placental development. Indeed, a recombinant WNT ligand was recently shown to promote trophoblast migration/invasion, however, the involved signalling cascades and their target genes have not been elucidated. The aim was to investigate signal transduction via canonical WNT-signalling or phosphatidylinositide 3-kinase (PI3K)/AKT-signalling, their cross-talk as well as trophoblast-specific protease expression in trophoblastic SGHPL-5 cells and primary 1st trimester extravillous trophoblasts (EVT). WNT3A-dependent activation/phosphorylation of AKT (pAKT) and the down-stream kinase glycogen synthase kinase (GSK)-3β were determined by Western blotting (WB). WNT3A-induced canonical WNT-signalling was analysed by luciferase reporter assay (TOPFlash) and nuclear recruitment of β-catenin. Trophoblast migration was studied using transwell assays and villous explant cultures. MMP2 expression/activation was investigated by qRT-PCR and WB/gelatine-zymography of supernatants. All experiments were performed +/– inhibitors of AKT-signalling or canonical WNT-signalling using LY294002 (PI3K inhibitor) or recombinant Dickkopf-1 (DKK1). WNT3A induced pAKT, luciferase expression of the canonical WNT reporter (P < 0.05) as well as accumulation of nuclear β-catenin. Inhibition of PI3K abolished WNT-dependent pAKT, pGSK-3β and cell migration, but did not affect TOPFlash activity or appearance of nuclear β-catenin. Inhibition through DKK1 did not influence pAKT and pGSK-3β, but decreased WNT reporter activity, nuclear β-catenin and cell migration. Both inhibitors decreased WNT3A-induced MMP2 expression in SGHPL-5 cells and pure EVTs (P < 0.05). WNT3A activates PI3K/AKT as well as canonical WNT-signalling through distinct receptors in invasive trophoblasts, since DKK1 did not activate the particular kinase. Although cross-talk between PI3K/AKT and canonical WNT-signalling has been observed in some cell types, these pathways seem to act independently in trophoblasts. However, both pathways promote Wnt-dependent migration and expression of MMP2. This is the 1st study identifying MMP2 as a novel target gene of canonical WNT-signalling in trophoblast.