Wingless (Wnt)-3A Induces Trophoblast Migration and Matrix Metalloproteinase-2 Secretion through Canonical Wnt Signaling and Protein Kinase B/AKT Activation

Abstract

Invasion of human trophoblasts is promoted through activation of wingless (Wnt) signaling, suggesting a role of the pathway in placental development and morphogenesis. However, details on the process such as involvement of canonical and/or noncanonical Wnt signaling cascades as well as their target genes are largely unknown. Hence, signal transduction via canonical Wnt signaling or phosphatidylinositide 3-kinase (PI3K)/AKT and their cross talk as well as trophoblast-specific protease expression were investigated in trophoblastic SGHPL-5 cells and primary extravillous trophoblasts purified from first-trimester placentas. Western blot analyses revealed that the recombinant Wnt ligand Wnt-3A increased phosphorylation of AKT and the downstream kinase glycogen synthase kinase (GSK)-3beta as well as accumulation of activated, nuclear beta-catenin. In accordance, luciferase expression of a canonical Wnt/TCF reporter and cell migration in first-trimester villous explant cultures and of SGHPL-5 cells were stimulated. Chemical inhibition of PI3K abolished Wnt-dependent phosphorylation of AKT and GSK-3beta and trophoblast motility but did not affect appearance of activated beta-catenin or Wnt/TCF reporter activity. In contrast, inhibition of the canonical pathway through soluble Dickkopf-1 did not influence AKT and GSK-3beta phosphorylation but reduced Wnt reporter activity, accumulation of active beta-catenin, and cell migration. Both inhibitors decreased Wnt-3A-induced secretion of pro- and active matrix metalloproteinase-2 from SGHPL-5 cells and pure EVT. The data suggest that Wnt-3A may activate canonical Wnt signaling and PI3K/AKT through distinct receptors. The two signaling cascades act independently in trophoblasts; however, both pathways promote Wnt-dependent migration and the release of matrix metalloproteinase-2, which has been identified as novel Wnt target in invasive trophoblasts.

Notch1 controls development of the extravillous trophoblast lineage in the human placenta

“…Primary CTBs were incubated under 20% (vol/vol) oxygen (ctrl), 5% (vol/vol) oxygen, or 20% (vol/vol) oxygen/ 50 μM CoCl 2 . Protein lysates were prepared after an additional 24 h. Luciferase activity and β-galactosidase activity were determined as previously published (72).…”

“…Whole-cell lysates were prepared using standard protocols as recently described (72). Villous explants were additionally homogenized for 20 s at 3,640 × g with a Precellys 24 (PeqLab).…”

The role of Wnt signaling members in the uterus and embryo during pre-implantation and implantation

“…Thus, it was suggested that, migration in primary explant cultures was induced by Wnt3a in the presence of either Dkk1 or the PI3K inhibitor [86]. Incubation with recombinant Wnt3a increased phosphorylation of AKT at Ser473 as well as Wnt-dependent phosphorylation of GSK-3B at Ser9 was noticed.…”

“…Incubation with recombinant Wnt3a increased phosphorylation of AKT at Ser473 as well as Wnt-dependent phosphorylation of GSK-3B at Ser9 was noticed. As, Dkk1 failed to inhibit Wntinduced phosphorylation of AKT or GSK-3B, it was suggested that a noncanonical Wnt receptor/ pathway may affect the particular kinases [86].…”

“…Wnt3a also stimulated pro-MMP-2 accumulation and this accumulation was inhibited by Dkk1 or LY294002 in extravillous trophoblast. Since Wnt3a was not effective in alteration of MMP-2 mRNA levels, it was suggested that Wnt3a has posttranscriptional effects on elevation of MMP-2 concentration [86].…”