Activation of the Canonical Wingless/T-Cell Factor Signaling Pathway Promotes Invasive Differentiation of Human Trophoblast

Abstract

The molecular mechanisms governing invasive differentiation of human trophoblasts remain largely elusive. Here, we investigated the role of Wnt-␤-catenin-T-cell factor (TCF) signaling in this process. Reverse transcriptase-polymerase chain reaction and Western blot analyses demonstrated expression of Wnt ligands, frizzled receptors, LRP-6, and TCF-3/4 transcription factors in total placenta and different trophoblast cell models. Immunohistochemistry of placental tissues and differentiating villous explant cultures showed that expression of TCF-3/4 strongly increased in invading trophoblasts. Some of these cells also accumulated dephosphorylated ␤-catenin in the nucleus. Wnt3A treatment of primary cytotrophoblasts and SGHPL-5 cells induced activity of TCF-luciferase reporters. Accordingly, the ligand provoked interaction of TCF-3/4 with ␤-catenin as assessed in electrophoretic mobility shift assays (EMSAs) and upregulation of Wnt/TCF target genes as observed by Western blot analyses. Wnt3A stimulated trophoblast migration and invasion through Matrigel, which could be blocked by addition of Dickkopf-1, mediating inhibition of canonical Wnt signaling. Dickkopf-1 also reduced basal migration, invasion, and proliferation of cytotrophoblasts, suggesting expression of endogenous Wnt ligand(s). Immunohistochemistry revealed that the percentage of extravillous trophoblasts containing nuclear ␤-catenin was significantly higher in placentas of complete hydatidiform mole pregnancies as compared to normal placentas. Thus, canonical Wnt signaling may promote invasive trophoblast differentiation, and exaggerated activation of the pathway could contribute to trophoblastic hyperplasia and local invasion.

Complex Expression Pattern of Wnt Ligands and Frizzled Receptors in Human Placenta and its Trophoblast Subtypes

“…Recombinant Wnt-5a did not induce the canonical Wnt reporter pSuper(8x)TopFlash in SGHPL-5 cells which however could be activated 10.3 fold by canonical Wnt-3a (Fig. 2) as also previously noticed [15]. In contrast, Wnt-5a suppressed Wnt-3a-mediated transcription to 63% and 67% at a concentration of 200 ng/ml and 150 ng/ ml, respectively, suggesting that Wnt-5a may antagonise canonical Wnt signalling in trophoblasts.…”

“…Utilisation of tissues was approved by the local ethical committee. Villous cytotrophoblasts of early (n = 5) and late gestation (n = 6) with a purity of at least 98% were isolated by Kliman method and immuno-depletion of HLA-I-positive cells as previously mentioned [15,16]. Primary trophoblasts were seeded on plastics at a density of 5 × 10 5 cells/cm 2 and cultivated in DMEM containing 10% fetal calf serum (FCS, Pall, East Hills, NY, USA) for 24 h. Villous fibroblasts of first trimester placentae (n = 4) were also isolated by trypsinisation and gradient centrifugation (between = 25% and 35% Percoll) and passaged two times in DMEM supplemented with 10% FCS.…”

“…SGHPL-5 cells were grown to a confluence of 80% and co-transfected with luciferase plasmids containing either multimeric LEF/TCF cognate sequences (pSuper(8x)TopFlash) or mutated binding sites (pSuper(8x)FopFlash) and CMV-β-Gal vectors as described recently [15]. Briefly, cells cultivated in 24 wells were incubated with 1.5 μg luciferase reporter plasmid and 0.5 μg pCMV-β-Gal in 1.5 μl lipofectamine 2000 (Invitrogen, Carlsbad, CA).…”

“…In addition, endometrial expression of Wnt3 and Dickkopf-1 (Dkk1), which inhibits canonical Wnt signalling by disrupting binding of LRP-5/6 to the Wnt/FZD complex, was observed [14], suggesting paracrine mechanisms regulating implantation and/or trophoblast invasion. The TCF family members TCF-3 and TCF-4 are predominantly expressed in invasive trophoblasts and activation of the canonical Wnt signalling pathway promotes trophoblast migration and invasion [15]. The fact that Dkk-1 also blocked basal proliferation and invasion of trophoblast cultures suggested that canonical Wnt signalling could be regulated in an autocrine manner [15].…”

“…The TCF family members TCF-3 and TCF-4 are predominantly expressed in invasive trophoblasts and activation of the canonical Wnt signalling pathway promotes trophoblast migration and invasion [15]. The fact that Dkk-1 also blocked basal proliferation and invasion of trophoblast cultures suggested that canonical Wnt signalling could be regulated in an autocrine manner [15]. Therefore, we here investigated the full range of Wnt and FZD mRNAs of the human placenta and its different cell types and performed initial experiments on the potential role of Wnt-5a in trophoblasts.…”