ABSTRACT
Previous studies showed that CCN3 is deregulated in early-onset pre-eclampsia (PE), a pregnancy disease associated with impaired trophoblast invasion, which leads to reduced fetal oxygen and nutrition support. Recently, we identified the glycosylated (g-CCN3) and the non-glycosylated (ng-CCN3) form of matricellular CCN3 as key factors in regulation of trophoblast proliferation and invasion. While Jeg3 cells revealed a decreased proliferation upon stimulation with both forms of CCN3, enhanced migration and invasion properties were only found for ng-CCN3. Here, we focused on the signalling cascades mitogen-activated protein kinase (MAPK), PI3 kinase/Akt and Notch/p21 for mediating the dual function of CCN3 on trophoblast proliferation versus migration in Jeg3 cells upon stimulation with g- and ng-recombinant CCN3 (g/ng-rCCN3). Analysis of the CCN3-mediated signalling pathways showed that ng-rCCN3 stimulated migration properties by activating the Akt as well as the MAPK pathway. Moreover, cell migration stimulated by ng-rCCN3 was mediated via Akt and integrin α5ß1 but not the antiproliferative effect of CCN3. There was evidence that the Notch pathway might contribute to the antiproliferative properties of both forms of CCN3 by an increase in Notch1 expression and its target gene, the cell cycle inhibitor p21. Our data showed that the presence of both forms of CCN3 is accompanied by a balance of trophoblast proliferation and migration/invasion properties, which are triggered by different signalling pathways. Thus, a deregulated expression of g/ng-CCN3 could lead to an imbalance in proliferation versus invasion, and might contribute to the shallow trophoblast invasion observed in PE.
Subject(s)
MAP Kinase Signaling System , Nephroblastoma Overexpressed Protein/physiology , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Notch/metabolism , Signal Transduction , Trophoblasts/cytology , Cell Movement , Cell Proliferation , Female , Humans , Models, Biological , Nephroblastoma Overexpressed Protein/metabolism , Trophoblasts/metabolismABSTRACT
BACKGROUND: Recently we have shown that the matricellular CCN3 protein expressed in invasive extravillous trophoblast cells (EVTs) is decreased in early-onset pre-eclampsia and is regulated by oxygen tension. Pathogenesis of pre-eclampsia relies on a shallow invasion of EVTs into the spiral arteries, which leads to hypoxia accompanied by uteroplacental insufficiency. Here we investigated the function of glycosylated and non-glycosylated CCN3 protein on cell growth as well as migration and invasion properties of the malignant trophoblast cell line Jeg3 which is a widely used model for the invasive trophoblast. METHODS AND RESULTS: Stable transfection of Jeg3 choriocarcinoma cells with full length CCN3 resulted in high expression of secreted glycosylated and cellular non-glycosylated CCN3. These cells revealed significantly reduced growth in cell numbers combined with a significantly increased migratory and invasive capacity. Matrix metalloprotease (MMP)-2 and MMP-9 activities were enhanced dependent on CCN3 expression, which could be confirmed by CCN3 knockdown studies. Using recombinant glycosylated and non-glycosylated CCN3, we revealed that CCN3 decreased growth in Jeg3 cell numbers independent of its glycosylation status, whereas only non-glycosylated CCN3 was able to enhance migration and invasion properties. CONCLUSIONS: The present results suggest that CCN3 protein regulates the decrease in Jeg3 cell numbers independent of its glycosylation status, whereas migratory and invasive properties are influenced only by non-glycosylated CCN3. An impaired balance in the expression of glycosylated and non-glycosylated CCN3 could contribute to the shallow invasion of EVTs observed in pre-eclampsia.
