Glycodelin-a suppressed trophoblast invasion by down-regulating urokinase plasminogen activator and extracellular signal regulated kinases activities

Abstract

INTRODUCTION: Invasion of the extravillous cytotrophoblast into the maternal endometrium is crucial to placenta formation and is strictly regulated by paracrine and autocrine interactions. Dysregulation of the process results in a wide spectrum of abnormal pregnancies, like preeclampsia, choriocarcinoma and intrauterine growth restriction. One of the invasive phenotypes of extravillous cytotrophoblast is production of matrix degrading enzyme, including urokinase plasminogen activator (uPA). Extracellular signal regulated kinase (ERK), a member of the mitogen activated protein kinase (MAPK) family is known to control trophoblast invasion and migration. Glycodelin-A (GdA), a glycoprotein abundantly synthesized by the decidua but not the trophoblast, is upregulated during first trimester coinciding with the time of trophoblast invasion. We hypothesize that decidual-derived glycodelin-A is a paracrine regulator fine-tuning the invasion of human trophoblast. MATERIAL & METHODS: Trans-well invasion assay was used to examine the effect of GdA and MEK (MAPK kinase) inhibitors (PD98059 & U0126) on the invasiveness of an immortalized first trimester extravillous cytotrophoblast (TEV-1) and a choricarcinoma (JEG-3) cell line. The RNA expression, proteinase activity and secretion of uPA were determined by real time quantitative PCR, quantitative urokinase activity assay and ELISA, respectively. Western blotting analysis was employed to examine the effect of GdA on ERK activities. RESULTS: Transwell invasion assay showed that GdA, but not deglycosylated GdA, significantly reduced the invasiveness of the cells. At 30 pmol/ml, it inhibited 47.7±8.7% of TEV-1 and 27.1±4.7% of JEG-3 invasion. The suppressive effect of GdA on invasion was associated with a suppression of uPA proteinase activity. The transcription and secretion of uPA were also significantly reduced by 72.0±4.3% and 36.13±8.8% respectively after GdA (30 pmol/ml) treatment. Western blotting analysis showed that GdA suppressed the phosphorylated ERK level in a dose dependent manner. The involvement of ERK in the trophoblast invasion was supported by the observation that MEK inhibitors PD98059 and U0126 that reduced phosphorylation of ERK also significantly suppressed the invasiveness of trophoblast. A dose-dependent down-regulation of uPA mRNA was also noted by the real time quantitative PCR after treatment with these inhibitors at concentrations that did not affect cell viability. CONCLUSIONS: This is the first report demonstrating a suppression of trophoblast invasion and uPA transcription through down regulating ERK activities in trophoblast cell lines. Thus, GdA is a potential paracrine regulator at the fetal-maternal interface limiting the extent of trophoblast invasion. The results may have implication in pregnancy complications such as preeclampsia, which is characterized by shallow invasion. Consistently, trophoblast cells from preeclamptic women have reduced uPA proteolytic activities.