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Invasiveness of trophoblast and choriocarcinoma cells is in part mediated via leukemia inhibitory factor- (LIF-) induced activation of signal transducer and activator of transcription 3 (STAT3). The regulation of STAT3 phosphorylation at its ser727 binding site, possible crosstalk with intracellular MAPK signaling, and their functional implications are the object of the present investigation. JEG-3 choriocarcinoma cells were cultured in presence/absence of LIF and the specific ERK1/2 inhibitor (U0126). Phosphorylation of signaling molecules (p-STAT3 (ser727 and tyr705) and p-ERK1/2 (thr 202/tyr 204)) was assessed per Western blot. Immunocytochemistry confirmed results, but also pinpointed the location of phosphorylated signaling molecules. STAT3 DNA-binding capacity was studied with a colorimetric ELISA-based assay. Cell viability and invasion capability were assessed by MTS and Matrigel assays. Our results demonstrate that LIF-induced phosphorylation of STAT3 (tyr705 and ser727) is significantly increased after blocking ERK1/2. STAT3 DNA-binding capacity and cell invasiveness are enhanced after LIF stimulation and ERK1/2 blockage. In contrast, proliferation is enhanced by LIF but reduced after ERK1/2 inhibition. The findings herein show that blocking ERK1/2 increases LIF-induced STAT3 phosphorylation and STAT3 DNA-binding capacity by an intranuclear crosstalk, which leads to enhanced invasiveness and reduced proliferation.

Leukaemia inhibitory factor (LIF) and oncostatin M (OSM) are pleiotropic cytokines present at the implantation site that are important for the normal development of human pregnancy. These cytokines share the cell membrane receptor subunit gp130, resulting in similar functions. The aim of this study was to compare the response to LIF and OSM in several trophoblast models with particular regard to intracellular mechanisms and invasion. Four trophoblast cell lines with different characteristics were used: HTR-8/SVneo, JEG-3, ACH-3P and AC1-M59 cells. Cells were incubated with LIF, OSM (both at 10ngmL(-1)) and the signal transducer and activator of transcription (STAT) 3 inhibitor S3I-201 (200µM). Expression and phosphorylation of STAT3 (tyr(705)) and extracellular regulated kinase (ERK) 1/2 (thr(202/204)) and the STAT3 DNA-binding capacity were analysed by Western blotting and DNA-binding assays, respectively. Cell viability and invasiveness were assessed by the methylthiazole tetrazolium salt (MTS) and Matrigel assays. Enzymatic activity of matrix metalloproteinase (MMP)-2 and MMP-9 was investigated by zymography. OSM and LIF triggered phosphorylation of STAT3 and ERK1/2, followed by a significant increase in STAT3 DNA-binding activity in all tested cell lines. Stimulation with LIF but not OSM significantly enhanced invasion of ACH-3P and JEG-3 cells, but not HTR-8/SVneo or AC1-M59 cells. Similarly, STAT3 inhibition significantly decreased the invasiveness of only ACH-3P and JEG-3 cells concomitant with decreases in secreted MMP-2 and MMP-9. OSM shares with LIF the capacity to activate ERK1/2 and STAT3 pathways in all cell lines tested, but their resulting effects are dependent on cell type. This suggests that LIF and OSM may partially substitute for each other in case of deficiencies or therapeutic interventions.

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