PP007. Effects of STAT1 suppression on ERK1/2 in trophoblastic cells

Abstract

Migration and trophoblast invasion are controlled functionally along with the active participation of cytokines and growth factors. Two important intracellular signaling pathways are the Janus kinase/signal transducer and activator of transcription (JAK-STAT) and extracellular regulated kinase1/2 (ERK1/2). These pathways have been associated with the regulation of gene expression, cellular proliferation, differentiation, angiogenesis, embryo development and invasion in tumor and trophoblast cells. The aim of our study is to characterize and analyze the regulation and crosstalks of STAT1 and ERK1/2 in trophoblast cells and the identification of activating cytokines. The trophoblast derived cell line HTR-8/svneo and a choriocarcinoma cell line (JEG-3) were stimulated with interleukin-6 (IL-6), IL-11, granulocyte-macrophage colony-stimulating factor (GMC-SF), leukemia inhibitory factor (LIF) or oncostatine M (OSM). The the expression and phosphorylation of STAT1(tyr705) and ERK1/2 were analyzed by gel electrophoresis and Western blotting. Expression of STAT1 was inhibited by administration of 50μM fludarabine (2-fluoro-ara-AMP) for 2, 4, 8, 24, 48 or 72h or by using small interfering RNA (siRNA). The full activation of STAT1 was assessed by using an STAT1 DNA-binding assay. Finally, proliferation and invasion assays were performed (Grant Deutscher Akademischer Austausch Dienst A/10172477). LIF and OSM induce STAT1 and ERK1/2 phosphorylation in HTR-8 and JEG-3 cells. Fludarabine inhibits the so induced phosphorylation of STAT1 when administered 48 or 72h before stimulation. Simultaneously, ERK phosphorylation increases. In contrast, silencing of STAT1 by application of specific siRNA induces reduction of ERK1/2 phosphorylation. Fludarabine reduces STAT1 DNA-binding capacity. LIF and OSM increase proliferation. Silencing of STAT1 slightly decreases invasiveness of analyzed cells. STAT1 in trophoblast cells can be activated by placental cytokines. Suppression of STAT1 by fludarabine or siRNA influences activity of ERK1/2 which indicates a crosstalk between both pathways. Current studies will clarify the reason for the different effects on ERK1/2 in trophoblastic cells.