Blocking Epidermal Growth Factor Receptor Signalin… /AKT and Induces Apoptosis

We identified a major peptide signaling target of EGF/EGFR pathway and explored the consequences of blocking or activating this pathway in the first trimester extravillous trophoblast cells, HTR-8/SVneo. A global analysis of protein phosphorylation was undertaken using novel technology (Kinexus Kinetworks) that utilizes SDS-polyacrylamide minigel electrophoresis and multi-lane immunoblotting to permit specific and semiquantitative detection of multiple phosphoproteins. Forty-seven protein phosphorylation sites were queried, and the results reported based on relative phosphorylation at each site. EGF- and Iressa-(gefitinib, ZD1839, an inhibitor of EGFR) treated HTR-8/SVneo cells were subjected to immunoblotting and flow cytometry to confirm the phosphoprotein screen and to assess the effects of EGF versus Iressa on cell cycle and apoptosis. EGFR mediates the phosphorylation of important signaling proteins, including PKBα/AKT. This pathway is likely to be central to EGFR-mediated trophoblast survival. Furthermore, EGF treatment induces proliferation and inhibits apoptosis, while Iressa induces apoptosis. 1. Introduction Epidermal growth factor (EGF) and its receptor (EGFR) are known to play critical roles in cell growth, differentiation, angiogenesis, and inhibition of apoptosis. EGF is a multifunctional 53 amino-acid peptide known for its ability to stimulate proliferation and differentiation in the placenta as well as many other cells and tissues [1]. EGF is important in embryonal implantation and trophoblast differentiation; it is critical for normal placental endocrine function [2]. EGF and its membrane-bound receptor, EGFR, are expressed in the placenta and can be found at all stages of differentiation; however, the signaling events activated by this pathway in the trophoblast are not fully understood. Nevertheless, growth factors such as EGF are believed to be intimately involved with the control of trophoblast physiology, and abnormal levels of EGFR have been reported in the placenta from pregnancies complicated by intrauterine growth restriction (IUGR), preeclampsia, and diabetes mellitus [3]. The association of EGF/EGFR with normal and aberrant placental function makes the study of downstream signaling pathways activated by this growth factor an important area of investigation. EGFR signals through a series of protein kinases. These are important transducers of information in human cells with the ability to regulate cellular functions by catalyzing the direct phosphorylation of target proteins [4]. Protein kinases operate within complex systems