Epidermal growth factor stimulates proliferation and migration of porcine trophectoderm cells through protooncogenic protein kinase 1 and extracellular-signal-regulated kinases 1/2 mitogen-activated protein kinase signal transduction cascades during early pregnancy

Abstract

For successful implantation and establishment of early epitheliochorial placentation, porcine conceptuses require histotroph, including nutrients and growth factors, secreted by or transported into the lumen of the uterus. Epidermal growth factor (EGF), an essential component of histotroph, is known to have potential growth-promoting activities on the conceptus and uterine endometrium. However, little is known about its effects to transactivate cell signaling cascades responsible for proliferation, growth and differentiation of conceptus trophectoderm. In the present study, therefore, we determined that EGFR mRNA and protein were abundant in endometrial luminal and glandular epithelia, stratum compactum stroma and conceptus trophectoderm on days 13–14 of pregnancy, but not in any other cells of the uterus or conceptus. In addition, primary porcine trophectoderm (pTr) cells treated with EGF exhibited increased abundance of phosphorylated (p)-AKT1, p-ERK1/2 MAPK and p-P90RSK over basal levels within 5min, and effect that was maintained to between 30 and 120min. Immunofluorescence microscopy revealed abundant amounts of p-ERK1/2 MAPK and p-AKT1 proteins in the nucleus and, to a lesser extent, in the cytoplasm of pTr cells treated with EGF as compared to control cells. Furthermore, the abundance of p-AKT1 and p-ERK1/2 MAPK proteins was inhibited in control and EGF-treated pTr cells transfected with EGFR siRNA. Compared to the control siRNA transfected pTr cells, pTr cells transfected with EGFR siRNA exhibited an increase in expression of IFND and TGFB1, but there was no effect of expression of IFNG. Further, EGF stimulated proliferation and migration of pTr cells through activation of the PI3K-AKT1 and ERK1/2 MAPK-P90RSK cell signaling pathways. Collectively, these results support the hypothesis that EGF coordinately activates multiple cell signaling pathways critical to proliferation, migration and survival of trophectoderm cells that are critical to development of porcine conceptuses during implantation and placentation.