The human placenta is an invasive structure in which highly proliferative, migratory, and invasive extravillous trophoblast (EVT) cells migrate and invade the uterus and its vasculature. Using in vitro propagated normal first-trimester EVT cells and immortalized EVT cells, which share all of the phenotypic and functional characteristics of the normal EVT cells, it has been shown that migration/invasion of human EVT cells is stringently regulated by many growth factors, their binding proteins, extracellular matrix (ECM) components, and some adhesion molecules in an autocrine/paracrine manner at the fetal-maternal interface in human pregnancy. Transforming growth factor beta (TGF-beta), decorin (a proteoglycan in the ECM), and melanoma cell adhesion molecule (Mel-CAM) inhibit, and insulin-like growth factor II (IGF-II), IGF-binding protein 1 (IGFBP-1), and endothelin 1 (ET-1) stimulate EVT cell migration/invasion. Inhibition of EVT cell migration by TGF-beta has been suggested to be due to upregulation of integrins, which make the cells more adhesive to the ECM. Its antiinvasive action is due to an upregulation of tissue inhibitor of matrix metalloprotease 1 (TIMP-1) and plasminogen activator inhibitor (PAI-1) and a downregulation of urokinase-type plasminogen activator (uPA). Molecular mechanisms of inhibition of migration/invasion of EVT cells by decorin and Mel-CAM remain to be identified. IGF-II action has been shown to be mediated by IGF type I receptors (IGF-RII) independently of IGF type I receptors (IGF-RI) and IGFBPs. This action of IGF-II appears to involve inhibitory G proteins and phosphorylation of mitogen-activated protein kinase (MAPK) (extracellular signal-regulated protein kinases 1 and 2 (ERK-1 and ERK-2)). IGFBP-1 stimulation of EVT cell migration appears to occur by binding its Arg-Gly-Asp (RGD) domain to alpha5beta1 integrin, leading to phosphorylation of focal adhesion kinase (FAK) and MAPK (ERK-1 and ERK-2). These studies may improve our understanding of diseases related to abnormal placentation, viz. hypoinvasiveness in preeclampsia and hyperinvasiveness in trophoblastic neoplasms.

We have earlier shown that migration and invasiveness of first trimester human extravillous trophoblast cells are stimulated by IGF-II, independently of IGF type 1 receptor and that migration stimulation is the primary reason for increased extravillous trophoblast cell invasiveness induced by IGF-II. In the present study we examined the functional role of IGF type II receptor in IGF-II stimulation of extravillous trophoblast cell migration and the underlying signal transduction pathways including the participation of inhibitory G protein(s) and MAPK. The migratory ability of a well characterized in vitro propagated human first trimester extravillous trophoblast cell line expressing the phenotype of extravillous trophoblast cells in situ was quantitated with a Transwell migration assay under different experimental conditions. We found that the extravillous trophoblast cells expressed an abundance of IGF type 2 receptor as detected by immunostaining and Western blots, and recombinant human IGF-II promoted their migration in a dose- and time-dependent manner. Both polyclonal and monoclonal IGF type 2 receptor-blocking antibodies blocked migration-stimulating effects of IGF-II. Two synthetic IGF-II analogs ([Leu27]IGF-II, which can bind to IGF type 2 receptor and IGF-binding proteins, but not IGF type 1 receptor, and [QAYL-Leu27]IGF-II, which can bind to IGFR-II, but neither IGFR-I nor IGF-binding proteins) both stimulated extravillous trophoblast cell migration to levels higher than those induced by wild-type IGF-II. These results reveal that IGF-II action was mediated by IGF type 2 receptor, independently of IGF type 1 receptor and IGF-binding proteins. Treatment of extravillous trophoblast cell membrane preparations with IGF-II decreased adenylyl cyclase activity in a concentration-dependant manner, indicating the participation of inhibitory G proteins in IGF-II action. This was substantiated further with the findings that increasing intracellular cAMP using forskolin or (Bu)2cAMP inhibited basal extravillous trophoblast cell migration and blocked IGF-II stimulation of migration. IGF-II treatment rapidly stimulated phosphorylation of MAPK (ERK-1 and -2), which was blocked by pretreatment of extravillous trophoblast cells with the MAPK kinase (MEK) inhibitor PD98059. Treatment with this inhibitor also blocked extravillous trophoblast cell migration in the presence or absence of IGF-II. These results, taken together, reveal that IGF-II stimulates extravillous trophoblast cell migration by signaling through IGF type 2 receptor, involving inhibitory G proteins and activating the MAPK pathway.

A highly migratory subpopulation of the human placental trophoblast, known as the extravillous trophoblast (EVT), invades the uterus and its vasculature, to establish adequate exchange of key molecules between the maternal and fetal circulations. During their formation, EVT cells selectively acquire alpha 5 beta 1 integrin. We had shown that alpha 5 beta 1 is required for their migratory function, and that EVT cell migration is stimulated by insulin-like growth factor-binding protein (IGFBP)-1 produced by the uterine decidua. The present study examined whether this stimulation is dependent on binding of the Arg-Gly-Asp (RGD) domain of IGFBP-1 to an RGD binding site on the alpha 5 beta 1 integrin, followed by activation of focal adhesion kinase (FAK) and stimulation of the mitogen-activated protein kinase (MAPK) pathway. IGFBP-1 treatment increased migration of EVT cells, whereas an anti-alpha 5 beta 1 integrin antibody blocked migration regardless of IGFBP-1 treatment. Migration stimulation by IGFBP-1 was abrogated by pretreatment with a Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), but not a Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) hexapeptide, and by mutation of the RGD domain of IGFBP-1 to Trp-Gly-Asp (WGD). IGFBP-1 treatment caused a rapid localization of immunoreactive FAK to cellular lamellipodia, a rapid increase in phosphorylation of FAK and extracellular-signal regulated kinases 1 and 2. Preincubation of EVT cells with Herbimycin A, a tyrosine kinase inhibitor, abrogated IGFBP-1 effects; whereas an MAPK kinase inhibitor, PD 98059, reduced migration regardless of IGFBP-1 treatment. These results indicate that IGFBP-1 stimulation of EVT cell migration occurs by binding of its RGD domain to the alpha 5 beta 1 integrin, leading to activation of FAK and stimulation of MAPK pathway.