
<ArticleTitle>Stimulation of human extravillous trophoblast migration by <prot>IGF-II</prot> is mediated by <prot>IGF type 2 receptor</prot> involving inhibitory G protein(s) and phosphorylation of <prot>MAPK</prot>.</ArticleTitle>
<AbstractText>We have earlier shown that migration and invasiveness of first trimester human extravillous trophoblast cells are stimulated by <prot>IGF-II</prot>, independently of <prot>IGF type 1 receptor</prot> and that migration stimulation is the primary reason for increased extravillous trophoblast cell invasiveness induced by <prot>IGF-II</prot>. In the present study we examined the functional role of <prot>IGF type II receptor</prot> in <prot>IGF-II</prot> stimulation of extravillous trophoblast cell migration and the underlying signal transduction pathways including the participation of inhibitory G protein(s) and <prot>MAPK</prot>. 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 <prot>IGF type 2</prot> receptor as detected by immunostaining and Western blots, and recombinant human <prot>IGF-II</prot> promoted their migration in a dose- and time-dependent manner. Both polyclonal and monoclonal <prot>IGF type 2 receptor</prot>-blocking antibodies blocked migration-stimulating effects of <prot>IGF-II</prot>. Two synthetic <prot>IGF-II</prot> analogs ([Leu27]<prot>IGF-II</prot>, which can bind to <prot>IGF type 2 receptor</prot> and IGF-binding proteins, but not <prot>IGF type 1 receptor</prot>, and [QAYL-Leu27]IGF-II, which can bind to <prot>IGFR-II</prot>, but neither <prot>IGFR-I</prot> nor IGF-binding proteins) both stimulated extravillous trophoblast cell migration to levels higher than those induced by wild-type <prot>IGF-II</prot>. These results reveal that IGF-II action was mediated by <prot>IGF type 2 receptor</prot>, independently of <prot>IGF type 1 receptor</prot> and IGF-binding proteins. Treatment of extravillous trophoblast cell membrane preparations with <prot>IGF-II</prot> decreased adenylyl cyclase activity in a concentration-dependant manner, indicating the participation of inhibitory G proteins in <prot>IGF-II</prot> 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 <prot>IGF-II</prot> stimulation of migration. <prot>IGF-II</prot> treatment rapidly stimulated phosphorylation of <prot>MAPK</prot> (ERK-1 and -2), which was blocked by pretreatment of extravillous trophoblast cells with the<prot> MAPK kinase</prot> (<prot>MEK</prot>) inhibitor PD98059. Treatment with this inhibitor also blocked extravillous trophoblast cell migration in the presence or absence of <prot>IGF-II</prot>. These results, taken together, reveal that <prot>IGF-II</prot> stimulates extravillous trophoblast cell migration by signaling through <prot>IGF type 2 receptor</prot>, involving inhibitory G proteins and activating the <prot>MAPK</prot> pathway.</AbstractText>
