IGF1 plays a central role in placental growth. IGF delivered from maternal circulation binds to the IGF1 receptor (IGF1R) in syncytium resulting in the activation of Akt; inhibition of this pathway affects cytotrophoblast turnover. However, the route(s) by which IGF1 enters placenta and how its signal is delivered into the two trophoblast compartments is unknown. We have utilised quantum dots (QDs) to deliver/track IGF1 binding and internalisation in first trimester placenta (EP) and BeWo cells and investigate the role of endocytosis in IGF1 signalling in trophoblast.
EP (812 weeks) explants and BeWo cells were exposed to clathrin or caveolin dependent endocytosis inhibitors CPMZ (50 μM) or m-β-CD (5 mM) respectively for 1 h and subsequently treated with QDIGF1 (50 nM) or IGF1 (20 nM) for 530 min. The effect of IGF1 on intracellular signalling was investigated by western blotting and immunocytochemistry.
IGF1 and QDIGF1 induced phosphorylation of IGF1R and Akt in both EP and BeWo cells. In EP, IGF1R phosphorylation was observed on the syncytial microvillous membrane (5 min), and later in cytotrophoblasts (1030 min), but pAkt was observed only in cytotrophoblasts (1030 min). Initially, QDs co-localised with clathrin and early endosomes in the apical syncytioplasm, but co-localisation was lost with deeper penetration of the ligand. In EP, CPMZ but not m-β-CD decreased IGF-stimulated increase of pAkt reflecting absence of caveolin in the syncytial layer. In BeWo cells, QDIGF co-localised with caveolin and m-β-CD, but not CPMZ, affected Akt activation.
IGF binding at the syncytial surface leads to clathrin-dependent internalisation and remarkably rapid Akt activation in cytotrophoblasts. Unlike villous trophoblasts, BeWo cells use caveolin for IGF internalisation reflecting differences in the cell membrane protein profile of distinct placental models. Further studies are required to identify specific functions of IGF1R/Akt signalling in respective compartments of the EP.