Trophoblast adhesion of the peri-implantation mouse blastocyst is regulated by integrin signaling that targets phospholipase C

Integrin signaling modulates trophoblast adhesion to extracellular matrices during blastocyst implantation. Fibronectin (FN)-binding activity on the apical surface of trophoblast cells is strengthened after elevation of intracellular Ca2+ downstream of integrin ligation by FN. We report here that phosphoinositide-specific phospholipase C (PLC) mediates Ca2+ signaling in response to FN. Pharmacological agents used to antagonize PLC (U73122) or the inositol phosphate receptor (Xestospongin C) inhibited FN-induced elevation of intracellular Ca2+ and prevented the upregulation of FN-binding activity. In contrast, inhibitors of Ca2+ influx through either voltage-gated or non-voltage-gated Ca2+ channels were without effect. Inhibition of protein tyrosine kinase activity by genistein, but not G-protein inhibition by suramin, blocked FN-induced intracellular Ca2+ signaling and upregulation of adhesion, consistent with involvement of PLC-γ. Confocal immunofluorescence imaging of peri-implantation blastocysts demonstrated that PLC-γ2, but not PLC-γ1 nor PLC-β1, accumulated near the outer surface of the embryo. Phosphotyrosine site-directed antibodies revealed phosphorylation of PLC-γ2, but not PLC-γ1, upon integrin ligation by FN. These data suggest that integrin-mediated activation of PLC-γ to initiate phosphoinositide signaling and intracellular Ca2+ mobilization is required for blastocyst adhesion to FN. Signaling cascades regulating PLC-γ could, therefore, control a critical feature of trophoblast differentiation during peri-implantation development.