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

Abstract

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 Ca(2+) downstream of integrin ligation by FN. We report here that phosphoinositide-specific phospholipase C (PLC) mediates Ca(2+) 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 Ca(2+) and prevented the upregulation of FN-binding activity. In contrast, inhibitors of Ca(2+) influx through either voltage-gated or non-voltage-gated Ca(2+) channels were without effect. Inhibition of protein tyrosine kinase activity by genistein, but not G-protein inhibition by suramin, blocked FN-induced intracellular Ca(2+) signaling and upregulation of adhesion, consistent with involvement of PLC-gamma. Confocal immunofluorescence imaging of peri-implantation blastocysts demonstrated that PLC-gamma2, but not PLC-gamma1 nor PLC-beta1, accumulated near the outer surface of the embryo. Phosphotyrosine site-directed antibodies revealed phosphorylation of PLC-gamma2, but not PLC-gamma1, upon integrin ligation by FN. These data suggest that integrin-mediated activation of PLC-gamma to initiate phosphoinositide signaling and intracellular Ca(2+) mobilization is required for blastocyst adhesion to FN. Signaling cascades regulating PLC-gamma could, therefore, control a critical feature of trophoblast differentiation during peri-implantation development.

Embryo–epithelium interactions during implantation at a glance

“…In mice, binding of fibronectin, vitronectin or OPN to the blastocyst stimulates the trafficking of integrins to the mouse trophectodermal surface and the assembly of focal contact-like structures, as indicated by the presence of the focal adhesion markers vinculin and talin (Chaen et al, 2012;Schultz and Armant, 1995;Schultz et al, 1997;Yelian et al, 1995). Integrin signalling modulates trophoblast adhesion through the activation of phospholipase Cγ to initiate phosphoinositide signalling and intracellular Ca 2+ mobilisation Wang et al, 2007). Accordingly, on substrates containing extracellular matrix ligands, trophoblast outgrowth ensues (Sutherland et al, 1988(Sutherland et al, , 1993.…”

Phosphatidylinositol 3-kinase signaling in mammalian preimplantation embryo development

“…Phospholipase C-g (PLC-g) is a PH domain protein and may be important in the context of Paf-mediated calcium signaling. The two PLC-g genes (Plcg1 and 2; Li et al 2007b) and immunodetectable PLC-g are expressed in the early embryo (Li et al 2007b, Wang et al 2007 (Emerson et al 2000). Selective inhibition of PLC inhibits Paf-induced calcium transients (Emerson et al 2000).…”

Intracellular Ca2+ Signaling and Preimplantation Development

“…Unlike the pathway downstream of HBEGF , inhibition of voltage gated Ca 2+ channels or non-voltage gated Ca 2+ channels to prevent Ca 2+ influx fails to prevent the production of a Ca 2+ transient or the increased adhesiveness of trophoblast cells after exposure to fibronectin (Wang et al 2007). However, the PLC inhibitor U73122 or the ITPR antagonist Xestospongin C are each capable of preventing intracellular Ca 2+ release and the change in adhesiveness of trophoblast cells upon fibronectin treatment.…”

“…This mechanism is more similar to Ca 2+ mobilization by GPCR agonists. The PLCβ family is activated by G-proteins (Rebecchi and Pentyala 2000), but the G-protein inhibitor suramin does not interfere with the stimulation of trophoblast adhesion by exposure to fibronectin (Wang et al 2007). However, the tyrosine kinase inhibitor genistein interferes with both Ca 2+ release and increased adhesiveness, suggesting that a tyrosine kinase-dependent PLC isoform could regulate Ca 2+ release.…”