RECOMBINANT ENTACTIN PROMOTES MOUSE PRIMARY TROPHOBLAST CELL-ADHESIONAND MIGRATION THROUGH THE ARG-GLY-ASP (RGD) RECOGNITION SEQUENCE

In vitro culture of mouse blastocysts during the period coinciding wit h implantation has revealed that primary trophoblast cells can adhere and migrate in serum-free medium when provided with certain extracellu lar matrix components, including fibronectin and laminin. Tightly asso ciated with laminin is the glycoprotein, entactin, that may play an im portant role in basement membrane assembly and cell attachment. Mouse blastocysts were studied using this in vitro model to determine whethe r entactin was capable of mediating trophoblast invasive activity. Alt hough entactin has never been shown to promote cell migration, we repo rt here that recombinant entactin supported blastocyst outgrowth in a dose-dependent manner, with a maximal effect at 20-50 mug/ml. The abil ity of trophoblast cells to adhere and migrate on entactin was specifi cally inhibited by anti-entactin antibody, but not by antibodies raise d against laminin. The synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, tha t contains the Arg-Gly-Asp (RGD) integrin recognition site, reversibly inhibited entactin-mediated blastocyst outgrowth in a dose-dependent manner, but had no effect on laminin-mediated outgrowth. The synthetic peptide, Gly-Phe-Arg-Gly-Asp-Gly-Gln, that comprises the actual RGD-c ontaining sequence within entactin, promoted trophoblast outgrowth whe n immobilized on the substratum. Furthermore, a mutated recombinant en tactin, altered to contain a Glu in place of Asp at the RGD site, prov ided no trophoblast cell adhesive activity. We conclude that entactin promotes trophoblast outgrowth through a mechanism mediated by the RGD recognition site, and that it may play an important role during invas ion of the endometrial basement membrane at implantation.