Induction of adherent activity in mastocytoma P-815 cells by the cooperation of two prostaglandin E₂ receptor subtypes, EP3 and EP4

In this study, we investigated the role of PGE₂ in mouse mastocytoma P-815 cell adhesion to extracellular matrix proteins (ECMs) in vitro. We report that PGE₂ accelerated ProNectin F™ (a proteolytic fragment of fibronectin)-mediated adhesion, which was abolished by addition of the GRGDS peptide, an inhibitor of the RDG binding site of ProNectin F™. We show that the cAMP level and cAMP-regulated protein kinase (PKA) activity are critical mediators of this PGE₂ effect, because the cell-permeable cAMP analogue 8-Br-cAMP accelerated P-815 cell adhesion to ProNectin F™ and the pharmacological inhibitor of PKA, H-89, blocked PGE₂-mediated adhesion. Consistent with mRNA expression of the G_scoupled EP4- and G_i-coupled EP3-PGE receptor subtypes, P-815 cell adhesion was accelerated by treatment with a selective EP4 agonist, ONO-AE1-329, but not a selective EP1/EP3 agonist, sulprostone. However, simultaneous treatment with ONO-AE1-329 and sulprostone resulted in augmentation of both the cAMP level and cell adhesion. The augmentation of EP3-mediated cAMP synthesis was dose-dependent, without affecting the half-maximal concentration for EP4-mediated G_s-activity, which was inhibited by a G_i inhibitor, pertussis toxin. In conclusion, these findings suggest that PGE₂ accelerates RGD-dependent adhesion via cooperative activation between EP3 and EP4 and contributes to the recruitment of mast cells to the ECM during inflammation.