Phosphorylation of glycogen synthase kinase-3 and stimulation of T-cell factor signaling following activation of EP2 and EP4 prostanoid receptors by prostaglandin E2

Recently we have shown that the FP, prostanoid receptor, a G-protein-coupled receptor that couples to Galpha(q), activates T-cell factor (Tcf)/Iymphoid enhancer factor (Lef)-mediated transcriptional activation (Fujino, H., and Regan, J. W. (2001) J. Biol. Chem. 276, 1248912492). We now report that the EP2 and EP4 prostanoid receptors, which couple to Galpha(s), also activate Tcf/Lef signaling. By using a Tcf/Lef-responsive luciferase reporter gene, transcriptional activity was stimulated similar to10-fold over basal by I h of treatment with prostaglandin E-2 (PGE(2)) in HEK cells that were stably transfected with the human EP2 and EP4 receptors. This stimulation of reporter gene activity was accompanied by a PGE(2)-dependent increase in the phosphorylation of both glycogen synthase kinase-3 (GSK-3) and Akt kinase. H-89, an inhibitor of protein kinase A (PKA), completely blocked the agonist-dependent phosphorylation of GSK-3 in both EP2- and EP4-expressing cells. However, H-89 pretreatment only blocked PGE(2)-stimulated Lef/Tef reporter gene activity by 20% in EP4-expressing cells compared with 65% inhibition in EP2-expressing cells. On the other hand wortmannin, an inhibitor of phosphatidylinositol 3-kinase, had the opposite effect and inhibited PGE(2)-stimulated reporter gene activity to a much greater extent in EP4-expressing cells as compared with EP2-expressing cells. These findings indicate that the activation of Tcf/Lef signaling by EP2 receptors occurs primarily through a PKA-dependent pathway, whereas EP4 receptors activate Tcf/Lef signaling mainly through a phosphatidylinositol 3-kinase-dependent pathway. This is the first indication of a fundamental difference in the signaling potential of EP2 and EP4 prostanoid receptors.