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_B prostanoid receptor, a G-protein-coupled receptor that couples to Gα_q, activates T-cell factor (Tcf)/lymphoid enhancer factor (Lef)-mediated transcriptional activation (Fujino, H., and Regan, J. W. (2001) J. Biol. Chem. 276, 12489-12492). We now report that the EP₂ and EP₄ prostanoid receptors, which couple to Gα_s, also activate Tcf/Lef signaling. By using a Tcf/Lef-responsive luciferase reporter gene, transcriptional activity was stimulated ∼10-fold over basal by 1 h of treatment with prostaglandin E₂ (PGE₂) in HEK cells that were stably transfected with the human EP₂ and EP₄ receptors. This stimulation of reporter gene activity was accompanied by a PGE₂-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 PGE₂-stimulated Lef/Tcf 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 PGE₂-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.