Abstract
Recently we have shown that the FP(B) prostanoid receptor, a G-protein-coupled receptor that couples to Galpha(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 EP(2) and EP(4) 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 approximately 10-fold over basal by 1 h of treatment with prostaglandin E(2) (PGE(2)) in HEK cells that were stably transfected with the human EP(2) and EP(4) 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 EP(2)- and EP(4)-expressing cells. However, H-89 pretreatment only blocked PGE(2)-stimulated Lef/Tcf reporter gene activity by 20% in EP(4)-expressing cells compared with 65% inhibition in EP(2)-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 EP(4)-expressing cells as compared with EP(2)-expressing cells. These findings indicate that the activation of Tcf/Lef signaling by EP(2) receptors occurs primarily through a PKA-dependent pathway, whereas EP(4) 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 EP(2) and EP(4) prostanoid receptors.
Highlights
We have shown that the FPB prostanoid receptor, a G-protein-coupled receptor that couples to G␣q, activates T-cell factor (Tcf)/lymphoid enhancer factor (Lef)-mediated transcriptional activation
We show that stimulation of EP2 receptors by prostaglandin E2 (PGE2) can activate a Tcf/Lef signaling pathway by a mechanism that mainly involves the phosphorylation of glycogen synthase kinase-3 (GSK-3) by protein kinase A (PKA)
The EP2 and EP4 prostanoid receptors are G-proteincoupled receptors (GPCR) that are linked to the stimulation of cAMP/PKA signaling through the sequential activation of G␣s and adenylyl cyclase
Summary
T-cell factor; GPCR, G-proteincoupled receptor; Lef, lymphoid enhancer factor; GSK-3, glycogen synthase kinase 3; PKA, cAMP-dependent protein kinase A; PGE2, prostaglandin E2; PI3 kinase, phosphatidylinositol 3-kinase; DMEM, Dulbecco’s modified Eagle’s medium; MES, 4-morpholineethanesulfonic acid; HIV, human immunodeficiency virus; BSA, bovine serum albumin. The first example of the activation of this signaling pathway by a wild type GPCR and its cognate ligand was recently made when we demonstrated that prostaglandin F2␣ acting through the FPB prostanoid receptor could decrease the phosphorylation of cytoplasmic -catenin and stimulate Tcf/Lef-mediated transcriptional activation [3]. A key enzyme in the -catenin/Tcf signaling pathway is glycogen synthase kinase-3 (GSK-3) This enzyme, which forms a complex with adenomatous polyposis coli and axin, is responsible for the phosphorylation and subsequent degradation of cytosolic -catenin. We show that stimulation of EP2 receptors by PGE2 can activate a Tcf/Lef signaling pathway by a mechanism that mainly involves the phosphorylation of GSK-3 by PKA. Stimulation of EP4 receptors by PGE2 can activate a Tcf/ Lef signaling pathway, but the mechanism is more complex and involves the activation of both PI3 kinase and PKA
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