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 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.
Fujino
Hiromichi
H
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721-0207, USA.
West
Kimberly A
KA
Regan
John W
JW
D000730:Androstadienes / Q000494:pharmacology; D001667:Binding, Competitive; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D051821:Lymphoid Enhancer-Binding Factor 1; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011869:Radioligand Assay; D018078:Receptors, Prostaglandin E / Q000378:metabolism*; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058309:Receptors, Prostaglandin E, EP4 Subtype; D015398:Signal Transduction; D013449:Sulfonamides; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D051821:Lymphoid Enhancer-Binding Factor 1; C545644:PTGER2 protein, human; C545650:PTGER4 protein, human; D018078:Receptors, Prostaglandin E; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058309:Receptors, Prostaglandin E, EP4 Subtype; D013449:Sulfonamides; D014157:Transcription Factors; D008156:Luciferases; D019869:Phosphatidylinositol 3-Kinases; D038341:Glycogen Synthase Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D015232:Dinoprostone; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D000077191:Wortmannin