Phosphorylation of Glycogen Synthase Kinase-3 and Stimulation of T-cell Factor Signaling following Activation of EP 2 and EP 4 Prostanoid Receptors by Prostaglandin E 2 *

Recently we have shown that the FP B prostanoid re- ceptor, a G-protein-coupled receptor that couples to G (cid:1) q , activates T-cell factor (Tcf)/lymphoid enhancer fac- tor (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 G (cid:1) s , also activate Tcf/Lef sig -naling. By using a Tcf/Lef-responsive luciferase reporter gene, transcriptional activity was stimulated (cid:1) 10-fold over basal b y 1 h oftreatment 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 -de-pendent 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 pretreat- ment 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.

An exciting connection is starting to emerge between the T-cell factor (Tcf) 1 /␤-catenin signaling pathway and G-proteincoupled receptors (GPCR). The potential for G-proteins to mediate this connection was suggested recently when it was shown that constitutively active G␣ 12 and G␣ 13 can interact with E-cadherin to cause the release of ␤-catenin and subsequent stimulation of Tcf/lymphoid enhancer factor (Lef) transcriptional activation (1). It has also been shown that a chimeric receptor constructed from the ligand binding and transmembrane domains of the ␤ 2 -adrenergic receptor and the cytoplasmic domains of rat Frizzled-1 can stimulate Tcf/Lef transcriptional activation through a mechanism that appears to involve signaling through G␣ q and/or G␣ o (2). 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 F 2␣ acting through the FP B prostanoid receptor could decrease the phosphorylation of cytoplasmic ␤-catenin and stimulate Tcf/Lef-mediated transcriptional activation (3). Interestingly the FP A isoform, which only differs from the FP B by having an additional 46 amino acids in its carboxyl terminus, was nearly inactive with respect to activation of ␤-catenin/Tcf signaling even though both isoforms can stimulate inositol phosphate signaling to a similar degree (4,5).
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. Direct inhibition of GSK-3 or disruption of the GSK-3-adenomatous polyposis coli-axin complex prevents the phosphorylation of cytoplasmic ␤-catenin resulting in stabilization and translocation to the nucleus where it can alter gene expression through interactions with members of the Tcf/ Lef family of transcriptional factors (6,7). One well characterized mechanism for inhibiting the kinase activity of GSK-3 is through phosphorylation. For example stimulation of the frizzled receptor by the Wnt ligand leads to the phosphorylation and inhibition of GSK-3␤ and thereby promotes ␤-catenin/Tcf signaling (7). Similarly, activation of phosphatidylinositol 3-kinase (PI3 kinase) can result in the phosphorylation and activation of Akt kinase (also known as protein kinase B) which can then phosphorylate and inhibit GSK-3␤. More recently, it has been found in vitro that cAMP-dependent protein kinase (PKA) can directly phosphorylate GSK-3␤ and inhibit its kinase activity (8,9). In addition it is known that PKA can indirectly phosphorylate and activate Akt kinase, which could provide an indirect mechanism for the inhibition of GSK-3␤ by PKA (10).
Given the ability of PKA to inhibit the activity of GSK-3, and the well known regulation of cAMP formation by GPCRs, we were interested in the potential activation of Tcf/Lef transcriptional activation by the EP 2 and EP 4 prostanoid receptors. The EP 2 and EP 4 receptors are two of the four subtypes of receptors for prostaglandin E 2 (PGE 2 ) (11,12). Both the EP 2 and EP 4 receptors are coupled to G␣ s and can activate adenylyl cyclase * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed. Tel.: 520-626-2181; Fax: 520-626-2466; E-mail: regan@pharmacy.arizona.edu. 1 The abbreviations used are: Tcf, T-cell factor; GPCR, G-proteincoupled receptor; Lef, lymphoid enhancer factor; GSK-3, glycogen synthase kinase 3; PKA, cAMP-dependent protein kinase A; PGE 2 , prostaglandin E 2 ; PI3 kinase, phosphatidylinositol 3-kinase; DMEM, Dulbecco's modified Eagle's medium; MES, 4-morpholineethanesulfonic acid; HIV, human immunodeficiency virus; BSA, bovine serum albumin. and increase intracellular cAMP formation. Prior to the molecular cloning of these receptors, it was thought that the stimulation of adenylyl cyclase by PGE 2 was mediated by a single EP receptor subtype. Molecular cloning revealed, however, two receptor subtypes that were the products of separate genes (13). The EP 2 and EP 4 receptors encoded by these genes only shared ϳ30% amino acid homology even though they shared the same endogenous ligand and apparent second messenger pathway. We now show that stimulation of EP 2 receptors by PGE 2 can activate a Tcf/Lef signaling pathway by a mechanism that mainly involves the phosphorylation of GSK-3 by PKA. Stimulation of EP 4 receptors by PGE 2 can also activate a Tcf/ Lef signaling pathway, but the mechanism is more complex and involves the activation of both PI3 kinase and PKA.

EXPERIMENTAL PROCEDURES
Stable Transfectants-Cell lines stably expressing the EP 2 or EP 4 receptors were prepared using HEK-293 EBNA cells (Invitrogen) and the mammalian expression vector pCEP4 (Invitrogen). Briefly, DNA sequences corresponding to the encoding regions of the human EP 2 receptor (13) and human EP 4 receptor (14) were subcloned into pCEP4, and 20 g of each purified plasmid was used to transfect one 10-cm plate of HEK-293 EBNA cells. Selection with hygromycin B and clonal expansion were done as described previously in detail (15) for the preparation of FP receptor-expressing cell lines. Clones expressing the human EP 2 and EP 4 receptor isoforms were identified based on immunofluorescence microscopy using EP 2 and EP 4 receptor-specific antibodies (16) and PGE 2 -stimulated cAMP formation. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) containing 10% fetal bovine serum, 250 g/ml geneticin, 100 g/ml gentamicin, and 200 g/ml hygromycin B.
Whole Cell Radioligand Binding Assay-Cells were cultured in 10-cm plates and were incubated for 1 h at 37°C with final concentrations of 0.1% dimethyl sulfoxide (Me 2 SO, vehicle) or 1 M PGE 2 . They were then trypsinized, centrifuged at 500 ϫ g for 2 min, and resuspended at a concentration of 10 7 cells/ml in ice-cold MES buffer consisting of 10 mM MES (pH 6.0), 0.4 mM EDTA, and 10 mM MnCl 2 .
[ 3 H]PGE 2 binding was performed using 100 l of sample added to a final assay volume of 200 l containing 2.5 nM [ 3 H]PGE 2 (Amersham Biosciences) or 2.5 nM [ 3 H]PGE 2 plus increasing concentrations of unlabeled PGE 2 . Samples were incubated for 1 h at room temperature and were filtered through Whatman GF/C glass filters to terminate the incubation. Filters were then washed five times with ice-cold MES buffer, and radioactivity was measured by liquid scintillation counting.
cAMP Assay-Cells were cultured in 10-cm plates and were washed once with fresh DMEM containing 0.1 mg/ml isobutylmethylxanthine (Sigma). Cells were then treated with either vehicle or 1 M PGE 2 for 1 h at 37°C in DMEM containing isobutylmethylxanthine, after which the media were removed and the cells were placed on ice. One ml of TE buffer (50 mM Tris-HCl, 4 mM EDTA (pH 7.5)) was added, and the cells were scraped off and transferred to microcentrifuge tubes. The samples were boiled for 8 min, placed on ice, and centrifuged for 1 min at 14,000 rpm. Fifty l of the supernatants (representing ϳ10 4 cells) was added to new tubes containing 50 l of [ 3 H]cAMP (PerkinElmer Life Sciences) and 100 l of 0.06 mg/ml PKA (Sigma product P5511). The mixture was vortexed and incubated on ice for 2 h, followed by the addition of 100 l of TE buffer containing 2% bovine serum albumin (BSA) and 26 mg/ml powdered charcoal. After vortexing and centrifugation for 1 min at 14,000 rpm, 100-l aliquots of the supernatants were removed for liquid scintillation. The amount of cAMP present was calculated from a standard curve prepared using cold cAMP and was expressed as pmol per 10 4 cells.
Western Blotting-Sixteen hours prior to the immunoblotting experiments, cells were switched from their regular culture medium to Opti-MEM (Invitrogen) containing 250 g/ml geneticin and 100 g/ml gentamicin. Cells were then incubated at 37°C with this same media containing 1 M PGE 2 for the times indicated in the figures. In some cases cells were pretreated with either vehicle (0.1% Me 2 SO) or inhibitors (10 M H-89, Calbiochem or 100 nM wortmannin, Sigma) for 15 min at 37°C. Cells were scraped into a lysis buffer consisting of 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0), 1% Nonidet P-40, 0.5% sodium deoxycholate, 10 mM sodium fluoride, 10 mM disodium pyrophosphate, 0.1% SDS, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 g/ml leupeptin, and 10 g/ml aprotinin and transferred to microcentrifuge tubes. The samples were rotated for 30 min at 4°C and were centrifuged at 16,000 ϫ g for 15 min. Aliquots of the supernatants containing ϳ100 g of protein were electrophoresed on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes as described previously (3). Membranes were incubated in 5% non-fat milk for 1 h and were then washed and incubated for 16 h at 4°C in 0.5% non-fat milk containing either anti-phospho-GSK-3␣/␤ antibody (Cell Signaling, catalog number 9331), anti-phospho-GSK-3␤ antibody (Cell Signaling, catalog number 9336), anti-GSK-3␤ antibody (Transduction Laboratories, catalog number G22320), or 5% BSA containing anti-phospho-Akt 4E2 antibody (Cell Signaling, catalog number 9276) or 5% BSA containing anti-Akt antibody (Cell Signaling, catalog number 9272). All antibodies were used at a dilution of 1:1,000. Membranes were washed three times and incubated for 1 h at room temperature in 0.5% non-fat milk for GSK-3 antibodies or in 0.2% non-fat milk for Akt antibodies, with a 1:10,000 dilution of the appropriate secondary antibodies conjugated with horseradish peroxidase. After washing three times, immunoreactivity was detected by chemiluminescence as described previously (17). To ensure equal loading of proteins, the membranes were stripped and reprobed with anti-GSK-3␤ antibodies or anti-Akt antibodies under the same conditions as described above.
Tcf/Lef Reporter Gene Assay-Cells, grown in 6-well plates, were transiently transfected using FuGENE-6 (Roche Molecular Biochemicals) and 1.25 g/well of either the TOP flash or FOP flash reporter plasmids (Upstate Biotechnologies, Inc.) as described previously (3). Cells were pretreated with either vehicle (0.1% Me 2 SO) or inhibitors (10 M H-89 or 100 nM wortmannin) for 15 min at 37°C followed by treatment with either vehicle or 1 M PGE 2 for 1 h at 37°C. Cells were then rapidly washed three times each with 1 ml/well of Opti-MEM and then incubated for 16 h at 37°C in 2 ml of Opti-MEM containing 250 g/ml geneticin, 100 g/ml gentamicin. Cell lysates were prepared and luciferase activity was measured using a Turner TD-20/20 luminometer as described previously (3) using 1 g of protein/sample. Measurements were corrected for background activity by subtraction of the FOP flash values from the corresponding TOP flash values.

EP 2 and EP 4 Receptor Expression and PGE 2 -stimulated
cAMP Formation-HEK cells stably expressing the human EP 2 and EP 4 prostanoid receptors were prepared and used for the characterization of the signal transduction properties of these receptors. Fig. 1 EP 2 and EP 4 receptors are G␣ s -coupled receptors and are known to activate adenylyl cyclase; therefore, the ability of PGE 2 to stimulate cAMP formation was examined in these cells. As shown in Fig. 1, panel B, treatment of untransfected HEK cells with 1 M PGE 2 for 1 h had negligible effects on cAMP formation as compared with treatment with vehicle; however, in EP 2 receptor-transfected cells there was a 71-fold stimulation and in EP 4 receptor-transfected cells a 10-fold stimulation of cAMP formation following treatment with PGE 2 as compared with the vehicle control. It is notable that the maximal levels of cAMP formation for the EP 4 receptor-transfected cells are so much lower as compared with the EP 2 receptor-transfected cells even though both receptors are expressed at similar levels and the affinity of PGE 2 is significantly higher for EP 4 receptors as compared with the EP 2 receptors. PGE 2 -stimulated Phosphorylation of GSK-3 and Akt in EP 2 and EP 4 Receptor-transfected HEK Cells-The kinase activities of GSK-3 and Akt have recently been shown to be regulated following in vitro phosphorylation by PKA (8 -10). To explore the signaling potential between these kinases and the activation of adenylyl cyclase stimulatory GPCRs, we examined the PGE 2 -dependent phosphorylation of GSK-3 and Akt in untransfected HEK cells and in HEK cells transfected with the human EP 2 and EP 4 prostanoid receptors. For these experiments (Fig. 2) cells were treated with 1 M PGE 2 for the times indicated and were then lysed, subjected to SDS-PAGE, and immunoblotted with antibodies that specifically recognized either GSK-3␤ or Akt, the phosphorylated forms of GSK-3 (pGSK-3␣; pGSK-3␤), or the phosphorylated form of Akt (pAkt). As shown in Fig. 2, panel A, in both EP 2 and EP 4 receptor-transfected cells GSK-3␣ was phosphorylated within 5 min following exposure to PGE 2 and remained phosphorylated for 60 min. The control HEK cells also showed some PGE 2 -dependent phosphorylation of GSK-3␣, but it was considerably weaker and may reflect the small amount of specific [ 3 H]PGE 2 binding that is present. Densitometric analysis of the phosphorylation of GSK-3␣ at 60 min, compared with time 0, showed a 7-fold increase in EP 2 receptor-transfected cells and a 4.5-fold increase in EP 4 receptor-transfected cells. To ensure equal loading of proteins, the blots shown in panel A were stripped and re-probed with antibodies to GSK-3␤, and as shown in panel B nearly identical amounts of GSK-3␤ were present throughout the time course of treatment and among the three cell lines. Panel C shows the results obtained using antibodies directed against phospho-Akt. In all the cell lines there was a detectable level of phospho-Akt present at the zero time point, and in both the EP 2 and EP 4 receptor-transfected cell lines there was an increase in Akt phosphorylation after 60 min of treatment with PGE 2 . Densitometric analysis showed this to be ϳ2-fold for both EP 2 and EP 4 receptor-transfected cells. To ensure equal loading of proteins, the blots shown in panel C were stripped and re-probed with antibodies to Akt, and as shown in panel D similar amounts of Akt were present throughout the time course. Fig. 2 we showed that the stimulation of EP 2 and EP 4 receptors by PGE 2 resulted in increased phosphorylation of GSK-3␣ and Akt. Given that both of these receptors couple to G␣ s and are known to activate cAMP/PKA signaling pathways, we decided to examine the effects of H-89, an inhibitor of PKA, on the PGE 2 -stimulated phosphorylation of GSK-3 and Akt in EP 2 and EP 4 receptor-transfected cells. In addition, because phosphorylation of GSK-3 is known to stabilize ␤-catenin and promote Tcf/Lef-mediated transcriptional activation, we examined the potential of PGE 2 to stimulate the luciferase activity in EP 2 and EP 4 receptor-transfected cells using a Tcf/Lef-responsive luciferase reporter gene. For these experiments the cell lines were pretreated with either vehicle or 10 M H-89 for 15 min followed by treatment with either vehicle or 1 M PGE 2 for 1 h. The upper part of Fig. 3 shows the results of immunoblot analysis that was done in the same manner as described for Fig. 2. Fig. 3, panel A, shows that following pretreatment of EP 2 and EP 4 receptor-transfected cells with H-89 there was a complete block of PGE 2 -stimulated phosphorylation of GSK-3␣, suggesting the direct involvement of PKA in this process. There was also a notable decrease in the phosphorylation of GSK-3␤ following H-89 treatment in all the cell lines. On the other hand, Fig. 3, panel C, shows that H-89 pretreatment increased the phosphorylation of Akt and actually enhanced the PGE 2 -stimulated phosphorylation of Akt in all the cell lines. The bottom part of Fig. 3 shows PGE 2 -stimu- lated Tcf/Lef luciferase reporter gene activity in untreated cells and in cells that were pretreated with H-89 under the same conditions as used above for the immunoblotting experiments. In the absence of H-89 pretreatment, 1 M PGE 2 produced a 12-fold stimulation of luciferase activity in EP 2 receptor-transfected cells and a 7-fold stimulation in EP 4 receptor-transfected cells over the vehicle-treated controls. After pretreatment with H-89, however, PGE 2 -stimulated luciferase activity was decreased by 65% in EP 2 receptor-transfected cells but was only decreased by 20% in EP 4 receptor-transfected cells. Therefore, H-89 inhibited PGE 2 -stimulated Tcf/Lef reporter gene activity much more effectively in EP 2 receptor-transfected cells as compared with EP 4 receptor-transfected cells, even though it was equally effective at blocking GSK-3␣ phosphorylation in both cell lines.

PGE 2 Stimulation of Tcf/Lef Reporter Gene Activity in EP 2 and EP 4 Receptor-transfected Cells, Differential Effects of H-89 and Wortmannin on This and on PGE 2 -stimulated Phosphorylation of GSK-3 and Akt-In
The results shown in Figs. 2 and 3 indicate that the phosphorylation of Akt following PGE 2 treatment of EP 2 and EP 4 receptor-expressing cells is not a direct effect of PKA and suggest the involvement of additional kinases. One such candidate is PI3 kinase because Akt is known to have roles both in the phosphorylation of GSK-3␤ and as a substrate for PI3 kinase. Therefore, we examined the effects of wortmannin, an inhibitor of PI3 kinase, on the PGE 2 -stimulated phosphorylation of GSK-3 and Akt and on the PGE 2 stimulation of Tcf/Lef reporter gene activity in EP 2 and EP 4 receptor-transfected cells. For these experiments the cells were pretreated with either vehicle or 100 nM wortmannin for 15 min followed by treatment with either vehicle or 1 M PGE 2 for 1 h. The upper part of Fig. 4 shows the results of immunoblot analysis that was done in the same manner as described in Figs. 2 and 3. Fig. 4, panel A, shows that wortmannin pretreatment decreased the phosphorylation of GSK-3␣ in the vehicle-treated cells and produced a marked 62% inhibition of PGE 2 -stimulated GSK-3␣ phosphorylation in the EP 4 receptor-transfected cells, but only a modest 14% inhibition in the EP 2 receptor-transfected cells. In addition wortmannin pretreatment inhibited the phosphorylation of GSK-3␤ in all the cell lines and, most interestingly, revealed a clear PGE 2 -dependent stimulation of GSK-3␤ phosphorylation in both the EP 2 and EP 4 receptor-transfected cells. Fig. 4, panel C, shows that wortmannin pretreatment abolished both the basal and PGE 2 -stimulated phosphorylation of Akt in all the cell lines. The bottom part of Fig. 4 shows PGE 2 -stimulated Tcf/Lef luciferase reporter gene activity in untreated cells and in cells that were pretreated with 100 nM wortmannin as above. The data for untreated cells are the same as that shown in Fig.  3 and shown the robust PGE 2 stimulation of luciferase activity in both the EP 2 and EP 4 receptor-transfected cells. In a clear distinction from the results obtained with H-89, however, pretreatment with wortmannin produced a significantly greater inhibition of PGE 2 -stimulated Tcf/Lef reporter luciferase activity in EP 4 receptor-transfected cells (61%) as compared with the inhibition obtained in EP 2 receptor-transfected cells (27%). These findings suggest a significant PI3 kinase-mediated con-

FIG. 2. Immunoblots of the time course of PGE 2 -stimulated phosphorylation of GSK-3 and Akt in untransfected HEK cells and in HEK cells transfected with either the EP 2 or EP 4 prostanoid receptors.
Cells were incubated with 1 M PGE 2 for the indicated times and were subjected to immunoblot analysis as described under "Experimental Procedures." Panel A, immunoblotting with antibodies against phospho-GSK-3␣ and -3␤ whose specificity, according to the manufacturer, is to serine 21 and serine 9, respectively. Panel B, immunoblotting with antibodies against GSK-3␤. Panel C, immunoblotting with antibodies against phospho-Akt whose specificity is to serine 473. Panel D, immunoblotting with antibodies against Akt. These results are representative of more than three independent experiments with each antibody and condition.

FIG. 3. The effects of H-89 on PGE 2 -stimulated phosphorylation of GSK-3 and Akt (immunoblots, panels A-D) and on stimulation of Tcf/Lef-responsive luciferase reporter gene activity (histograph) in untransfected HEK cells or HEK cells transfected with either the EP 2 or EP 4 prostanoid receptors.
Cells were pretreated with either vehicle or 10 M H-89 for 15 min followed by either vehicle (v) or 1 M PGE 2 (P) for 1 h at 37°C and were then immediately subjected to immunoblot analysis or were washed to remove PGE 2 and incubated for 16 h after which luciferase activity was measured as described under "Experimental Procedures." Panels A-D are exactly as described in Fig. 2 and represent the immunostaining of phospho-GSK-3␣ and -3␤ (panel A), total GSK-3␤ (panel B), phospho-Akt (panel C), and total Akt (panel D). Immunoblotting results are representative of three experiments with each antibody and condition. Luciferase data are the means Ϯ S.E. of two measurements from a representative experiment that was repeated three times. tribution to the PGE 2 stimulation of Tcf/Lef reporter gene activity in the EP 4 receptor-transfected cells. DISCUSSION The EP 2 and EP 4 prostanoid receptors are GPCRs that are linked to the stimulation of cAMP/PKA signaling through the sequential activation of G␣ s and adenylyl cyclase. PGE 2 is the endogenous ligand for both of these receptors and the fact that these receptors represented two unique subtypes were not fully appreciated until the molecular cloning of the EP 2 receptor in 1994 (13). Comparison of the pharmacology of this receptor with a previously cloned adenylyl cyclase stimulatory EP receptor led to recognition of the EP 4 subtype (7,8). Structurally, these receptors have less in common than one might think given their similarities with respect to ligand binding and second messenger coupling. The EP 4 receptor is bigger, 488 amino acids versus 358, most of which is accounted for by a significantly longer carboxyl-terminal domain, 155 amino acids versus 34.
It is of considerable interest to understand the physiological and/or pathophysiological significance of the EP 2 and EP 4 prostanoid receptors. Nishigaki et al. (18) have found that these subtypes differ with respect to agonist-mediated desensitization. Thus, when transfected into Chinese hamster ovary cells the EP 4 subtype underwent short term desensitization in response to treatment with PGE 2 , whereas the EP 2 receptor did not. Related to this, Desai et al. (19) found that when transfected into HEK cells the EP 4 receptor underwent rapid agonist-mediated internalization, and again, the EP 2 did not. In the present study we have also found that the EP 4 receptor subtype is much more sensitive to the regulatory effects of agonist exposure and that pretreatment with 1 M PGE 2 for 1 h decreased EP 4 receptor number by ϳ70%, but only decreased EP 2 receptor number by ϳ30%. Rapid desensitization may also partially account for the markedly lower amount of agoniststimulated cAMP formation in EP 4 receptor-transfected cells as compared with EP 2 receptor-transfected cells. Thus, the maximal level of PGE 2 -stimulated cAMP formation in EP 4 receptor-transfected cells was only ϳ20% that achieved in EP 2 receptor-transfected cells, even though both receptors were expressed to nearly the same extent prior to agonist pretreatment. However, more rapid desensitization of the EP 4 receptor is not the only explanation for its lower stimulation of cAMP formation. It is plausible that EP 4 receptors are less efficiently coupled to adenylyl cyclase and/or they have additional pathways of signal transduction that do not involve the activation of cAMP/PKA signaling.
A major signaling pathway, which until recently was thought to be relatively unaffected by events in the cAMP/PKA pathway, is the Wnt signaling pathway. As reviewed in the Introduction, an important control point in this pathway involves the phosphorylation of GSK-3 which can serve to inhibit its kinase activity and promote ␤-catenin stabilization and Tcf/Lef transcriptional activation. There are two isoforms of GSK-3 designated as GSK-3␣ (51 kDa) and GSK-3␤ (47 kDa). At the amino acid level they share 85% homology, and both are phosphorylated by Akt as a consequence of Wnt signaling, at serine 9 in GSK-3␤ and at serine 21 in GSK-3␣ (20). PKA has been shown recently (8,9) to phosphorylate GSK-3␤ and GSK-3␣ at these same positions leading to the possibility of cross-talk between the Wnt and cAMP/PKA signaling pathways. A second mechanism for such cross-talk has been described in which PKA can indirectly activate Akt resulting in the phosphorylation and inhibition of GSK-3 (10). In our studies we have shown that the activation of the adenylyl cyclase stimulatory EP 2 and EP 4 prostanoid receptors leads to a rapid (within 5 min) agonistdependent phosphorylation of GSK-3␣ and a slower agonistdependent phosphorylation of Akt. Interestingly, the PKA inhibitor H-89 completely blocked the agonist-dependent phosphorylation of GSK-3␣, but it actually enhanced the phosphorylation of Akt, suggesting that the phosphorylation of GSK-3␣ is mediated directly by PKA, whereas the phosphorylation of Akt is mediated by another kinase that is negatively regulated by PKA. This other kinase is likely to be PI3 kinase, which is corroborated by our finding that wortmannin, an inhibitor of PI3 kinase, completely blocked the agonist-dependent phosphorylation of Akt.
Given the effects of EP 2 and EP 4 receptor activation on the phosphorylation of GSK-3␣, it is not surprising that we observed a stimulation of Tcf/Lef reporter gene activity following the incubation of these receptors with PGE 2 . What is surprising, however, is that the maximal stimulation of reporter gene activity is the same, or even higher, for the EP 4 receptor as compared with the EP 2 receptor, even though the EP 4 receptor only yielded ϳ20% of the maximal amount of cAMP formation as that obtained with the EP 2 receptor. Furthermore, PGE 2stimulated phosphorylation of GSK-3␣ in EP 2 receptor-transfected cells was approximately twice that obtained in EP 4 re-

FIG. 4. The effects of wortmannin on PGE 2 -stimulated phosphorylation of GSK-3 and Akt (immunoblots, panels A-D) and on stimulation of Tcf/Lef-responsive luciferase reporter gene activity (histograph) in untransfected HEK cells or HEK cells transfected with either the EP 2 or EP 4 prostanoid receptors.
Cells were pretreated with either vehicle or 100 nM wortmannin for 15 min followed by either vehicle (v) or 1 M PGE 2 (P) for 1 h at 37°C and were then immediately subjected to immunoblot analysis or were washed to remove PGE 2 and incubated for 16 h after which luciferase activity was measured as described under "Experimental Procedures." Panels A-D are exactly as described in Fig. 2 and represent the immunostaining of phospho-GSK-3␣ and -3␤ (panel A), total GSK-3␤ (panel B), phospho-Akt (panel C), and total Akt (panel D). Immunoblotting results are representative of three experiments with each antibody and condition. Luciferase data are the means Ϯ S.E. of two measurements from a representative experiment that was repeated three times. Note, the reporter gene experiments shown in this figure and Fig. 3 were done simultaneously; therefore, the luciferase activity data for cells that were not pretreated with either H-89 or wortmannin are the same in both figures.
ceptor-transfected cells, suggesting that the stimulation of Tcf/ Lef reporter gene activity should have been significantly greater for the EP 2 receptor. It is very relevant, therefore, that H-89 only inhibited PGE 2 -stimulated reporter gene activity by ϳ20% in EP 4 receptor-transfected cells in contrast to the 65% inhibition obtained in EP 2 receptor-transfected cells. Given the similar maximal stimulation of reporter gene activity by these receptors, the 20% inhibition of activity obtained with H-89 for the EP 4 receptor is exactly as one would predict based upon the relative ability of these receptors to stimulate cAMP formation and strongly suggests that stimulation of Tcf/Lef reporter gene activity by the EP 4 receptor involves an additional signaling pathway.
As suggested above, based upon the inhibition of Akt phosphorylation by wortmannin, this additional pathway appears to involve PI3 kinase. This premise is also supported by the differential effects of wortmannin on PGE 2 -stimulated Tcf/Lef reporter gene activity. Thus, in contrast to the results obtained with H-89, wortmannin had nearly the opposite effect and inhibited agonist-stimulated reporter gene activity to a much greater extent in EP 4 receptor-transfected cells than in EP 2 receptor-transfected cells. The putative involvement of PI3 kinase with EP 4 receptor signaling is further supported by the more obvious time course and extent of phosphorylation of Akt following the treatment of EP 4 receptor-transfected cells with PGE 2 (cf. Fig. 2). Therefore, although the phosphorylation of GSK-3␣ by activation of EP 4 receptors is entirely dependent on cAMP/PKA, the stimulation of Tcf/Lef reporter gene activity primarily involves activation of PI3 kinase and Akt. In contrast, both the phosphorylation of GSK-3␣ and the stimulation of reporter gene activity by EP 2 receptor activation are primarily dependent on cAMP/PKA.
One apparent discrepancy in our data, with respect to the putative involvement of PI3 kinase and Akt in the stimulation of Tcf/Lef reporter gene activity by the activation of EP 4 receptors, is that we did not observe enhanced phosphorylation of either GSK-3␣ or GSK-3␤ following pretreatment of EP 4 -expressing cells with H-89 (cf. Fig. 3). Thus, it would be reasonable to expect that one of these isoforms would show increased phosphorylation given the observed increase in Tcf/Lef reporter gene activity. This apparent discrepancy may be explained, however, by the 16-h time differential between the measurement of GSK-3 phosphorylation and the assay of luciferase reporter gene activity. Thus, GSK-3 phosphorylation is measured immediately after the 1-h incubation with PGE 2 , whereas the reporter gene activity is measured 16 h after PGE 2 treatment and washout. (Attempts to measure luciferase activity immediately after PGE 2 treatment were unsuccessful presumably because of the time required for de novo synthesis of the enzyme.) During this time it was therefore possible that in EP 4 -expressing cells there was a prolonged stimulation of Akt phosphorylation that resulted in the phosphorylation of GSK-3␤ and activation of Tcf/Lef signaling. As to why this is only observed in EP 4 -expressing cells may be related to the greater desensitization and internalization of the EP 4 receptor as compared with the EP 2 receptor (18,19) and is supported by our studies of the FP prostanoid receptor isoforms. Thus, as compared with the FP A isoform, the FP B isoform shows a markedly greater degree of functional desensitization of phosphoinositide and Ca 2ϩ signaling (17), and in this way resembles the differences between the EP 2 and EP 4 receptors. Interestingly, relative to the FP A isoform, the FP B isoform shows significantly prolonged activation of cellular shape change and activation of Tcf/Lef signaling following the removal of agonist (3). It is also possible, however, that there is a PI3 kinase-dependent, but GSK-3-independent, pathway leading to the activation of Tcf/Lef signaling, and further work will be needed to clarify these mechanisms.
Our present findings clearly establish the potential for the activation of novel signaling pathways by the EP 2 and EP 4 prostanoid receptors. Ultimately, this potential will need to be realized in a more physiological setting; however, there is evidence to suggest that such pathways may operate in vivo, particularly as it concerns the effects of PGE 2 on the immune system. For example, it is known that PGE 2 acting through an EP 4 receptor enhances the transcriptional activation that occurs during human immunodeficiency virus (HIV) infection (21). Interestingly, the HIV-long terminal repeat that drives this transcriptional activity contains a TCF-1␣ consensus region (22) offering a possible mechanism by which EP 4 receptor activation could modulate HIV-long terminal repeat transcriptional activity. Another effect of PGE 2 on immune system function, which is known to involve the activation of EP 2 and EP 4 receptors, concerns isotype switching and clonal expansion of B lymphocytes (23). These processes, which essentially represent cellular differentiation and proliferation, have been associated with increases in cAMP formation, but the downstream signaling pathways specifically mediating these effects are still obscure. Direct or synergistic influences of EP 2 and/or EP 4 receptor activation on PKA, GSK-3, PI3 kinase, and Tcf/Lef transcriptional activation would be compatible with potential regulation of cellular differentiation and proliferation.