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• W2058034672 abstract "Prostaglandin E2(PGE2) mediates its physiological effects by interactions with a subfamily of G-protein-coupled receptors known as EP receptors. These receptors consist of four primary subtypes named EP1, EP2, EP3, and EP4. The EP2 and EP4 subtypes are known to couple to Gαs and stimulate intracellular cyclic 3,5- adenosine monophosphate formation, whereas the EP1 and EP3 receptors are known to couple to Gαq and Gαi, respectively. Recently we found that EP2 and EP4 receptors can activate T-cell factor signaling; however, EP2 receptors did this primarily through a cAMP-dependent protein kinase-dependent pathway, whereas EP4 receptors primarily utilized a phosphatidylinositol 3-kinase (PI3K)-dependent pathway (Fujino, H., West, K. A., and Regan, J. W. (2002) J. Biol. Chem. 277, 2614–2619). We now report that PGE2 stimulation of EP4 receptors, but not EP2 receptors, leads to phosphorylation of the extracellular signal-regulated kinases (ERKs) through a PI3K-dependent mechanism. Furthermore, this activation of PI3K/ERK signaling by the EP4 receptors induces the functional expression of early growth response factor-1 (EGR-1). Under the same conditions induction of EGR-1 protein expression was not observed following PGE2 stimulation of EP2 receptors. These findings point to important differences in the signaling potential of the EP2 and EP4 receptors, which could be significant with respect to the potential involvement of EP4 receptors in inflammation and cancer. Prostaglandin E2(PGE2) mediates its physiological effects by interactions with a subfamily of G-protein-coupled receptors known as EP receptors. These receptors consist of four primary subtypes named EP1, EP2, EP3, and EP4. The EP2 and EP4 subtypes are known to couple to Gαs and stimulate intracellular cyclic 3,5- adenosine monophosphate formation, whereas the EP1 and EP3 receptors are known to couple to Gαq and Gαi, respectively. Recently we found that EP2 and EP4 receptors can activate T-cell factor signaling; however, EP2 receptors did this primarily through a cAMP-dependent protein kinase-dependent pathway, whereas EP4 receptors primarily utilized a phosphatidylinositol 3-kinase (PI3K)-dependent pathway (Fujino, H., West, K. A., and Regan, J. W. (2002) J. Biol. Chem. 277, 2614–2619). We now report that PGE2 stimulation of EP4 receptors, but not EP2 receptors, leads to phosphorylation of the extracellular signal-regulated kinases (ERKs) through a PI3K-dependent mechanism. Furthermore, this activation of PI3K/ERK signaling by the EP4 receptors induces the functional expression of early growth response factor-1 (EGR-1). Under the same conditions induction of EGR-1 protein expression was not observed following PGE2 stimulation of EP2 receptors. These findings point to important differences in the signaling potential of the EP2 and EP4 receptors, which could be significant with respect to the potential involvement of EP4 receptors in inflammation and cancer. prostaglandin E2 T-cell factor cAMP-dependent protein kinase phosphatidylinositol 3-kinase early growth response factor-1 tumor necrosis factor-α mitogen-activated protein kinases extracellular signal-regulated kinases Jun N-terminal kinase protein kinase C human embryonic kidney cells The EP2 and EP4 prostanoid receptors are two of the four primary receptor subtypes for prostaglandin E2(PGE2).1 Both of these receptors couple to Gαs and can activate adenylyl cyclase resulting in the increased formation of intracellular cyclic 3,5-adenosine monophosphate (cAMP). Prior to the molecular cloning of these receptors it was thought that the stimulation of adenylyl cyclase by PGE2 was mediated by a single receptor subtype that was defined pharmacologically as the EP2 subtype (1Bastien L. Sawyer N. Grygorczyk R. Metters K.M. Adams M. J. Biol. Chem. 1994; 269: 11873-11877Google Scholar). Thus, the first adenylyl cyclase stimulatory EP receptor to be cloned was thought to be the EP2 subtype, but it was subsequently redefined as the EP4 subtype (2Nishigaki N. Negishi M. Honda A. Sugimoto Y. Namba T. Narumiya S. Ichikawa A. FEBS Lett. 1995; 364: 339-341Google Scholar) when a second adenylyl cyclase stimulatory EP receptor was cloned, which had the expected pharmacology of the EP2 subtype (3Regan J.W. Bailey T.J. Pepperl D.J. Pierce K.L. Bogardus A.M. Donello J.E. Fairbairn C.E. Kedzie K.M. Woodward D.F. Gil D.W. Mol. Pharmacol. 1994; 46: 213-220Google Scholar). Surprisingly the EP2and EP4 receptors encoded by these cDNAs only shared ∼307 amino acid homology and were really no more related to each other than to other prostanoid receptor subtypes (4Pierce K.L. Regan J.W. Life Sci. 1998; 62: 1479-1483Google Scholar). In fact, the EP2 receptor shows a closer phylogenetic relationship to the DP and IP receptors than it does to the EP4 receptor. The human EP4 receptor is larger than the human EP2 (488 amino acids versus 358), which is almost entirely due to a significantly longer carboxyl-terminal domain (155 versus 34 amino acids). Recently we have shown that PGE2 stimulation of the EP2 receptors activates a T-cell factor (Tcf) signaling pathway by a mechanism that mainly involves the activation of cAMP-dependent protein kinase (PKA) (5Fujino H. West K.A. Regan J.W. J. Biol. Chem. 2002; 277: 2614-2619Google Scholar). PGE2stimulation of the EP4 receptors also activates Tcf signaling, but the mechanism is more complex and involves the activation of phosphatidylinositol 3-kinase (PI3K) (5Fujino H. West K.A. Regan J.W. J. Biol. Chem. 2002; 277: 2614-2619Google Scholar). The involvement of PI3K with EP4 receptor signaling has also been suggested in studies of human colorectal carcinoma cells (6Sheng H. Shao J. Washington M.K. DuBois R.N. J. Biol. Chem. 2001; 276: 18075-18081Google Scholar). Thus, PGE2 stimulation of endogenous EP4 receptors in LS-174 cells increased cellular proliferation and motility through a PI3K-dependent pathway. Given the known role of PI3K in carcinogenesis, it is interesting that a recent study of knockout mice suggested the potential involvement of EP4 receptors in colon cancer (7Mutoh M. Watanabe K. Kitamura T. Shoji Y. Takahashi M. Kawamori T. Tani K. Kobayashi M. Maruyama T. Kobayashi K. Ohuchida S. Sugimoto Y. Narumiya S. Sugimura T. Wakabayashi K. Cancer Res. 2002; 62: 28-32Google Scholar). Early growth response factor-1 (EGR-1) is a member of the zinc finger family of transcription factors and plays a key role in cell growth and differentiation. It is recognized as an immediate early gene product that regulates the expression of a number of downstream genes, such as tumor necrosis factor-α (TNF-α), interleukin-2, and p53 (8Silverman E.S. Collins T. Am. J. Pathol. 1999; 154: 665-670Google Scholar). The induction of EGR-1 expression is known to involve members of the family of mitogen-activated protein kinases (MAPKs). For example, the induction of EGR-1 expression by growth hormone was shown to depend on the phosphorylation and activation of extracellular signal-regulated kinases (ERKs), but not on the activation of either Jun N-terminal kinase (JNK) or p38 MAPK (9Hodge C. Liao J. Stofega M. Guan K. Carter-Su C. Schwartz J. J. Biol. Chem. 1998; 273: 31327-31336Google Scholar). Similarly the calmodulin antagonist, trifluoroperazine, induced EGR-1 expression through the activation of ERKs and the downstream transcription factor, Elk-1 (10Shin S.Y. Kim S.-Y. Kim J.-H. Min D.S. Ko J. Kang U.-G. Kim Y.S. Kwon T.K. Han M.Y. Kim Y.H. Lee Y.H. J. Biol. Chem. 2001; 276: 7797-7805Google Scholar). Previous studies have also shown regulation of EGR-1 expression by protein kinase C (PKC), possibly involving members of the family of prostanoid receptors. For example, in Swiss 3T3 fibroblasts increases in PGE2 were associated with a PKC-dependent increase in EGR-1 mRNA expression (11Danesch U. Weber P.C. Sellmayer A. J. Biol. Chem. 1994; 269: 27258-27263Google Scholar). Likewise, in mouse MC3T3 osteoblastic cell lines PGE2 was found to increase EGR-1 mRNA expression. This increase was also PKC-dependent, but did not appear to involve PKA since the induction of EGR-1 mRNA expression was not observed following stimulation with forskolin, an agent which increases the formation of intracellular cAMP (12Fang M.A. Noguchi G.M. McDougall S. Prostaglandins Leukot. Essen. Fatty Acids. 1996; 54: 109-114Google Scholar). These findings would appear to exclude the participation of the adenylyl cyclase stimulatory EP2 and EP4 receptors in the induction of EGR-1 mRNA expression. However, because neither of these studies included pharmacological characterizations, the conclusions that can be drawn are limited with respect to the specific prostanoid receptor subtypes mediating these effects. We now show that the EP4 prostanoid receptor, but not the EP2, can induce the functional expression of EGR-1 through a signaling pathway involving the sequential activation of PI3K and the ERKs. These results further strengthen the involvement of PI3K with EP4receptor signaling and clearly differentiate the signaling potential of the EP2 and EP4 receptors. Cell lines stably expressing the EP2 or EP4 receptors were prepared using HEK-293 EBNA cells and the mammalian expression vector pCEP4 (Invitrogen) as previously described (5Fujino H. West K.A. Regan J.W. J. Biol. Chem. 2002; 277: 2614-2619Google Scholar). Cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 107 fetal bovine serum, 250 ॖg/ml geneticin, 100 ॖg/ml gentamicin, and 200 ॖg/ml hygromycin B. 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 PGE2 for the times indicated in the figures. In some cases cells were pretreated with either vehicle (0.17 Me2SO) or 100 nm wortmannin (Sigma) for 15 min or 10 ॖm PD98059 (Calbiochem) for 10 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), 17 Nonidet P-40, 0.57 sodium deoxycholate, 10 mm sodium fluoride, 10 mmdisodium pyrophosphate, 0.17 SDS, 0.1 mmphenylmethylsulfonyl 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 × g for 15 min. Aliquots of the supernatants containing 20∼100 ॖg of protein were electrophoresed on 7.5 or 107 SDS-polyacrylamide gels and transferred to nitrocellulose membranes as described previously (5Fujino H. West K.A. Regan J.W. J. Biol. Chem. 2002; 277: 2614-2619Google Scholar). Membranes were incubated in 57 nonfat milk for 1 h and were then washed and incubated for 16 h at 4 °C with primary antibodies using the following conditions, which varied according to the antibody being used. For the ERKs, incubations were done in 37 nonfat milk containing either a 1:1,000 dilution of antiphospho-ERK1/2 antibody (9106, Cell Signaling); or mixture of a 1:500 dilution of anti-ERK1 antibody and a 1:10,000 dilution of anti-ERK2 antibody (sc-93 and sc-154, Santa Cruz Biotechnology). For JNK, incubations were done in 37 nonfat milk containing a 1:1,000 dilution of antiphospho-JNK antibody (9255, Cell Signaling) or 0.17 nonfat milk containing a 1:1,000 dilution of anti-JNK antibody (9252, Cell Signaling). For p38 MAPK, incubations were done in 57 bovine serum albumin containing a 1:1,000 dilution of antiphospho-p38 MAPK antibody (9211, Cell Signaling); or 17 nonfat milk containing a 1:1,000 dilution of anti-p38 MAPK antibody (sc-535-G, Santa Cruz Biotechnology). For EGR-1, incubations were done in 37 nonfat milk containing a 1:1,000 dilution of anti-EGR-1 antibody (sc-110, Santa Cruz Biotechnology). After incubating with the primary antibody, membranes were washed three times and incubated for 1 h at room temperature with a 1:10,000 dilution of the appropriate secondary antibodies conjugated with horseradish peroxidase using the same conditions as described above for each of the primary antibodies. After washing three times, immunoreactivity was detected by chemiluminescence as described previously (5Fujino H. West K.A. Regan J.W. J. Biol. Chem. 2002; 277: 2614-2619Google Scholar). To ensure equal loading of proteins, the membranes were stripped and re-probed with appropriate antibodies under the same conditions as described above. Cells, grown in 10-cm plates, were pretreated with either vehicle (0.17 Me2SO) or 10 ॖm PD98059 for 10 min at 37 °C followed by treatment with either vehicle or 1 ॖm PGE2for 60 min at 37 °C. Cells were washed, trypsinized, and centrifuged at 500 × g for 1 min, and the supernatant was removed. The pellet was resuspended in approximately three times the original pellet volume using buffer A, containing 10 mm HEPES-KOH (pH 7.9), 1.5 mm MgCl2, 10 mm KCl, and 0.5 mm dithiothreitol. The sample was transferred into a new tube, placed on ice for 15 min, and then centrifuged at 16,000 × g for 30 s at 4 °C. The supernatant was discarded, and the pellet was resuspended in ∼2 volumes of buffer A with seven rapid strokes of a 1-ml syringe with a 25-gauge needle. The sample was centrifuged at 16,000 × g for 5 min at 4 °C, and the supernatant was transferred to another tube (S100 fraction). The pellet was rinsed with buffer A and resuspended with buffer C, consisting of 20 mm HEPES-KOH (pH 7.9), 257 glycerol, 0.42 m NaCl, 1.5 mmMgCl2, 0.2 mm EDTA, and 0.5 mmdithiothreitol. After a 30-min incubation on ice, the sample was centrifuged at 16,000 × g for 5 min at 4 °C, and the supernatant (nuclear extract) was dialyzed for 2 h using a 3,000 molecular weight cutoff membrane (Pierce) against buffer D containing 20 mm HEPES-KOH (pH 7.9), 207 glycerol, 20 mm KCl, 2 mm MgCl2, 0.2 mm EDTA, and 0.5 mm dithiothreitol. A double-stranded DNA oligonucleotide probe was designed corresponding to the GC box-1 and GC box-2 sequences in the human prostaglandin E2 synthase gene promoter (13Naraba H. Yokoyama C. Tago N. Murakami M. Kudo I. Fueki M. Oh-ishi S. Tanabe T. J. Biol. Chem. 2002; 277: 28601-28608Google Scholar). The sense strand of this oligonucleotide sequence is 5′-GTGGGGCGGGGCGTGGGCGGTGCT-3′, where the underlined sequence represents the consensus binding site for EGR-1 (8Silverman E.S. Collins T. Am. J. Pathol. 1999; 154: 665-670Google Scholar). The sense and antisense oligonucleotides were annealed and labeled with [γ-32P]ATP (Amersham Biosciences) using T4 kinase (Invitrogen). The reaction mixture was applied to a spin column to remove free [γ-32P]ATP and run on an 87 native polyacrylamide gel to separate any single-stranded DNA from the double-stranded probe. The binding reaction was performed for 15 min at room temperature using ∼10,000 cpm of the probe and 10 ॖg of nuclear extract in 15 ॖl of a buffer containing 47 Ficoll 400, 1 mm EDTA (pH 8.1), 1 mm dithiothreitol, 4 mg/ml bovine serum albumin, 0.1 m KCl, and 2 ॖg of poly-(deoxyinosinic-deoxycytidylic) (Sigma). The mobility shift assay was done with a 57 native polyacrylamide gel followed by drying and visualization by autoradiography using Hyperfilm MP (Amersham Biosciences). The supershift assay was performed using 3 ॖg of the anti-EGR-1 antibody in which the antibody was incubated with the probe and the nuclear extract for an additional 15 min following the initial binding reaction. We have previously reported that EP4 prostanoid receptor can activate Tcf signaling by a mechanism involving PI3K (5Fujino H. West K.A. Regan J.W. J. Biol. Chem. 2002; 277: 2614-2619Google Scholar). Further characterization of this interaction between the EP4 receptor and PI3K was of interest given the important role of PI3K in both normal and aberrant cell signaling pathways. One of the downstream effectors of PI3K signaling is the activation of members of the MAPKs. To determine if EP4 receptors could potentially activate MAPK signaling, we examined the ability of PGE2 to stimulate the phosphorylation of three MAPKs in HEK-293 cells stably transfected with the human EP2 and EP4 prostanoid receptors. For these experiments untransfected HEK cells and cells stably expressing the EP2 and EP4 receptors were incubated with 1 ॖm PGE2 for various times and were then immunoblotted with antibodies to the phosphorylated and nonphosphorylated forms of ERK1 and ERK2, p54 JNK, and p38 MAPK. As shown in panel A of Fig. 1, treatment with PGE2 resulted in a time-dependent phosphorylation of ERKs 1 and 2 in EP4-expressing cells, but not in EP2-expressing cells or the control HEK cells. The PGE2-stimulated phosphorylation of ERKs in EP4 cells was maximal at 5–10 min and by 60 min had nearly returned to the original, unstimulated, levels (0 min). These same blots were stripped and re-probed with antibodies to the nonphosphorylated forms of ERK1 and ERK2, and as shown in panel B of Fig. 1 nearly identical amounts of ERKs 1 and 2 were present throughout the time course of treatment and among the three cell lines. Panels C–F of Fig. 1 show the corresponding results for p54 JNK and p38 MAPK, respectively. In contrast to the results obtained with the ERKs, there were no substantive differences between the EP2- and EP4-expressing cells with respect to the phosphorylation of either p54 JNK (panel C) or p38 MAPK (panel E). In both EP2- and EP4-expressing cells there was significant phosphorylation of p54 JNK even at the 0 time point, which was not observed in the control HEK cells (panel C). This appears to suggest some constitutive effect of EP2 and EP4 receptors on the phosphorylation of p54 JNK. In both EP2- and EP4-expressing cells there was a PGE2-dependent induction of the phosphorylation of p38 MAPK, which was maximal at ∼15 min and which nearly returned to the original, unstimulated, levels by 60 min (panel E). As with the ERKs, nearly identical amounts of p54 JNK (panel D) and p38 MAPK (panel F) were present throughout the time course of treatment and among the three cell lines. The PI3K inhibitor, wortmannin, was used to examine the potential involvement of PI3K on the phosphorylation of ERKs 1 and 2 following PGE2 stimulation of the EP4 receptor. For these experiments EP2- and EP4-expressing cells were pretreated with either vehicle or 100 nm wortmannin for 15 min followed by treatment with either vehicle or 1 ॖmPGE2 for 10 min. The cells were then subjected to immunoblot analysis using antibodies to the phosphorylated and nonphosphorylated forms of the ERKs. As shown in the upper panel of Fig. 2 stimulation with PGE2 in the absence of wortmannin pretreatment resulted in the phosphorylation of ERKs 1 and 2 in EP4 expressing cells, but not in EP2-expressing cells. However, following pretreatment with wortmannin there was a complete blockade of PGE2-stimulated phosphorylation of ERKs 1 and 2 in EP4-expressing cells, suggesting the direct involvement of PI3K in this process. The lower panel of Fig. 2 shows that nearly identical amounts of ERKs 1 and 2 were present under all conditions in both cell lines. Given the phosphorylation of ERKs 1 and 2 following PGE2 stimulation of the human EP4 prostanoid receptor, it was of interest to explore the possible modulation of downstream of effectors that could reflect this selective activation of the ERK signaling pathway. One such effector that is known to be induced following the activation of ERKs is the early growth response factor-1 (EGR-1). Immunoblot analysis was, therefore, used to examine the time course of expression of EGR-1 following treatment of EP2- and EP4-expressing cells with 1 ॖm PGE2. As shown in the upper panels of Fig. 3, there was a modest induction of EGR-1 expression in EP4-expressing cells following 30 min of incubation with PGE2, which was markedly increased at the 60-min time point. On the other hand, in EP2-expressing cells the expression of EGR-1 was barely detectable even at the 60-min time point. To check for the equal loading of protein these blots were stripped and re-probed with antibodies to ERKs 1 and 2. As shown in the lower panels of Fig. 3 nearly identical amounts of ERKs 1 and 2 were present in both cell lines and throughout the time course of treatment with PGE2. Pretreatment of EP2- and EP4-expressing cells with either the PI3K inhibitor, wortmannin, or the MAPK/ERK kinase inhibitor, PD98059, was used to examine the potential involvement of these kinases on the PGE2 induced expression of EGR-1. As shown in theupper panel of Fig.4A, the robust induction of EGR-1 expression in EP4-expressing cells following 60 min of exposure to 1 ॖm PGE2 was completely blocked by pretreatment with wortmannin. EGR-1 expression in EP2-expressing cells was not detectable either before or after treatment with PGE2, or following pretreatment with wortmannin. The lower panel of Fig. 4A shows that nearly equal amounts of ERKs 1 and 2 were present under all conditions and indicates that the observed differences in EGR-1 expression are not because of differences in overall protein expression or in the amount of protein loaded on the gels. The MAPK/ERK kinase inhibitor, PD98059, was then used to explore whether this specific induction of EGR-1 expression in EP4-expressing cells involved the sequential activation of PI3K and the ERKs. For these experiments EP2- and EP4-expressing cell lines were pretreated with either vehicle or 10 ॖm PD98059 for 10 min followed by treatment with either vehicle or 1 ॖm PGE2 for 60 min. As shown in the upper panel of Fig. 4B, in the absence of pretreatment with PD98059 there was a strong PGE2-mediated induction of EGR-1 expression in EP4-expressing cells that was completely absent in EP2-expressing cells. Following pretreatment with PD98059, however, there was a complete block of PGE2-mediated EGR-1 expression in EP4-expressing cells. To confirm equal loading of protein, this blot was stripped and re-probed with antibodies to ERKs 1 and 2. The lower panel of Fig.4B shows that nearly identical amounts of ERKs 1 and 2 were present under all conditions in both cell lines. Together these results strongly suggest that the sequential activation of PI3K and the ERKs is required for the induction of EGR-1 expression by PGE2 in EP4-expressing cells. The potential for PGE2 to stimulate EGR-1 mediated transcriptional activation was examined in EP2- and EP4-expressing cells using electrophoresis mobility shift assays. The probe used for these experiments consisted of an oligonucleotide designed from the promoter region of the human gene for prostaglandin E2 synthase containing a consensus binding site for EGR-1 (13Naraba H. Yokoyama C. Tago N. Murakami M. Kudo I. Fueki M. Oh-ishi S. Tanabe T. J. Biol. Chem. 2002; 277: 28601-28608Google Scholar). EP2- and EP4-expressing cells were pretreated with either vehicle or 10 ॖm PD98059 for 10 min followed by treatment with either vehicle or 1 ॖm PGE2 for 60 min. In Fig. 5 a comparison of the 舠free舡 lane with the other lanes shows that the probe migrated with relative mobilities of 0.26 and 0.40 under all conditions in which a nuclear extract was present. Following treatment with PGE2, however, there was a selective shift in the mobility of the probe in EP4-expressing cells, but not in EP2-expressing cells, yielding a unique band with a relative mobility of 0.16. The identity of the protein causing this shift was investigated in a supershift assay by an additional incubation with antibodies to EGR-1. As shown in the last two lanes of Fig. 5, incubation with the antibodies to EGR-1 diminished the intensity of the 0.16-band following treatment with PGE2 and resulted in the appearance of a new band with a mobility of 0.08. This supershift confirms the interaction of the probe with the EGR-1 following the treatment of EP4expressing cells with PGE2. Fig. 5 also shows that pretreatment of EP4-expressing cells with the MAPK/ERK kinase inhibitor, PD98059, prevented the PGE2 induced mobility shift and appearance of 0.16 band. Thus, as predicted from our previous experiments with PD98059, the PGE2-induced functional expression of EGR-1 involves the activation of a MAPK/ERK signaling pathway. We have previously shown that stimulation of EP2prostanoid receptors by PGE2 can activate a Tcf signaling pathway by a mechanism that mainly involves the activation of PKA (5Fujino H. West K.A. Regan J.W. J. Biol. Chem. 2002; 277: 2614-2619Google Scholar). PGE2 stimulation of EP4 prostanoid receptors can also activate a Tcf signaling pathway, but the mechanism is more complex and involves the activation of PI3K (5Fujino H. West K.A. Regan J.W. J. Biol. Chem. 2002; 277: 2614-2619Google Scholar). We now show that PGE2 stimulation of the EP4 receptor activates an additional signaling pathway involving PI3K. Thus, PGE2treatment of cells expressing EP4 receptor, leads to a PI3K-dependent phosphorylation of ERKs 1 and 2 followed by a de novo increase in the functional expression of EGR-1. The activation of this PI3K signaling cascade was unique for the EP4 receptor and was not observed in cells expressing EP2 receptors. One of the ways in which the EP2 and EP4receptors are known to differ is in the characteristics of their agonist induced desensitization and internalization. Thus, EP4 receptors undergo rapid, PGE2-mediated desensitization (14Nishigaki N. Negishi M. Ichikawa A. Mol. Pharmacol. 1996; 50: 1031-1037Google Scholar) and internalization (15Desai S. April H. Nwaneshiudu C. Ashby B. Mol. Pharmacol. 2000; 58: 1279-1286Google Scholar); whereas EP2receptors do not. Since the internalization of some G-protein-coupled receptors is associated with a transactivation of the MAPK pathway (16Rodriguez-Viciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Google Scholar,17Naga Prasad S.V. Barak L.S. Rapacciuolo A. Caron M.G. Rockman H.A. J. Biol. Chem. 2001; 276: 18953-18959Google Scholar), it may be supposed that such a mechanism could explain the present findings of selective activation of ERK signaling by the EP4 receptors. However, in a previous study using human EP4 receptors expressed in HEK-293 cells, it was found that the phosphorylation of ERKs 1 and 2 was independent of PGE2-mediated receptor internalization (18Desai S. Ashby B. FEBS Lett. 2001; 501: 156-160Google Scholar). Another way in which the EP2 and EP4 prostanoid receptors appear to differ is in their ability to stimulate intracellular cAMP formation. Thus, despite nearly identical levels of receptor expression, the maximal levels of PGE2-stimulated cAMP formation in EP4-expressing cells was only 207 of the level obtained in EP2-expressing cells (5Fujino H. West K.A. Regan J.W. J. Biol. Chem. 2002; 277: 2614-2619Google Scholar). However, under the same conditions the ability of PGE2 to stimulate Tcf signaling was ∼507 greater in EP4-expressing cells as compared with EP2-expressing cells (5Fujino H. West K.A. Regan J.W. J. Biol. Chem. 2002; 277: 2614-2619Google Scholar). This indicates that the lower amounts of PGE2-stimulated cAMP formation in EP4-expressing cells is because of less efficient coupling to this pathway and not because of an overall impairment in the signaling potential of these receptors. The more efficient coupling of the EP2 receptor to intracellular cAMP formation; however, may be significant with respect to the present findings. Thus, it has been reported that the phosphorylation of Raf kinase by PKA inhibits the activity of Raf kinase and subsequently decreases Raf mediated MAPK signaling (19Dhillon A.S. Pollock C. Steen H. Shaw P.E. Mischak H. Kolch W. Mol. Cell. Biol. 2002; 22: 3237-3246Google Scholar, 20Sidovar M.F. Kozlowski P. Lee J.W. Collins M.A. He Y. Graves L.M. J. Biol. Chem. 2000; 275: 28688-28694Google Scholar). In EP2-expressing cells, therefore, a robust activation of PKA may inhibit Raf kinase and block the phosphorylation and activation of ERKs. Our findings of a PGE2-mediated induction of EGR-1 expression by the EP4 receptor is interesting light of recent studies with knockout mice that show a potential involvement of the EP4 receptor with colon cancer and rheumatoid arthritis. For example EP4 knockout mice, but not EP2 knockout or control mice, show a reduced formation of preneoplastic lesions following treatment with azoxymethane, a known colon carcinogen (7Mutoh M. Watanabe K. Kitamura T. Shoji Y. Takahashi M. Kawamori T. Tani K. Kobayashi M. Maruyama T. Kobayashi K. Ohuchida S. Sugimoto Y. Narumiya S. Sugimura T. Wakabayashi K. Cancer Res. 2002; 62: 28-32Google Scholar). EP4 knockout mice, but not EP2 knockout or control mice, also show a significantly decreased incidence and severity of collagen antibody induced arthritis, an animal model of rheumatoid arthritis (21McCoy J.M. Wicks J.R. Audoly L.P. J. Clin. Invest. 2002; 110: 651-658Google Scholar). In addition, in both colon cancer and rheumatoid arthritis prostaglandin levels are elevated and both conditions benefit to some extent by treatment with inhibitors of the cyclooxygenases. In colon cancer, the expression of cyclin D1, a key regulator of cell cycle progression, is known to be regulated by Tcf signaling (22Tetsu O. McCormick F. Nature. 1999; 398: 422-426Google Scholar). However, it has also been reported that the expression of cyclin D1 is regulated by EGR-1 through a PI3K- and ERK-dependent pathway (23Guillemot L. Levy A. Raymondjean M. Rothhut B. J. Biol. Chem. 2001; 276: 39394-39403Google Scholar). Furthermore it has been shown that PGE2 synthase is up-regulated by the binding of EGR-1 to the promoter region of the mouse gene encoding PGE2 synthase (13Naraba H. Yokoyama C. Tago N. Murakami M. Kudo I. Fueki M. Oh-ishi S. Tanabe T. J. Biol. Chem. 2002; 277: 28601-28608Google Scholar). Signaling through an EP4receptor would have the potential, therefore, to increase the expression of cyclin D1 and PGE2 synthase through a PGE2-mediated induction of EGR-1 expression. Since the product of PGE2 synthase is PGE2 itself, the potential for a positive feedback loop is obvious. A similar situation could also explain the increased levels of PGE2 observed in rheumatoid arthritis. However, in this disease in addition to an up-regulation of the expression of PGE2 synthase, signaling through EP4 receptors could also potentially up regulate levels of TNF-α (21McCoy J.M. Wicks J.R. Audoly L.P. J. Clin. Invest. 2002; 110: 651-658Google Scholar) whose expression is under the control of EGR-1 (24Yao J. Mackman N. Edgington T.S. Fan S.-T. J. Biol. Chem. 1997; 272: 17795-17801Google Scholar). Inhibitors of TNF-α, such as etancercept and infliximab, are used therapeutically in the treatment of rheumatoid arthritis because they slow the progression of this disease (25Aeberli D. Oertle S. Mauron H. Reichenbach S. Fordi B. Villiger P.M. Swiss Med. Wkly. 2002; 132: 414-422Google Scholar). It is possible that the reduced incidence and severity of collagen antibody induced arthritis in EP4knockout mice (21McCoy J.M. Wicks J.R. Audoly L.P. J. Clin. Invest. 2002; 110: 651-658Google Scholar) is related to the loss of EP4receptor-mediated signaling through a PI3K/ERKs/EGR-1 pathway. Recently we have shown that the FPB prostanoid receptor can signal through a ॆ-catenin/Tcf signaling pathway (26Fujino H. Regan J.W. J. Biol. Chem. 2001; 276: 12489-12492Google Scholar) and interact with PI3K (27Fujino H. Srinivasan D. Regan J.W. J. Biol. Chem. 2002; 277: 48786-48795Google Scholar); and thus, has potential to be involved in the pathophysiology of colon cancer. It has also been reported that EP1 receptors (28Watanabe K. Kawamori T. Nakatsugi S. Ohta T. Ohuchida S. Yamamoto H. Maruyama T. Kondo K. Ushikubi F. Narumiya S. Sugimura T. Wakabayashi K. Cancer Res. 1999; 59: 5093-5096Google Scholar) and EP2 receptors (29Sonoshita M. Takaku K. Sasaki N. Sugimoto Y. Ushikubi F. Narumiya S. Oshima M. Taketo M.M. Nat. Med. 2001; 7: 1048-1051Google Scholar) may have roles in colon cancer. In addition, gene knockout studies with the cyclooxygenases indicate that the synthesis of prostaglandins by these enzymes contributes to the pathophysiology of colon cancer (30Chulada P.C. Thompson M.B. Mahler J.F. Doyle C.M. Gaul B.W. Lee C. Tiano H.F. Morham S.G. Smithies O. Langenbach R. Cancer Res. 2000; 60: 4705-4708Google Scholar). Interestingly, the expression of cyclooxygenase-2 in intestinal polyps appears to be under a positive feedback through the EP2receptor (29Sonoshita M. Takaku K. Sasaki N. Sugimoto Y. Ushikubi F. Narumiya S. Oshima M. Taketo M.M. Nat. Med. 2001; 7: 1048-1051Google Scholar). Furthermore, it is clear from the findings of Sonoshitaet al. (29Sonoshita M. Takaku K. Sasaki N. Sugimoto Y. Ushikubi F. Narumiya S. Oshima M. Taketo M.M. Nat. Med. 2001; 7: 1048-1051Google Scholar) that the effects of the cyclooxygenase-2 knockout on the number and size of intestinal polyps was greater than the effects of the EP2 knockout and appear to require the involvement of another prostanoid receptor. Potential involvement of the EP4 receptor could work in concert with the EP2 receptor to increase PGE2 levels by increased expression of both cyclooxygenase-2 and PGE2synthase. Our present findings with the EP4 receptors further strengthen an association of the prostaglandins and their receptors with cancer and inflammation; further knowledge of which could have practical therapeutic benefits." @default.
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• W2058034672 title "Prostaglandin E2 Induced Functional Expression of Early Growth Response Factor-1 by EP4, but Not EP2, Prostanoid Receptors via the Phosphatidylinositol 3-Kinase and Extracellular Signal-regulated Kinases" @default.
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