Induction of adherent activity in mastocytoma P-815 cells by the cooperation of two prostaglandin E2 receptor subtypes,EP3 and EP4

In this study, we investigated the role of PGE(2) in mouse mastocytoma P-815 cell adhesion to extracellular matrix proteins (ECMs) in vitro. We report that PGE(2) accelerated ProNectin F(TM) (a proteolytic fragment of fibronectin)-mediated adhesion, which was abolished by addition of the GRGDS peptide, an inhibitor of the RDG binding site of ProNectin F(TM). We show that the cAMP level and cAMP-regulated protein kinase (PKA) activity are critical mediators of this PGE(2) effect, because the cell-permeable cAMP analogue 8-Br-cAMP accelerated P-815 cell adhesion to ProNectin F(TM) and the pharmacological inhibitor of PKA, H-89, blocked PGE(2)-mediated adhesion. Consistent with mRNA expression of the G(s)-coupled EP4- and G(i)-coupled EP3-PGE receptor subtypes, P-815 cell adhesion was accelerated by treatment with a selective EP4 agonist, ONO-AE1-329, but not a selective EP1/EP3 agonist, sulprostone. However, simultaneous treatment with ONO-AE1-329 and sulprostone resulted in augmentation of both the cAMP level and cell adhesion. The augmentation of EP3-mediated cAMP synthesis was dose-dependent, without affecting the half-maximal concentration for EP4-mediated G(s)-activity, which was inhibited by a G(i) inhibitor, pertussis toxin. In conclusion, these findings suggest that PGE(2) accelerates RGD-dependent adhesion via cooperative activation between EP3 and EP4 and contributes to the recruitment of mast cells to the ECM during inflammation.     

PGE2 stimulates human brain natriuretic peptide expression via EP4 and p42/44 MAPK

Brain natriuretic peptide (BNP) produced by cardiac myocytes has antifibrotic and antigrowth properties and is a marker of cardiac hypertrophy. We previously showed that prostaglandin E2 (PGE2) is the main prostaglandin produced in myocytes treated with proinflammatory stimuli and stimulates protein synthesis by binding to its EP4 receptor. We hypothesized that PGE2, acting through EP4, also regulates BNP gene expression. We transfected neonatal ventricular myocytes with a plasmid encoding the human BNP (hBNP) promoter driving expression of a luciferase reporter gene. PGE2 increased hBNP promoter activity 3.5-fold. An EP4 antagonist reduced the stimulatory effect of PGE2 but not an EP1 antagonist. Because EP4 signaling can involve adenylate cyclase, cAMP, and protein kinase A (PKA), we tested the effect of H-89, a PKA inhibitor, on PGE2 stimulation of the hBNP promoter. H-89 at 5 muM decreased PGE2 stimulation of BNP promoter activity by 100%. Because p42/44 MAPK mediates the effect of PGE2 on protein synthesis, we also examined the role of MAPKs in the regulation of BNP promoter activity. PGE2 stimulation of the hBNP promoter was inhibited by a MEK1/2 inhibitor and a dominant-negative mutant of Raf, indicating that p42/44 MAPK was involved. In contrast, neither a p38 MAPK inhibitor nor a JNK inhibitor reduced the stimulatory effect of PGE2. Involvement of small GTPases was also studied. Dominant-negative Rap inhibited PGE2 stimulation of the hBNP promoter, but dominant-negative Ras did not. We concluded that PGE2 stimulates the BNP promoter mainly via EP4, PKA, Rap, and p42/44 MAPK.     

Prostaglandin E(2) receptors, EP2 and EP4, differentially modulate TNF-alpha and IL-6 production induced by lipopolysaccharide in mouse peritoneal neutrophils

The expression and function of prostaglandin (PG) E(2) receptors were examined in mouse neutrophils exudated into the peritoneal cavity by casein treatment. Expressions of the EP2 and EP4 receptors were detected in neutrophils by Northern blot, but those of EP1 and EP3 receptors were not detected by RT-PCR. EP2-selective agonist, ONO-AE1-259, and EP4-selective agonist, ONO-AE1-329, stimulated cAMP formation in the cells. PGE(2) affected the TNF-alpha and IL-6 production in lipopolysaccharide (LPS)-treated neutrophils; it suppressed the TNF-alpha production and enhanced the IL-6 production. The PGE(2) effects were mimicked by dibutyryl cAMP. This is the first study of the enhancement of IL-6 production by cAMP-elevating reagents in neutrophils. Using neutrophils from EP2- and EP4-deficient mice in combination with EP2- and EP4-selective agonists, it was found that the augmentation of IL-6 was mediated mainly by the EP2 receptor and the suppression of TNF-alpha by the EP4 receptor and partially by the EP2 receptor. These findings indicate that casein-induced peritoneal neutrophils express Gs-coupled PGE(2) receptors, EP2 and EP4, which might differentially regulate the LPS-induced production of TNF-alpha and IL-6.     

Prostaglandin E2 enhances interleukin-8 production via EP4 receptor in human pulmonary microvascular endothelial cells

Prostaglandin E(2) (PGE(2)) is a bioactive prostanoid implicated in the inflammatory processes of acute lung injury/acute respiratory distress syndrome. This study investigated whether PGE(2) can induce production of interleukin (IL)-8, the major chemokine for neutrophil activation, from human pulmonary microvascular endothelial cells (HPMVECs). PGE(2) significantly enhanced IL-8 protein production with increases in IL-8 mRNA expression and intracellular cAMP levels. HPMVECs expressed only EP4 receptor mRNA. The PGE(2) effects were mimicked by a selective EP4 receptor agonist, ONO-AE1-329, and inhibited by a selective EP4 receptor antagonist, ONO-AE3-208, or a protein kinase A inhibitor, Rp-adenosine 3',5'-cyclic monophosphorothioate triethylamine salt. The specific agonist for EP1, EP2, or EP3 receptor did not induce IL-8 production. PGE(2)-induced IL-8 production was accompanied by p38 phosphorylation and was significantly inhibited by a p38 inhibitor, SB-203580, but not by an ERK1/2 inhibitor, U-0126, or a JNK inhibitor, SP-600125. Additionally, PGE(2) increased cyclooxygenase-2 expression with no change in constitutive cyclooxygenase-1 expression, suggesting possible involvement of an autocrine or paracrine manner. In conclusion, PGE(2) enhances IL-8 production via EP4 receptor coupled to G(s) protein in HPMVECs. Activation of the cAMP/protein kinase A pathway, followed by p38 activation, is essential for these mechanisms. Because neutrophils play a critical role in the inflammation of acute lung injury/acute respiratory distress syndrome, IL-8 released from the pulmonary microvasculature in response to PGE(2) may contribute to pathophysiology of this disease.     

Prostaglandin E(2) inhibits IL-18-induced ICAM-1 and B7.2 expression through EP2/EP4 receptors in human peripheral blood mononuclear cells

Costimulatory molecules play important roles in immune responses. In the present study we investigated the effects of PGE(2) on the expression of ICAM-1, B7.1, and B7.2 on monocytes in IL-18-stimulated PBMC using FACS analysis. Addition of PGE(2) to PBMC inhibited ICAM-1 and B7.2 expression elicited by IL-18 in a concentration-dependent manner. We examined the involvement of four subtypes of PGE(2) receptors, EP1, EP2, EP3, and EP4, in the modulatory effect of PGE(2) on ICAM-1 and B7.2 expression elicited by IL-18, using subtype-specific agonists. ONO-AE1-259-01 (EP2R agonist) inhibited IL-18-elicited ICAM-1 and B7.2 expression in a concentration-dependent manner with a potency slightly less than that of PGE(2), while ONO-AE1-329 (EP4R agonist) was much less potent than PGE(2). The EP2/EP4R agonist 11-deoxy-PGE(1) mimicked the effect of PGE(2) with the same potency. ONO-D1-004 (EP1R agonist) and ONO-AE-248 (EP3R agonist) showed no effect on IL-18-elicited ICAM-1 or B7.2 expression. These results indicated that EP2 and EP4Rs were involved in the action of PGE(2). Dibutyryl cAMP and forskolin down-regulated ICAM-1 and B7.2 expression in IL-18-stimulated monocytes. As EP2 and EP4Rs are coupled to adenylate cyclase, we suggest that PGE(2) down-regulates IL-18-induced ICAM-1 and B7.2 expression in monocytes via EP2 and EP4Rs by cAMP-dependent signaling pathways. The fact that anti-B7.2 as well as anti-ICAM-1 Ab inhibited IL-18-induced cytokine production implies that PGE(2) may modulate the immune response through regulation of the expression of particular adhesion molecules on monocytes via EP2 and EP4Rs.     

Molecular cloning and functional characterization of the canine prostaglandin E2 receptor EP4 subtype.

Prostaglandin E2 (PGE2) is an important mediator of diverse biologic functions in many tissues and binds with high affinity to four cell surface, seven-transmembrane domain, G protein-coupled receptors (EP1-EP4). The EP4 receptor subtype has a long intracellular carboxy-terminal region and is functionally coupled to adenylate cyclase, resulting in elevated intracellular cyclic adenosine 5' monophosphate (cAMP) levels upon activation. To further study EP4 receptor subtype function, a canine kidney cDNA library was screened and three clones were isolated and sequenced. The longest clone was 3,103 bp and contained a single open reading frame of 1,476 bp, potentially encoding a protein of 492 amino acids with a predicted molecular weight of 53.4 kDa. Sequence analysis of this open reading frame reveals 89% identity to the human EP4 protein coding region at the nucleotide level and 90% identity when the putative canine and human protein sequences are compared. Northern blot analysis showed relatively high levels of canine EP4 expression in heart, lung and kidney, while Southern blot analysis of canine genomic DNA suggests the presence of a single copy gene. Following transfection of canine EP4 into CHO-KI cells, Scatchard analysis revealed a dissociation constant of 24 nM for PGE, while competition binding studies using 3H-PGE2 as ligand demonstrated specific displacement by PGE2 prostaglandin E, (PGE1), and prostaglandin A3 (PGA3). Treatment with PGE2 also resulted in increased levels of cAMP in transfected, but not in parental, CHO-KI cells. In contrast, butaprost, an EP2 selective ligand, and sulprostone, an EP1/EP3 selective ligand, did not bind to this receptor at the maximal concentration used (320 nM). To further investigate secondary signaling, the canine EP4 cDNA was truncated to produce an 1,117 bp fragment encoding a 356 amino acid protein lacking the intracellular carboxy-terminus. When transfected, this truncated cDNA produced a protein with a dissociation constant of 11 nM for PGE2 and a binding and cAMP accumulation profile similar to that of the full-length protein. Both full-length and truncated canine EP4 underwent short term PGE2-induced desensitization as shown by a lack of continuing cAMP accumulation after the initial PGE2 stimulation, suggesting no involvement of the C-terminal intracellular tail. This result is in contrast to that reported for the human EP4 receptor, where residues within the C-terminal intracellular tail were shown to mediate short term, ligand induced desensitization.     

Role of prostaglandin E(2) EP receptors and cAMP in the expression of connective tissue growth factor

Wild-type (WT) Rat-1 fibroblasts express undetectable quantities of the prostaglandin E(2) (PGE(2)) EP1, EP2, and EP3 receptor types and detectable amounts of the EP4 receptor. In the WT Rat-1, PGE(2) enhances connective tissue growth factor (CTGF) mRNA. PGE(2) does not stimulate cAMP production in these cells. However, forskolin induces cAMP production and ablates TGF beta-stimulated increases in CTGF mRNA. A similar pattern of CTGF expression in response to PGE(2) and forskolin is observed in neonatal rat primary smooth muscle cell cultures. When WT Rat-1 cells are stably transfected with the EP2 receptor, PGE(2) causes a sizable elevation in intracellular cAMP and ablates the TGF beta-stimulated increase in CTGF mRNA. PGE(2) does not have this effect on cells expressing the EP1, EP3, or EP4 receptor subtypes. These results demonstrate the importance of the EP2 receptor and cAMP in the inhibition of TGF beta-stimulated CTGF production and suggest a role for PGE(2) in increasing CTGF mRNA levels in the absence of the EP2 receptor. Involvement of inositol phosphate in this upregulation of CTGF expression by PGE(2) is doubtful. None of the cell lines containing the four EP transfectants nor the WT Rat-1 cells responded to PGE(2) with inositol phosphate production.     

Regulation of vascular endothelial cell growth factor expression in mouse mammary tumor cells by the EP2 subtype of the prostaglandin E2 receptor

Prostaglandin E(2) (PGE(2)), a major metabolite of the cyclooxygenase pathway in the mammary gland, induces angiogenesis during mammary tumor progression. To better define the molecular mechanisms involved, we examined the role of the G protein-coupled receptors (GPCR) for PGE(2) in mammary tumor cell lines isolated from MMTV-cyclooxygenase-2 (COX-2) transgenic mice. Expression of the EP2 subtype of the PGE(2) receptor was correlated with the tumorigenic phenotype and the ability to induce vascular endothelial growth factor (VEGF). Overexpression of EP2 by adenoviral transduction into EP2-null cells resulted in the induction of VEGF expression in response to PGE(2) and CAY10399, an EP2 receptor agonist. The induction of VEGF by the EP2 receptor did not require the hypoxia inducible factor (HIF)-1alpha pathway, MAP kinase pathway, or phosphoinositide-3-kinase/Akt pathway, but required the cAMP/protein kinase A pathway. These results suggest that EP2 receptor is a critical element for PGE(2) mediated VEGF induction in mouse mammary tumor cells.     

Prostaglandin E2 induces degranulation-independent production of vascular endothelial growth factor by human mast cells

Mast cells accumulate in large numbers at angiogenic sites, where they have been shown to express a number of proangiogenic factors, including vascular endothelial growth factor (VEGF-A). PGE(2) is known to strongly promote angiogenesis and is found in increased levels at sites of chronic inflammation and around solid tumors. The expression pattern of VEGF and the regulation of VEGF-A by PGE(2) were examined in cord blood-derived human mast cells (CBMC). CBMC expressed mRNA for five isoforms of VEGF-A and other members of the VEGF family (VEGF-B, VEGF-C, and VEGF-D) with strong expression of the most potent secretory isoforms. PGE(2) was a very strong inducer of VEGF-A(121/165) production by CBMC and also elevated VEGF-A mRNA expression. The amount of VEGF-A(121/165) protein production induced by PGE(2) was 4-fold greater than that induced by IgE-mediated activation of CBMC. Moreover, the response to PGE(2) as well as to other cAMP-elevating agents such as forskolin and salbutamol was observed under conditions that were not associated with mast cell degranulation. CBMC expressed substantial levels of the EP(2) receptor, but not the EP(4) receptor, when examined by flow cytometry. In contrast to other reported PGE(2)-mediated effects on mast cells, VEGF-A(121/165) production occurred via activation of the EP(2) receptor. These data suggest a role for human mast cells as a potent source of VEGF(121/165) in the absence of degranulation, and may provide new opportunities to regulate angiogenesis at mast cell-rich sites.     

Prostaglandin E2-EP4 receptor promotes endothelial cell migration via ERK activation and angiogenesis in vivo

Prostaglandin E2 (PGE(2)), a major product of cyclooxygenase, exerts its functions by binding to four G protein-coupled receptors (EP1-4) and has been implicated in modulating angiogenesis. The present study examined the role of the EP4 receptor in regulating endothelial cell proliferation, migration, and tubulogenesis. Primary pulmonary microvascular endothelial cells were isolated from EP4(flox/flox) mice and were rendered null for the EP4 receptor with adenoCre virus. Whereas treatment with PGE(2) or the EP4 selective agonists PGE(1)-OH and ONO-AE1-329 induced migration, tubulogenesis, ERK activation and cAMP production in control adenovirus-transduced endothelial EP4(flox/flox) cells, no effects were seen in adenoCre-transduced EP4(flox/flox) cells. The EP4 agonist-induced endothelial cell migration was inhibited by ERK, but not PKA inhibitors, defining a functional link between PGE(2)-induced endothelial cell migration and EP4-mediated ERK signaling. Finally, PGE(2), as well as PGE(1)-OH and ONO-AE1-329, also promoted angiogenesis in an in vivo sponge assay providing evidence that the EP4 receptor mediates de novo vascularization in vivo.     

Prostaglandin E2 stimulates cystogenesis through EP4 receptor in IMCD-3 cells

Previously, we demonstrated that prostaglandin E(2) (PGE(2)) induced cAMP and cyst formation through PGE(2) receptor-2 (EP2) activity in human autosomal-dominant polycystic kidney disease (ADPKD) epithelial cells. In this study, we determined the role of EP2 and EP4 receptors in mediating PGE(2) stimulation of cAMP signaling and cystogenesis in mouse renal epithelial cells using the inner medullary collecting duct-3 (IMCD-3) cell line. In contrast to human ADPKD cells, using novel EP2 and EP4 antagonists, we found that IMCD-3 cells expressed functional EP4 but not EP2, which stimulated cAMP formation and led to cyst formation in 3D culture system. The involvement of EP4 receptors in IMCD-3 cells was further supported by the specific effect of EP4 siRNA that inhibited PGE(2)-induced cystogenesis. We also observed different cellular localization of EP2 or EP4 receptors in IMCD-3 transfected cells. Collectively, our results suggest an important role of different expression of EP2 or EP4 receptors in the regulation of cystogenesis.     

Prostaglandin E2 aggravates gastric mucosal injury induced by histamine in rats through EP1 receptors

We demonstrated that prostaglandin (PG) E2 aggravates gastric mucosal injury caused by histamine in rats, and investigated using various EP agonists which EP receptor subtype is involved in this phenomenon. Rats were used after 18 hr fasting. Histamine (80 mg/kg) dissolved in 10% gelatin, was given s.c., either alone or in combination with i.v. administration of PGE2 or various EP agonists such as 17-phenyl PGE2 (EP1), butaprost (EP2), sulprostone (EP1/EP3), ONO-NT012 (EP3) and ONO-AE1-329 (EP4). The animals were killed 4 hr later, and the mucosa was examined for lesions. The mucosal permeability was determined using Evans blue (1%). Histamine alone induced few lesions in the gastric mucosa within 4 hr. PGE2 dose-dependently worsened the lesions induced by histamine, the response being inhibited by tripelennamine but not cimetidine. The effect of PGE2 was mimicked by 17-phenyl PGE2 and sulprostone, but not other EP agonists, including EP2, EP3, and EP3/EP4 agonists. The mucosal vascular permeability was slightly increased by histamine, and this response was markedly enhanced by co-administration of 17-phenyl PGE2 as well as PGE2. The mucosal ulcerogenic and vascular permeability responses induced by histamine plus PGE2 were both suppressed by pretreatment with ONO-AE829, the EP1 antagonist. These results suggest that PGE2 aggravates histamine-induced gastric mucosal injury in rats. This action of PGE2 is mediated by EP1 receptors and functionally associated with potentiation of the increased vascular permeability caused by histamine through stimulation of H1-receptors.     

Activation of prostaglandin E receptor EP4 subtype suppresses food intake in mice

Prostaglandin (PG) E2, a bioactive lipid produced in the brains of various mammals, decreases food intake after central administration. We examined which of four distinct subtypes of PGE2 receptors (EP1-EP4) mediated the anorexigenic action of PGE2 using highly selective ligands. PGE2 at a dose of 0.1-10 nmol/mouse decreased food intake after intracerebroventricular (i.c.v.) administration in a dose-dependent manner in fasted mice. A centrally administered EP4 agonist, ONO-AE1-329 at a dose of 1-10 nmol/mouse mimicked the anorexigenic action by PGE2. The anorexigenic action of PGE2 or EP4 agonist was ameliorated by EP4 antagonist ONO-AE3-208 at a dose of 10 nmol/mouse. Thus, activation of PGE2-EP4 signaling in the central nervous system suppresses food intake. The EP4 agonist at a dose of 10 nmol/mouse delayed gastric emptying and elevated blood glucose.