Abstract

1. Introduction

Prostaglandins (PGs) and thromboxane A₂ (TXA₂) are synthesized from arachidonic acid by cyclooxygenase (COX) and specific prostanoid synthases. Released in response to various physiological and pathological stimuli they play essential roles in maintaining body homeostasis. Prostaglandin E₂ (PGE₂) is involved in several biological processes such as pain, fever, regulation of vascular tone, renal function, mucosal integrity, inflammation, angiogenesis and tumor growth. PGE₂ often causes complex and divergent effects which can be attributed to its activation of four so-called E-type prostanoid receptors (EP1 to EP4). Studies conducted with EP receptor knock-out mice and the recent availability of highly selective pharmacological agonists and antagonists have allowed us to identify distinct — although sometimes overlapping — roles for EP receptor subtypes in PGE₂-regulated processes. In this review, we summarize the physiological roles of EP4, its molecular structure and intracellular signaling pathways, and how EP4 receptors might be targeted pharmacologically for the benefit of human disease, including vascular and renal diseases, inflammation, osteoporosis and cancer. Tables 1–3 provide an overview of currently described selective and non-selective agonists, and antagonists for EP4, respectively, and their affinity, dosing and biological effects.

2. Prostaglandin E₂ and its four receptors

Prostanoids are derived from arachidonic acid, a 20 carbon polyunsaturated fatty acid, which is usually found in phosphoglycerides of mammalian cell membranes (Fig. 1). Arachidonic acid is released by phospholipase A₂ from the cell membrane and is converted to PGG₂ and then reduced to PGH₂ by COX-1 or COX-2 (Smith & Dewitt, 1996; Park & Christman, 2006). In general COX-1 is constitutively active whose expression appears to be regulated developmentally, while COX-2 is usually absent from cells but its expression can be induced in response to different stimuli. However, COX-2 is also constitutively expressed in some tissues, such as the endothelium, kidney, gastrointestinal mucosa and brain (O'Banion, 1999; Grosser et al., 2006; Zidar et al., 2009). The endoperoxide intermediate PGH₂ is converted to the different prostanoids (PGE₂, PGI₂, TXA₂, PGD₂, PGF_2α) by specific synthases. In general, the expression pattern of the prostanoid synthases is specific for different cells and determines which prostanoid they will be producing in abundance. PGE₂ is generated at large amounts in fibroblasts, monocytes, and epithelial and endothelial cells by three enzyme isoforms, i.e. inducible microsomal PGE synthase-1, and constitutively expressed microsomal PGE synthase-2 and cytosolic PGE synthase (Fig. 1) (Kudo & Murakami, 2005).

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Fig. 1

PGE₂ biosynthesis and receptors. Arachidonic acid is liberated from membrane phospholipids by phospholipase A₂ enzyme activity. Arachidonic acid is converted to the endoperoxide PGG₂ and further reduced to PGH₂ by the action of cyclooxygenase 1 and 2 enzymes. PGE₂ is formed from PGH₂ by PGE synthases and binds to and activates four EP receptor subtypes, designated EP1 to EP4 receptors. These receptors are coupled to different G proteins leading to subsequent activation of specific signal transduction pathways. Besides PGE₂, other prostanoids are also formed from PGH₂, i.e. PGI₂, TXA₂, PGD₂ and PGF_2α. Additionally, PGE₂ can also be converted to PGA₂.

The various biological effects of PGE₂ are mediated by four EP receptors, which show differential patterns of tissue distribution. EP1 mRNA is ubiquitously expressed in murine tissues, while EP3 receptor mRNA levels are high in adipose tissues, pancreas, kidney and vena cava. EP4 mRNA is mainly expressed in the gastrointestinal tract, uterus, hematopietic tissues and skin, whereas EP2 receptor mRNA was found to be least abundant among EP receptors, with the highest expression occurring in the airways, ovary, bone marrow and olfactory epithelium (Regard et al., 2008). The EP receptors are coupled to different G proteins and accordingly activate diverse signaling pathways (Fig. 1) (Alfranca et al., 2006). EP1 signals predominantly via G_q, thereby elevating intracellular Ca²⁺ levels through the action of phospholipase Cβ followed by activation of protein kinase (PK) C. EP2 and EP4 are described as G_s coupled receptors, which activate adenylate cyclase and induce intracellular cyclic adenylyl monophosphate (cAMP) production, while EP3 is coupled to G_i and inhibits cAMP production (Alfranca et al., 2006). The human EP1 receptor is comprised of 402 amino acids, the EP2 from 358, the EP3 from 390 while the EP4 receptor is composed of 488 amino acids, additionally there are two alternative splice variants of the EP1 and eight variants of the EP3 (Regan, 2003). A similar tendency can be observed in murine EP1, EP2, EP3 and EP4 receptors, which consist of 405, 362, 366 and 513 amino acids, respectively (Negishi et al., 1993; Sugimoto & Narumiya, 2007).

3. E-type prostanoid receptor 4 structure, signaling and ligands

3.1. Structure

When first cloned in 1993 as a PGE₂ receptor that stimulated cAMP formation, the EP4 receptor was designated as “EP2” (Honda et al., 1993). After another cAMP-stimulating PGE₂ receptor had been discovered which was sensitive to butaprost (Regan et al., 1994), the butaprost-insensitive receptor which mediated vasorelaxation in piglet saphenous vein was renamed “EP4” (Coleman et al., 1994; Nishigaki et al., 1995; Breyer et al., 1996).

The EP4 gene contains three exons of which the first is non-coding. The initiation site was found to lack a TATA box, but it is GC-rich, and contains two CCAAT boxes and SP1-, AP2- and NF-κB-binding motifs (Foord et al., 1996). Across EP receptors in humans and mice, most amino acid conservation can be found in the transmembrane helices which form the binding pocket for the natural ligand PGE₂ (Fig. 2) (Stillman et al., 1998). Amino acid homology among the EP receptor subtypes is small, between single EP receptors not more than 30%. Even EP2 and EP4 receptors, which are both coupled to G_αs proteins and activate adenylate cyclase, share only 38% homology in humans and 31% in mice (Regan, 2003; Sugimoto & Narumiya, 2007). Regarding the interaction of PGE₂ with the EP4 receptor, a single threonine residue, T168, in the second extracellular loop was identified as being essential (Fig. 2) (Stillman et al., 1998). Furthermore, the very recent 3D modeling of the human EP4 receptor and in silico docking experiments for PGE₂ revealed the most likely binding sites of PGE₂ on the 3D structure of EP4. These pertinent amino acid residues are S103, T168, Y186, F191, L195, S285, and D311 (Fig. 2) (Margan et al., 2012). Radioligand binding studies suggested that the affinity of PGE₂ for EP4 receptors is higher (EC₅₀: 2.8 nM) than that for EP2 (EC₅₀: 19 nM) (Fujino et al., 2002).

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Fig. 2

The structure of the EP4 receptor and its activation of signaling modules. The 488 amino acid sequence of the human EP4 receptor is displayed with color code indicating the single residues conserved in all EPs, the binding sites for the native ligand PGE₂, regions responsible for desensitization and for interaction with EPRAP. Binding of an agonist induces G_αs-dependent activation of adenylate cyclase (AC), formation of cAMP and either activation of Epac (exchange factor activated by cAMP), activation of the PKA-independent AMP-activated protein kinase (AMPK) or activation of PKA and eNOS, or alternatively the transcription factor CREB (cAMP-responsive element-binding protein). Additionally, cAMP can act through inducible cAMP early repressor (ICER). EP4 receptor activation has been found to reduce retinoic acid secretion by ICER-mediated blockade of the expression of retinoic acid dehydrogenase These signals finally mediate vascular relaxation, angiogenesis and protection against cerebral ischemic injury. EP4 receptor becomes rapidly desensitized upon binding of G protein-coupled receptor kinases (GRK) to serine residues on the C-terminus. These residues are phosphorylated and attract β-arrestin initiating receptor internalization, and c-Src which leads to transactivation of EGFR and further activation of PI3K/ERK/Akt kinases. Alternatively, Pertussis toxin-sensitive G_αi can be activated which also may induce the activation of PI3K/ERK pathway. These latter signaling pathways enable migration and metastasis of colorectal carcinoma and inhibit the activation of eosinophils in allergic inflammation. The extended C-terminus of EP4 receptor allows interaction with EPRAP which in turn stabilizes the p105 subunit that prevents the activation of NF-κB and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase kinase 1/2 (MEK/ERK1/2), and inhibits transcription of pro-inflammatory cytokines in activated macrophages.

4. Immune modulation by E-type prostanoid receptor 4

COX inhibitors, also referred to as non-steroidal anti-inflammatory drugs (NSAIDs), are widely used for symptomatic treatment of inflammatory diseases, particularly due to their potent analgesic effect. However, their clinical use is considerably limited as they give rise to gastrointestinal, renal and cardiovascular complications, among others. Selective COX-2 inhibitors that had been developed to minimize adverse effects by sparing the homeostatic functions of COX-1 did not meet expectations due to increased cardiovascular risk (Grosser et al., 2010). This Janus head nature of prostaglandins is consistent with the numerous pro- and anti-inflammatory effects that PGE₂ can exert, which might be up to local concentrations of PGE₂ and expression patterns of its receptors. The anti-inflammatory actions of PGE₂ have been mainly ascribed to the EP4 receptor (Tang et al., 2012), although the EP2 receptor may play similar, redundant roles in many cellular and in vivo models. Nevertheless, the EP4 receptor might also act to promote inflammation, particularly in Th17-dependent diseases (Sheibanie et al., 2007; Sakata et al., 2010), and is widely considered as an important pro-tumorigenic molecule (Chell et al., 2006).

The EP4 receptor is expressed on most immune cell types, and functional responses mediated by EP4 receptors have been reported in B- and T-lymphocytes, natural killer cells, dendritic cells, eosinophils, monocytes and macrophages (Norel, 2007; Kalinski, 2012). PGE₂ and EP4 receptors are indispensable for monocyte-derived dendritic cells and Langerhans cells to attain a migratory phenotype (Luft et al., 2002; Kabashima et al., 2003), which is a prerequisite for them to evade into lymph nodes and subsequently present antigens there. EP2/EP4-mediated expression of matrix metalloprotease-9 in dendritic cells appears to be crucial for their migration (Yen et al., 2011). Concomitantly, cytokine release from these cells is inhibited while survival is enhanced by the EP4 receptor (Luft et al., 2002; Jing et al., 2003; Baratelli et al., 2005).

Recently, PGE₂ was recognized to positively modulate the differentiation and activity of Th17 cells. EP4 receptor stimulation accentuates LPS-induced IL-23 secretion from immature dendritic cells and supernatants from these cells enhance IL-17 production in activated T cells (Sheibanie et al., 2007). Naïve T cells upregulate IL-23 and IL-1 receptors in response to EP2/EP4 signaling and PGE₂ synergizes with these cytokines in promoting the typical Th2 phenotype, whereby EP4 receptor stimulation particularly leads to IL-10 downregulation (Boniface et al., 2009; Napolitani et al., 2009). In vivo, the EP4 antagonist ONO-AE3-208 ameliorated experimental autoimmune encephalomyelitis or contact hypersensitivity, and reduced IL-17 production in regional lymph nodes (Yao et al., 2009). In contrast, human plasmacytoid dendritic cells produce less interferon-α following EP2/EP4 stimulation by PGE₂ and lose their Th1 co-stimulatory capacity (Fabricius et al., 2010).

Dendritic cells that mature in the presence of PGE₂ preferably drive Th2 polarization, and produce Th2-attracting chemokines, which might argue for a crucial role of EP4 receptors in allergic sensitization (Kubo et al., 2004; McIlroy et al., 2006). This notion is further supported by the observation that PGE₂ promotes the differentiation of B-cells and the class-switch to IgE through EP2 and EP4 receptors (Fedyk & Phipps, 1996). In contrast, PGE₂ suppresses antigen-specific responses, such as proliferation and cytokine secretion of human Th1- and Th2 cells through EP2/EP4 signals (Okano et al., 2006), suggesting that PGE₂ might limit allergic responses upon allergen re-exposure. PGE₂ might also take part in systemic immune suppression following UV irradiation of the skin, an effect that was due to accumulation of self-tolerant, DEC205-expressing, dendritic cells and CD4⁺/Foxp3⁺ regulatory T cells in peripheral lymph nodes, but was not observed in mice treated with an EP4 antagonist (Soontrapa et al., 2011).

5. Airways

In the pulmonary environment, PGE₂ attenuates inflammatory responses and reduces tissue injury of the airways (Vancheri et al., 2004). Additionally, COX-1 and -2 deficient mice show increased allergic responses in the airways (Gavett et al., 1999; Nakata et al., 2005). Consequently, selective COX-1 and -2 inhibitors enhanced allergic inflammation and airway hyperresponsiveness (Peebles et al., 2002). PGE₂ was also shown to play a bronchoprotective role in patients with asthma (Pavord et al., 1993; Melillo et al., 1994; Aggarwal et al., 2010). Similarly, intratracheal administration of PGE₂ diminished the ovalbumin-induced early and late phase airway responses in rats (Martin et al., 2002). Isolated tracheal and bronchial preparations from mice, rats, guinea pigs, monkeys and humans were all relaxed by PGE₂, and this effect was mediated by the EP4 receptor only in human and rat airways, but by the EP2 receptor in the other species (Buckley et al., 2011; Benyahia et al., 2012). These important observations — EP4 receptor-mediated inhibitory effect on eosinophil effector functions and direct bronchodilator action — might contribute to the development of next generation bronchodilators as an alternative to the widely used β₂ adrenoceptor agonists, which fail to treat the underlying tissue inflammation.

Consistent with its bronchoprotective action, PGE₂ inhibited bronchial smooth muscle cell proliferation and migration through EP4 (Sastre et al., 2011; Aso et al., 2013). Moreover, EP4 receptor activation caused apoptosis in human adult lung fibroblasts by reducing the expression of survivin, an inhibitor of apoptosis and attenuating Akt activity (Huang et al., 2009), and partially mediated the inhibitory effect of PGE₂ on the migration of human fetal fibroblasts in a cAMP/PKA/PI3K-dependent manner (Y.J. Li et al., 2011). Therefore, EP4 agonists might be a useful therapeutic intervention for airway inflammatory diseases such as asthma and chronic bronchitis due to their bronchoprotective, antifibrotic and anti-inflammatory effects. In contrast, blockade of EP4 receptors reversed the senescence of lung fibroblasts from COPD patients, suggesting a potential usefulness of EP4 receptor antagonists in COPD (Dagouassat et al., 2013).

6. Gastrointestinal tract

It is long known that NSAIDs contribute to worsening of gastrointestinal mucosal damage and inflammation in humans, and are a major risk factor for ulcer disease and gastrointestinal hemorrhage (Kaufmann & Taubin, 1987; Bjarnason et al., 1993; Felder et al., 2000; Laine et al., 2008). Experimental gastric damage as induced by the COX inhibitor indomethacin or by a mixture of ethanol plus HCl in mice and rats are ameliorated by PGE₂ and an EP1 agonist (Suzuki et al., 2001; Takeuchi et al., 2001), while duodenal and intestinal lesions are prevented by EP3 and EP4 agonists (Kunikata et al., 2001; Kunikata et al., 2002). Part of the EP4 receptor-mediated intestinal mucosal protection might be due to stimulation of duodenal bicarbonate and mucus secretion (Takeuchi et al., 1997; Araki et al., 2000; Aihara et al., 2007), its anti-apoptotic effect on epithelial cells (Hoshino et al., 2003), vasodilation (Hattori et al., 2008) and vascular endothelial growth factor (VEGF) release to promote angiogenesis and mucosal healing (Hatazawa et al., 2007).

Consistent with the anti-inflammatory action of prostaglandins in the gut, COX-1 and -2 deficient mice show augmented colitis induced experimentally by oral administration of dextran sodium sulfate (DSS) (Morteau et al., 2000). The involvement of EP receptors was addressed by inducing colitis with 3% DSS in mice deficient for DP, EP1, EP2, EP3, FP, IP or TP receptor (Kabashima et al., 2002). Only EP4 receptor deficient mice developed severe colitis, similar to mice treated with the EP4-selective antagonist ONO AE3-208. Switching off the EP4 receptor abolished the mucosal barrier function and led to edema formation, damage of the epithelial layer and infiltration of immune cells, e.g. neutrophils and CD4⁺ T cells in the colon. Two different EP4-selective agonists, ONO AE1-734 and AGN205203, reversed the pathology of DSS colitis in wild-type mice, an effect that was strongly suppressed by an EP4 antagonist. On the cellular level, EP4 receptor stimulation protected against colon epithelial apoptosis, prevented goblet cell depletion, and promoted the regeneration of the epithelial layer. A substantial observation was the upregulation of EP4 mRNA following DSS-induced colitis in mice and rats (Kabashima et al., 2002; Nitta et al., 2002; Jiang et al., 2007).

These results were at variance with a study that addressed the role of EP4 receptors in a different, more severe, model of colitis induced by trinitrobenzene sulfonic acid. EP4 receptor stimulation by PGE₁-OH or misoprostol, a non-selective EP receptor agonist, aggravated colitis and reduced survival rate, which was attributed to EP4 receptor-mediated IL-23 secretion from dendritic cells leading to accumulation of IL-17-producing cells in the colon (Sheibanie et al., 2007). These seemingly contradicting findings of EP4 receptors being beneficial in one colitis model, but deleterious in the other more severe model, might be due to the inhibitory role of EP4 receptors in fibroblast migration, thereby hampering wound healing and tissue repair (Rieder et al., 2010).

A clinical pilot study recently addressed the protective effect of the EP4 receptor agonist ONO-4819CD, a prodrug, in a small group of patients (four patients in the active treatment group, three in the placebo group) suffering from mild-to-moderate ulcerative colitis (Nakase et al., 2010). A tendency towards improvement of clinical symptoms as well as histological scores was noted in EP4 agonist-treated patients.

Disruption of the intestinal mucosal barrier is characteristic in several gastrointestinal diseases including ischemia–reperfusion, inflammatory bowel disease and NSAID-induced gastropathy (DeMeo et al., 2002). PGE₂ was shown to induce gastrointestinal anion secretion as a mechanism of mucosal barrier protection (Bunce & Spraggs, 1990). PGE₂ stimulated chloride secretion in ischemia-injured porcine ileal mucosa, which was followed by significant increase in transepithelial electrical resistance referring to recovery of epithelial barrier function (Moeser et al., 2004). The chloride channel ClC-2 was identified as the principal path for this response and showed co-expression with occludin in epithelial tight junctions (Moeser et al., 2004). Lubiprostone, a PGE₁ derivative, was also shown to enhance gastrointestinal mucosal barrier function and was characterized as a selective activator of ClC-2, acting very similar to PGE₂ (Moeser et al., 2007). Lubiprostone is currently approved for the treatment of chronic constipation in the U.S.A., Switzerland, United Kingdom and Japan. In rat and human gut preparations lubiprostone was found to interact with EP1 and EP4 receptors (Bassil et al., 2008) and to increase the expression of ClC-2 and cystic fibrosis transmembrane conductance regulator (CFTR). Moreover, lubiprostone induced contraction of villi and proximal colonic plicae and increased mucus exocytosis in goblet cells (Jakab et al., 2012). Similar to its gastrointestinal effects, lubiprostone was shown to stimulate tracheal submucosal gland secretion in pigs, sheep and humans without producing bronchoconstriction (Joo et al., 2009). An EP4-selective antagonist (L-161982) prevented the lubiprostone-induced chloride transport and submucosal gland secretion in sheep airways, and an EP4 agonist (L-902688) mimicked the effects of lubiprostone (Cuthbert, 2011). In a human airway epithelial cell line, EP4 receptor stimulation increased chloride efflux through CFTR (Joy & Cowley, 2008). Consequently, lubiprostone and other EP4 agonists could find therapeutic application in diseases with reduced airway fluid secretion.

7. Vasculature

The EP4 receptor has been first identified owing to its role in the PGE₂-induced relaxation of the piglet saphenous vein (Coleman et al., 1994). In this first report, the EP1, EP2 and EP3 receptors were ruled out as being responsible for the vasodilator effect of PGE₂ and, thus, the existence of a fourth EP receptor was proposed. Today, it is generally accepted that activation of EP1 and EP3 receptor subtypes induces vasoconstriction, whereas the EP2 and EP4 receptors expressed on smooth muscle cells mediate vasodilation (Norel, 2007). The EP4 receptor was further established as an important regulator of vascular tone in isolated perfused rat kidneys where the PGE₂-induced relaxation of renal afferent arterioles was EP4 receptor-dependent (Tang et al., 2000). Coupling to G_αs and the subsequent formation of cAMP determined the observed vasorelaxation. A different signaling pathway was proposed to mediate the vasorelaxing effect of EP4 receptors in aortic rings of mice where increased eNOS activity and intracellular cGMP accumulation were found (Hristovska et al., 2007). The PGE₂-induced relaxation was mimicked by an EP4 agonist (ONO AE1-329), and reversed by an EP4 receptor antagonist (ONO AE3-208). Consistent with these findings, relaxation was attenuated significantly in aortic rings from EP4⁻/⁻ mice but remained unaffected in EP2⁻/⁻ mice. Moreover, the in vivo hypotensive response to PGE₂ was attenuated in eNOS-deficient mice (Hristovska et al., 2007). EP4-selective agonists also induced relaxation of the human pulmonary vein, an effect that was prevented by blockade of EP4 receptors. In contrast, the pulmonary artery was not relaxed after EP4 receptor activation. Accordingly, smooth muscle cells of pulmonary vein express more EP4 mRNA than cells isolated from pulmonary artery (Foudi et al., 2008).

7.4. Endothelial function

PGE₂ along with PGI₂ has been shown to increase human pulmonary artery endothelial barrier function. Particularly, PGE₂ was shown to reduce dextran permeability of resting and thrombin-stimulated human pulmonary artery endothelial cells and to enhance endothelial electrical resistance via cAMP/PKA/Rap1/Rac signaling (Birukova et al., 2007). In a recent study we have identified the EP4 receptor as being responsible for the enhanced endothelial barrier function in response to PGE₂ (Konya et al., 2013). Using human pulmonary microvascular endothelial cells as a model system, we observed that only an EP4 receptor agonist (ONO AE1-329), but not EP1, EP2 and EP3 agonists mimicked the barrier enhancing effect of PGE₂ (Fig. 3). Similar responses were recorded in human coronary artery endothelial cells. The barrier enhancement was totally abolished by an EP4 antagonist (ONO AE3-208) and by preventing actin polymerization. On the cellular level PGE₂ and the EP4 agonist induced cortical actin polymerization and enhanced endothelial junctional expression of VE-cadherin (Fig. 3). Interestingly, the classical cAMP/PKA signaling module, eNOS or Rac1, PI3K or p38 kinases were not involved in this system. Additionally, EP4 receptor activation facilitated wound healing of pulmonary microvascular endothelial monolayers. Endothelial E-selectin upregulation, neutrophil adhesion and transmigration, induced by TNF-α pretreatment of endothelial cells, were also inhibited by endothelial EP4 receptor stimulation. The reduction of E-selectin expression depended on PI3K/PKC kinases. However, the exact mechanism of EP4 receptor-mediated endothelial barrier function awaits further investigation. Taken together, these observations indicate that EP4 agonists offer a potential novel approach to diseases with increased vascular permeability and neutrophil extravasation (Birukova et al., 2007; Konya et al., 2013).

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Fig. 3

The EP4 receptor promotes endothelial barrier function. EP4 receptor activation induces rapid cortical actin polymerization and VE-cadherin expression in the endothelial junctions, which results in markedly increased electrical resistance of the cell monolayer. Furthermore, E-selectin upregulation, and leukocyte adhesion and transendothelial migration as induced by TNF-α treatment is prevented by EP4 receptor activation. EP4 receptor-mediated E-selectin down-regulation is PI3K/PKC-dependent; however, none of the generally used EP4 receptor signaling modules (cAMP, eNOS, Rac1, PI3K, p38, ERK1/2) are involved in the barrier-enhancing effect of EP4 receptor. In contrast, the cAMP/PKA/p38 MAP kinase pathway mediates the EP4 receptor-stimulated release of IL-8 in endothelial cells.

By contrast, another recent report suggested that human pulmonary microvascular endothelial cells show enhanced release of the chemokine IL-8 upon stimulation with PGE₂ or ONO AE1-329 for 24 h (Aso et al., 2012). This pro-inflammatory action of EP4 receptors was mediated by cAMP/PKA and p38 MAP kinase but without involving ERK1/2 (Fig. 3). Whether the EP4 receptor-induced IL-8 release from endothelial cells actually results in neutrophil recruitment to the tissue has not yet been addressed, but similar EP4 receptor-mediated expression of proinflammatory cytokines was observed in airway and colon epithelial cell lines (Dey et al., 2009; T. Li et al., 2011). On a similar line, EP4 receptor activation in murine cerebrovascular endothelial cells upregulated intercellular adhesion molecule-1 (ICAM-1) expression (Park et al., 2013).

8. Kidney

PGE₂ plays diverse and important roles in the regulation of renal function (Hao & Breyer, 2008). Accordingly, the kidney is a major target of adverse effects of COX inhibitors particularly in the elderly, which include salt retention, hypertension, and blunted renal function (Harirforoosh & Jamali, 2009). Among the EP receptors, EP4 receptor (and to some extent EP2) is key to renal homeostasis by regulating cell proliferation, vascular tone and renin secretion. Cultured juxtaglomerular cells show pronounced renin secretion in response to PGE₂ and EP4 agonists (Friis et al., 2005). This response appears to be mediated by cAMP-dependent hyperpolarization and inhibition of Ca²⁺ currents that paradoxically inhibit renin-containing granule exocytosis. In vivo, low salt diet stimulates kidney renin mRNA levels and plasma renin concentrations in wild-type mice but not in EP4 knock-out mice or in mice treated with the EP4 antagonist ONO-AE3-208 (Poschke et al., 2012). Similar results were obtained in mice treated with furosemide (Nusing et al., 2005; Facemire et al., 2011). Vascular tone in the kidney also depends on EP4 receptors: the PGE₂-induced vasodilatation at low concentrations is abolished, while EP1-mediated vasoconstriction at higher concentrations of PGE₂ is augmented, in EP4-deficient mice (Purdy & Arendshorst, 2000; Schweda et al., 2004).

PGE₂ and EP receptors including EP4 play important roles in kidney development, as suggested by a reduced glomerular size in EP4-deficient mice (Frolich et al., 2012). In vitro, PGE₂ acting via EP4 receptors augmented the survival of glomerular epithelial cells deprived of serum (Aoudjit et al., 2006), and an EP4 receptor agonist stimulated proliferation and decreased apoptosis of renal epithelial cells (Yamamoto et al., 2010). PGE₂ and EP4 receptors have been suggested to be involved in autosomal dominant polycystic kidney disease, as they induce proliferation and chloride secretion in polycystin-1 deficient epithelial cells, while inhibiting proliferation in polycystin-1 expressing cells (Liu et al., 2012).

Several other pathologies of the kidney are counterbalanced by the PGE₂/EP4 axis, suggesting that EP4 agonists might be a useful therapeutic option in renal disease. In a mouse model of renal diabetes insipidus, EP4 receptor stimulation with ONO AE1-329 compensated the loss of vasopressin V2 receptors probably by activating aquaporin-2 in tubule epithelial cells (Li et al., 2009; Olesen et al., 2011). The same EP4 receptor agonist protected against anti-glomerular basement membrane antibody-associated nephritis in mice (Nagamatsu et al., 2006). In a murine model of ureteral obstruction, the development of renal fibrosis, the accumulation of macrophages, and the formation of proinflammatory and profibrotic cytokines were significantly augmented in the kidneys of EP4 knock-out mice and suppressed by an EP4 agonist, ONO-4819 (Nakagawa et al., 2012). In a mercury chloride-induced rat model of acute kidney failure, a systemically administered EP4 agonist (CP-044,519-02) reduced serum creatinine levels and increased the survival rate of the animals (Vukicevic et al., 2006). Similarly, treatment with the EP4 agonist delayed the progression of chronic renal failure as induced by 5/6 nephrectomy (Vukicevic et al., 2006).

In contrast, podocyte-specific overexpression of EP4 receptors significantly aggravated, while deletion of EP4 receptors in podocytes ameliorated, the decline of renal function in 5/6 nephrectomized mice (Stitt-Cavanagh et al., 2010). Interestingly, there is a positive feedback in PGE₂ secretion, as COX-2 expression is induced in podocytes following EP4 stimulation through a PKA-independent mechanism (Faour et al., 2008), and COX-2 and EP4 receptors are found upregulated in compromised or regenerating tubule epithelial cells (Yamamoto et al., 2010; Nakagawa et al., 2012). EP4 receptor expression in glomeruli is increased following salt deprivation as are PGE₂-evoked cAMP and renin responses in juxtaglomerular cells (Jensen et al., 1999). The EP4 receptor-induced stimulation of sodium absorption prompted by an activated renin/angiotensin II/aldosterone system is counterbalanced by the natriuretic action of EP1 receptors and diuretic action of EP3 receptors in the collecting duct (Fleming et al., 1998; Guan et al., 1998; Tamma et al., 2003).

9. Bone and cartilage

Osteoporosis is a bone degenerative disease of the elderly, induced by the imbalance of bone resorption by osteoclasts and bone formation by osteoblasts. Such loss of bone density increases the incidence of bone fractures. Among prostaglandins, PGE₂ shows the most prominent activity in bone, being involved both in bone formation and resorption (Norrdin et al., 1990). The dual role of prostaglandins in bone dynamics is also reflected by epidemiological data showing that chronic use of COX-2 inhibitors is associated with reduced bone mineral density in males, while the opposite effect is observed in postmenopausal women not taking hormone replacement therapy (Richards et al., 2006). In clinical practice, COX inhibitors are being used to control ectopic bone formation, but their use in the treatment of fracture pain has raised concern with respect to a potential delay of fracture healing (Vuolteenaho et al., 2008).

Bone resorption is carried out by osteoclasts, which originate from bone marrow-derived mononuclear cells. EP1 and EP4 receptors were found to be highly expressed in a mouse osteoblastic cell line, and stimulation of EP4 receptors by 11-deoxy-PGE₁, a purported EP4 receptor agonist, promoted osteoblast differentiation (Suda et al., 1996). PGE₂ was found to induce in vitro osteoclast formation from bone marrow cells in the presence of primary murine osteoblasts, which was mimicked by 11-deoxy-PGE₁. Additionally, osteoclast formation in response to PGE₂, but also to TNFα, IL-1β or LPS, was ablated in cells isolated from EP4 deficient mice (Sakuma et al., 2000). In agreement with these data, an EP4 antagonist was found to attenuate LPS-induced osteoclast formation and bone destruction in rat periodontal tissue (Oka et al., 2012).

In contrast, osteoclast differentiation from human peripheral blood monocytes is blocked by EP4 receptor activation (Take et al., 2005). At the same time, EP4 receptors also seem to play a role in the bone anabolic effect of PGE₂. COX-2 deficient mice presented with impaired healing of fractures, which was reversed by EP4 agonist treatment (Xie et al., 2009). EP4-deficient mice showed impaired bone formation in vivo in response to PGE₂, while other EP receptor-deficient mice exhibited unchanged callus formation (Yoshida et al., 2002). The effect of PGE₂ was mimicked by the EP4 agonist ONO AE1-329. Consistently, the EP4 agonist also induced the formation of mineralized nodules in bone marrow cultures from wild-type mice, but not EP4 deficient mice. Moreover, the same study reported that the EP4 agonist ONO 4819CD promoted de novo bone formation in experimental rat models of osteoporosis, induced by ovarectomy and immobilization, respectively (Yoshida et al., 2002). The protective effect of EP4 receptor stimulation against immobilization-induced bone loss is in line with the observation that mechanical strain applied to human osteoblasts down-regulates sclerostin, a potent inhibitor of bone formation, via COX-2 induction and EP4 receptor/ERK signaling (Galea et al., 2011).

Very recently, novel dual-action bone-targeting drug conjugates, consisting of EP4 receptor agonists and a bisphosphonate, alendronic acid, were reported (Arns et al., 2012). Radiolabeled conjugates of these drugs were administered to rats and were found to be taken up into the bone and released subsequently in a sustained manner, suggesting an attractive novel approach to modulating bone metabolism.

PGE₂ and EP4 receptors may play a similar, dual role in cartilage homeostasis and arthritis. In cartilage of osteoarthritis patients PGE₂ acting via EP4 receptors attenuated chondrocyte expression of connective tissue growth factor (Masuko et al., 2010), inhibited proteoglycan synthesis and promoted matrix degradation (Attur et al., 2008), while it also stimulated the release of osteoclast-activating factor RANKL (Moreno-Rubio et al., 2010).

Moreover, the EP4 receptor seems to be involved in inflammatory pain in experimental arthritis models and to promote Th1 differentiation, Th17 cell expansion, and IL-23 secretion by activated dendritic cells (Yao et al., 2009; Chen et al., 2010). Consequently, EP4 antagonists might have analgesic and anti-inflammatory effects in arthritis (Omote et al., 2002; Okumura et al., 2008; Colucci et al., 2010). Ultimately, EP4 knock-out mice were found to be resistant to type-II collagen antibody-induced arthritis (McCoy et al., 2002). In a positive feed-back loop, EP4 receptors are upregulated in arthritic tissue (Kurihara et al., 2001).

On the other hand, EP4 receptor activation in chondrocytes has been linked to PGE₂-induced inhibition of matrix metalloprotease expression (Fushimi et al., 2007; Nishitani et al., 2010). In an ex vivo model of human pannus formation, i.e. aberrant proliferation of synovial tissue leading to joint destruction, endogenous PGE₂ via activating its EP4 receptor, inhibited pannus growth and osteoclast activity (Shibata-Nozaki et al., 2011). These findings might be consistent with a crucial role of EP4 receptors in promoting acute inflammatory responses and pain in arthritis, but also with limiting cartilage and bone destruction in the chronic phase.

10. Cancer

The role of prostaglandins has extensively been studied in cancer, and COX-2 has emerged as a potential therapeutic target in some tumors. COX-2 expression has been found to correlate with cancer progression in experimental models and in many types of cancers, and pharmacological inhibition or genetic ablation of COX-2 reduces tumor cell proliferation and metastasis (Cebola & Peinado, 2012). Moreover, long-term administration of COX inhibitors such as acetylsalicylic acid can exert significant prophylactic effects against certain cancers (Harris, 2009). The significant adverse effects of COX inhibitors need to be considered, though, and might be avoided by selectively addressing the respective receptors for prostaglandins instead of non-selectively inhibiting prostaglandin production.

Revisiting the roles of different EP receptors in cancer development, a large body of evidence supports EP4 receptors to predominantly mediate the overall pro-tumorigenic action of PGE₂. The potential involvement of EP4 receptors has been described in many different types of cancer. Activation of EP4 may confer diverse cellular responses, such as promoting angiogenesis, proliferation, motility and metastasis, or delaying apoptosis, of tumor cells. These notions are based on studies using selective EP4 antagonists and agonists as well as mice that have been made deficient in EP4 receptors. In agreement with these findings, EP4 receptors are expressed on sprouting angiogenic cells, immune cells and the tumor cells itself, which are activated by PGE₂ in an autocrine or paracrine manner.

For instance, EP4 receptor expression was observed to increase during transition of colorectal adenoma to carcinoma (Chell et al., 2006) and EP4 receptor over-expression favored an anchorage-independent phenotype in otherwise anchorage-dependent human colorectal adenoma cells (Chell et al., 2006; Hawcroft et al., 2007). Consistently, the EP4 antagonist ONO AE3-208 attenuated colony forming capacity. PGE₂ stimulates migration of colon cancer cells via EP4/β-arrestin 1/c-Src signaling that transactivates and phosphorylates the epidermal growth factor receptor (Buchanan et al., 2006). EP4 receptor over-expression resulted in the suppression of apoptosis in a cAMP/CREB-dependent manner, and enhanced anchorage-independent growth, of a human colon cancer cell line (Hawcroft et al., 2007). In a different colon carcinoma cell line EP4 receptor was found to drive in vivo growth by augmenting tumor vascularization, and delaying apoptosis (Pozzi et al., 2004), while colon carcinogenesis was inhibited in mice lacking EP4 but not EP2 receptors, and by the EP4 antagonist ONO AE2-227 (Mutoh et al., 2002). Interestingly, over-expression of k-Ras, an oncogene involved in malignant transformation of many tumors, facilitated COX-2 and EP4 receptor expression in colon carcinoma cells (Wu et al., 2010).

EP4 mRNA levels were also found elevated in cervical carcinoma compared with normal cervix (Sales et al., 2001), and expression of COX-2 and EP4 receptors in transitional cell carcinoma of the upper urinary tract was associated with metastasis and shorter survival of patients (Miyata et al., 2005). COX-2 derived PGE₂ stimulated the migration and CCR7 expression of breast cancer cells via EP4 receptors linked to PKA and Akt signaling (Timoshenko et al., 2003; Pan et al., 2008). In a murine breast cancer model, EP4 receptors promoted lymphangiogenesis and ensuing metastasis (Xin et al., 2012). In breast cancer patients, expression of the EP4 receptor correlated with enhanced lymphatic invasion (Pan et al., 2008), and the EP4 antagonists, AH23848 and ONO AE3-208, attenuated lung metastasis of these tumor cells in mice, while ONO-AE2-227 prevented their osteolytic activity in vitro (Ohshiba et al., 2003; Ma et al., 2006). Similarly, EP4 receptor stimulation resulted in cAMP/EPAC-mediated activation of RAP small GTPase and augmented migration of renal carcinoma cells (Wu et al., 2011). Ovarian cancer cells responded to activation of PGE₂/EP4 receptor signaling with VEGF expression and tumor cell invasion, by activating tumor-associated matrix metalloproteinases (Spinella et al., 2004). EP4 receptors facilitated pulmonary metastasis of lung carcinoma cells injected intravenously in mice and liver metastasis after intrasplenic injection of colon cancer cells (Yang et al., 2006). Treatment of mice with ONO AE3-208 or siRNA knock-down of EP4 receptor in the tumor cells ameliorated tumor growth and metastasis.

In mouse skin, overexpression of EP4 receptors enhanced non-melanoma skin tumorigenesis (Rundhaug et al., 2011), and PGE₂ acting via EP4 receptors stimulated the migration of human melanoma cells (Singh et al., 2011). Consistently, the EP4 antagonist ONO AE3-208 prevented melanoma-induced bone metastasis and osteolysis in mice (Takita et al., 2007). Other types of cancer that might be sensitive to EP4 receptor stimulation include mycosis fungoides (Kopp et al., 2010), pancreatic adenocarcinoma (Funahashi et al., 2008), esophageal adenocarcinoma (Ogunwobi et al., 2006; Jimenez et al., 2010), endometrium cancer (Catalano et al., 2011), prostate cancer (Terada et al., 2010; Miao et al., 2012), neuroblastoma (Rasmuson et al., 2012), and glioblastoma (Kambe et al., 2009).

In addition to directly stimulating tumor cell proliferation and motility, EP4 receptor-mediated PGE₂ signals can down-regulate immune responses, thereby hampering tumor-limiting mechanisms. In detail, tumor-derived PGE₂ stimulating EP4 receptors curb natural killer cell and γδ T cell activity (Kundu et al., 2009; Martinet et al., 2010; Holt et al., 2011), and cytokine secretion from macrophages (Qian et al., 2011) and microglia (Nakano et al., 2008). Dendritic cell maturation is blocked and their differentiation is diverted towards a phenotype referred to as myeloid-derived suppressor cells (Obermajer et al., 2011a, 2011b). Importantly, in a positive feedback loop PGE₂ induced further COX-2 leading to exaggerated PGE₂ biosynthesis in immature myeloid cells (Ogunwobi et al., 2006; Obermajer et al., 2011a). Similarly, PGE₂/EP4 receptor-mediated induction of Foxp3/CD4/CD25⁺ T regulatory cells was shown to result in enhanced tumor burden in mice injected with lung cancer cells (Sharma et al., 2005). Conversely, PGE₂ supported the recruitment of tumor-promoting macrophages to mouse gastric tumors thereby enhancing tumor growth (Oshima et al., 2011).

Taken together, several studies have demonstrated that EP4 antagonists might have beneficial effects on carcinogenesis and tumor progression. Recent studies, however, suggest that the EP4 receptor might also act as tumor suppressor, for instance in human B cells (Murn et al., 2008). The EP4 receptor provides a negative feedback signal to proliferation in response to B cell receptor activation, suppressing growth in a PGE₂-dependent manner. B cell receptor activation upregulates EP4 receptors on immature B cells and promotes their apoptosis (Prijatelj et al., 2011). In B cell lymphomas, EP4 receptors are down-regulated and growth suppression is diminished (Murn et al., 2008). In another study, activation of EP4 receptors inhibited the growth of four different human gastric carcinoma cell lines (Okuyama et al., 2002).

11. Miscellaneous

In addition to what is described above, EP4 receptors may be useful therapeutic targets in various other organs, including eye, inner ear, brain or female reproductive tract. Topical application of the EP4 agonist prodrug PF-04475270 lowered ocular pressure through its active metabolite CP-734432 in normotensive dogs (Prasanna et al., 2009). However, conjunctival hyperemia and corneal neovascularization were also observed (Aguirre et al., 2009). EP4 knock-out mice exhibit a slight hearing deficit at baseline and are more susceptible to acoustic trauma as reflected by increased loss of outer hair cells. An EP4 antagonist recapitulated these effects while an EP4 agonist prevented hearing loss in wild-type mice (Hamaguchi et al., 2012). EP2 and EP4 agonists stimulated the production of VEGF in the spiral ganglion suggesting a beneficial effect in diseases like acute sensorineural hearing loss (Hori et al., 2010).

EP4 receptors are also involved in mediating various effects exerted by PGE₂ in female sexual organs. Blockade of EP4 receptors inhibited the proliferation and invasion of human endometriotic cells, and induces their apoptosis (Banu et al., 2009; Lee et al., 2011). PGE₂ stimulated follicle maturation in immature rat ovaries through upregulation of IL-8 and this was mimicked by an EP4 agonist (El-Nefiawy et al., 2005). An EP4 agonist also stimulated ovulation in mice (Xiao et al., 2008). In contrast, PGE₂ delayed nuclear maturation and fertilization of mouse and monkey oocytes, probably through EP2 and/or EP4 receptors (Duffy et al., 2010). Endogenous PGE₂ mediated LPS-induced cervical ripening in rabbits, which was reversed by an EP4 antagonist (Fukuda et al., 2007). An EP4 antagonist likewise prevented bladder hyperactivity induced by cyclophosphamide or PGE₂ in rats (Chuang et al., 2012).

Finally, EP4 receptors are capable of modulating central nervous activity. EP4 receptor activation in a murine model of Alzheimer's disease, EP4 deficiency or the EP4 antagonist ONO-AE3-208 decreased amyloid-β levels in the brain and improved the behavioral performance of the animals (Hoshino et al., 2012). By contrast, intracerebroventricular administration of PGE₂ or selective EP1 or EP4 agonists to mice caused anxiolytic-like effects in the elevated plus-maze and open field test, which was not observed in EP4-deficient animals, but could be reversed after blockade of serotonin 5-HT1A, dopamine D1 and GABA_A receptors (C. Suzuki et al., 2011). In a similar fashion, intracerebroventricular administration of PGE₂ and the EP4 agonist, ONO-AE1-329, suppressed food intake in mice, and this effect was prevented by EP4 antagonism. Accordingly, the EP4 agonist also delayed gastric emptying, but elevated blood glucose levels (Ohinata et al., 2006). EP4 receptors are expressed in enteroendocrine cells and stimulate the release of incretins, such as glucagon-like peptide-1, in vitro and in vivo in mice (Coskun et al., 2013). A potential role of EP4 receptors in metabolic disease is further supported by enhanced adipogenesis from mouse embryonic fibroblasts of EP4-deficient mice and by the inhibitory effect of PGE₂ acting via EP4 receptors and peroxisome proliferator-activated receptor-γ (Inazumi et al., 2011).

12. Conclusion

EP4 receptors are the most widely expressed PGE₂ receptors in the body, and research over the past ten years or so has unraveled that numerous novel, or well-recognized biological effects of PGE₂ can be attributed to EP4 receptor activation (Fig. 4). Among these are several anti-inflammatory roles, modulation of tissue development and regeneration, and regulation of vascular tone and hemostasis. In contrast to these EP4 receptor-mediated, assumingly beneficial actions of PGE₂, pathogenic roles of EP4 receptors have been implicated in cancer growth and metastasis, and in some inflammatory diseases. As such, both EP4 receptor agonists and antagonists might become useful novel drug classes. Accordingly, the pharmaceutical industry has launched numerous EP4 drug development programs, and a variety of EP4 receptor agonists and antagonists have become available for experimental studies and further clinical development. While systemic administration of EP4 receptor antagonists might be well tolerated, agonists are better administered locally, thereby avoiding systemic side effects. Moreover, in consideration of the sometimes redundant biological roles of EP2 and EP4 receptors, dual EP2/EP4 antagonists might be of advantage. Finally, ligand-biased selectivity of EP4 receptor signaling may exist for G_αs, G_αi and β-arrestin-dependent pathways and should be taken into consideration when interpreting biological effects of different EP4 ligands.

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Fig. 4

The effect of EP4 receptor activation in different diseases and cellular processes. Promotion of a respective response is shown by while indicates inhibitory modulation.

Conflict of interest

The authors declare to have no conflict of interest.

Acknowledgment

References