PGE Receptor Subtypes Research Articles

Prostaglandin E2 induced contraction of human intercostal arteries is mediated by the EP3 receptor

Arterial vascularization of the spinal cord may be mechanically or functionally altered during thoraco-abdominal surgery/intravascular procedures. Increased arterial pressure has been shown to restore spinal perfusion and function probably by increasing the blood flow through the intercostal arteries. The regulation of human intercostal artery (HICA) vascular tone is not well documented. Prostaglandin (PG)E2 concentration is increased during inflammatory conditions and has been shown to regulate vascular tone in many preparations. In this context, the pharmacological response of HICA to PGE2 and the characterization of the PGE2 receptor subtypes (EP1, EP2, EP3 or EP4) involved are of importance and that is the aim of this study. Rings of HICA were prepared from 29 patients and suspended in organ baths for isometric recording of tension. Cumulative concentration–response curves were performed in these preparations with various EP receptor agonists in the absence or presence of different receptor antagonists or inhibitors. PGE2 induced the contraction of HICA (Emax=7.28±0.16g; pEC50 value=0.79±0.18; n=17); contractions were also observed with the EP3 receptor agonists, sulprostone, 17-phenyl-PGE2, misoprostol or ONO-AE-248. In conclusion, PGE2 induced vasoconstriction of HICA via EP3 receptor subtypes and this result was confirmed by the use of selective EP receptor antagonists (L-826266, ONO-8713, SC-51322) and by a strong detection of EP3 mRNA. These observations suggest that in the context of perioperative inflammation, increased PGE2 concentrations could trigger vasoconstriction of HICA and possibly alter spinal vascularization.

PGE2 receptor (EP4) agonists: Potent dilators of human bronchi and future asthma therapy?

BackgroundAsthma and chronic obstructive pulmonary disease are characterized by inappropriate constriction of the airway smooth muscle. In this context, the physiological response of the human airways to selective relaxant agonists like PGE2 is highly relevant. The aim of this study was thus to characterize the PGE2 receptor subtypes (EP2 or EP4) involved in the relaxation of human bronchial preparations. MethodsHuman bronchial preparations cut as rings were mounted in organ baths for isometric recording of tension and a pharmacological study was performed using selective EP2 or EP4 ligands. ResultsIn the presence of a thromboxane TP receptor antagonist and indomethacin, PGE2 induced the relaxation of human bronchi (Emax = 86 ± 04% of papaverine response; pEC50 value = 7.06 ± 0.13; n = 6). This bronchodilation was significantly blocked by a selective EP4 receptor antagonist (GW627368X, 1 and 10 μmol/L) with a pKB value of 6.38 ± 0.19 (n = 5). In addition, the selective EP4 receptor agonists (ONO-AE1-329; L-902688), but not the selective EP2 receptor agonist (ONO-AE1-259), induced potent relaxation of bronchial preparations pre-contracted with histamine or anti-IgE. ConclusionPGE2 and EP4 agonists induced potent relaxations of human bronchial preparations via EP4 receptor. These observations suggest that EP4 receptor agonists could constitute therapeutic agents to treat the increased airway resistance in asthma.

C6orf176: a novel possible regulator of cAMP-mediated gene expression

cAMP mediates diverse cellular signals including prostaglandin (PG) E(2)-mediated intraocular pressure (IOP)-lowering activity in human ocular ciliary smooth muscle cells (hCSM). We have identified gene regulatory networks and key genes upon activation of the cAMP pathway in hCSM, using novel agonists highly selective for PGE(2) receptor subtypes EP2 or EP4, which are G protein-coupled receptors well known to activate cAMP signaling. Here we describe a novel, EP2/EP4-induced, primate-specific gene of hitherto unknown function, also known as C6orf176 (chromosome 6 open reading frame 176) and recently reclassified as noncoding RNA in NCBI's database. Its expression, as determined by quantitative real-time RT-PCR (qRT-PCR), is dramatically upregulated (>2,000-fold) subsequent to transduction of EP2/EP4/Gs/cAMP signaling not only in hCSM, but also in HEK cells overexpressing the recombinant receptors. Moreover, activation of other IOP lowering, Gs-coupled prostanoid receptors, such as DP1 and IP, as well as a direct activator of adenylyl cyclase, forskolin, also substantially upregulated C6orf176 in hCSM, while FP and TP, which are Gq-coupled prostanoid receptor subtypes, did not. Novel transcript variants carrying open reading frames, derived from an at least 67 kb genomic locus on chromosome 6q27 with putative alternative transcription start sites, were identified. Transcriptional upregulation of transcript variants as well as of two genes expressed in antisense orientation that partially overlap the transcribed C6orf176 region was observed, to varying degrees, subsequent to induction of cAMP signaling using various agonists. Small interfering RNA-mediated C6orf176 gene silencing experiments showed modulation of several cAMP-responsive genes. These transcriptional activities identify C6orf176 as a potential biomarker and/or therapeutic target in context with diseases linked to deregulated cAMP signaling. Also, the cAMP-inducible C6orf176 gene locus could be useful as a model system for studying transcriptional regulation by chromatin and RNA polymerase II.

Towards developing new strategies to reduce the adverse side-effects of nonsteroidal anti-inflammatory drugs

The antipyretic and analgesic actions of nonsteroidal anti-inflammatory drugs (NSAIDs) are caused by the inhibition of prostaglandin E(2) (PGE(2)), thromboxane A(2) and prostacyclin (PGI(2)) production. Accumulating evidence suggests that the inhibition of PGE(2) production can cause adverse side-effects of NSAIDs on fluid and blood pressure regulation, such as hypertension and edema formation. Since both cyclooxygenase (COX)-1 and COX-2 isoforms contribute to the production of PGE(2), selective COX-2 inhibitors are not free of these adverse side-effects although they may be less severe. Four subtypes of PGE(2) receptors have been identified. The antipyretic action of blunted PGE(2) production is mediated predominantly by a reduced input to the prostaglandin E receptor 3 (EP(3)) pathway, whereas the analgesic action is mediated predominantly by a reduced input to the EP(1) pathway and perhaps by contributions from the other EP receptors. Accordingly, some of the adverse side-effects might be moderated by combined use of NSAIDs with selective EP(2) or EP(4) agonists that do not block the antipyretic or analgesic actions of NSAIDs that are mediated by reduced activation of EP(1) or EP(3) receptors. Moreover, EP(2) receptor-deficient mice had salt-sensitive hypertension and EP(4) receptor blockade moderated salt and water excretion and both EP(2) and EP(4) agonists had renoprotective effects. This suggests that strategies to maintain activation of EP(2) and EP(4) receptors during NSAID administration may not only reduce adverse effects but might confer additional benefits. In conclusion, enhancing EP(2) and EP(4) receptor activity by administration of selective agonists during the administration of NSAIDs has the potential to permit treating fever, inflammation and pain but with marginal adverse effects on fluid or blood pressure regulation.

Differential reactivity of human mammary artery and saphenous vein to prostaglandin E2: Implication for cardiovascular grafts

Human internal mammary arteries (IMA) and saphenous veins (SV) are frequently used for coronary artery bypass graft surgery. Intra- and postoperatively, the bypass grafts are exposed to inflammatory conditions, under which there is a striking increase in the synthesis of prostaglandin E(2) (PGE(2) ). In this context, the physiological response of these vascular grafts to PGE(2) is highly relevant. The aim of this study was thus to characterize the PGE(2) receptor subtypes (EP(1) , EP(2) , EP(3) or EP(4) ) involved in modulation of the vascular tone in these two vessels. Rings of IMA and SV were prepared from 48 patients. The rings were mounted in organ baths for isometric recording of tension, and a pharmacological study was performed, together with associated reverse transcriptase PCR and immunohistochemistry experiments. PGE(2) induced contractions of IMA (E(max) = 1.43 ± 0.20 g; pEC(50) = 7.50 ± 0.10); contractions were also observed with the EP(3) receptor agonists, sulprostone, 17-phenyl-PGE(2) , misoprostol or ONO-AE-248. In contrast, PGE(2) induced relaxation of the precontracted SV (E(max) =-0.22 ± 0.02 g; pEC(50) = 7.14 ± 0.09), as did the EP(4) receptor agonist, ONO-AE1-329. These results were confirmed by the use of selective EP receptor antagonists (GW627368X, L-826266, ONO-8713, SC-51322) and by molecular biology and immunostaining. PGE(2) induced potent and opposite effects on the human vascular segments used for grafting, namely vasoconstriction of the IMA and vasodilatation of the SV via EP(3) and EP(4) receptors respectively. These observations suggest that EP(3) and EP(4) receptors could constitute therapeutic targets to increase vascular graft patency.

Discovery of an 8-Aza-5-thiaProstaglandin E1 Analog as a Highly Selective EP4 Receptor Agonist

For the purpose of discovering an orally available EP4 subtype-selective agonist, a series of 8-aza prostaglandin E(1) (PGE(1)) analogs were synthesized and evaluated for their affinity for PGE(2) receptor subtypes. Additionally, the structure-activity relationships of these compounds were studied. Among the tested compounds, the 8-aza PGE(1) analog 6 and 8-aza-5-thiaPGE(1) analog 12 had highly potent EP4 receptor affinity, good functional activity, and excellent subtype-selectivity. Furthermore, these analogs demonstrated good stability in human liver microsomes. As a result, we concluded that these two series of 8-aza PGE(1) analogs could be promising chemical leads for an orally available EP4 subtype-selective agonist.

A novel role of prostaglandin E2 in neuropathic pain

Neuropathic pain produced by damage to or dysfunction of the nervous system is a common and severely disabling state that affects millions of people worldwide. Recent evidence indicates that activated microglia are key cellular intermediaries in the pathogenesis of neuropathic pain and that ATP serves as the mediator. However, the in vivo mechanism underlying the retention of activated microglia in the injured region has not yet been completely elucidated. Prostaglandin E(2) (PGE(2)) is the principal proinflammatory prostanoid and plays versatile roles by acting via four PGE receptor subtypes, EP1-EP4. In the present study, we investigated the role of PGE(2) in spinal microglial activation in relation to neuropathic pain by using genetic and pharmacological methods. Mice deficient in microsomal prostaglandin E synthase-1 impaired the activation of microglia and the NMDA-nitric oxide (NO) cascade in spinal neurons in the dorsal horn and did not exhibit mechanical allodynia after peripheral nerve injury. The intrathecal injection of indomethacin, a nonsteroidal anti-inflammatory drug, ONO-8713, a selective EP1 antagonist, or 7-nitroindole, a neuronal NO synthase inhibitor, attenuated mechanical allodynia and the increase in activated microglia observed in the established neuropathic-pain state. We further demonstrated that ATP-induced microglial migration was blocked in vitro by PGE(2) via EP2 and by S-nitrosoglutathione, an NO donor. Taken together, the present study suggests that PGE(2) participated in the maintenance of neuropathic pain in vivo not only by activating spinal neurons, but also by retaining microglia in the central terminals of primary afferent fibers via EP2 subtype and via EP1-mediated NO production.

Prostaglandin E 2 2 Signaling and Bacterial Infection Recruit Tumor-Promoting Macrophages to Mouse Gastric Tumors

Helicobacter pylori infection induces an inflammatory response, which can contribute to gastric tumorigenesis. Induction of cyclooxygenase-2 (COX-2) results in production of prostaglandin E(2) (PGE(2)), which mediates inflammation. We investigated the roles of bacterial infection and PGE(2) signaling in gastric tumorigenesis in mice. We generated a germfree (GF) colony of K19-Wnt1/C2mE mice (Gan mice); these mice develop gastric cancer. We examined tumor phenotypes, expression of cytokines and chemokines, and recruitment of macrophages. We also investigated PGE(2) signaling through the PGE(2) receptor subtype 4 (EP4) in Gan mice given specific inhibitors. Gan mice raised in a specific pathogen-free facility developed large gastric tumors, whereas gastric tumorigenesis was significantly suppressed in GF-Gan mice; reconstitution of commensal flora or infection with Helicobacter felis induced gastric tumor development in these mice. Macrophage infiltration was significantly suppressed in the stomachs of GF-Gan mice. Gan mice given an EP4 inhibitor had decreased expression of cytokines and chemokines. PGE(2) signaling and bacterial infection or stimulation with lipopolysaccharide induced expression of the chemokine C-C motif ligand 2 (CCL2) (which attracts macrophage) in tumor stromal cells or cultured macrophages, respectively. CCL2 inhibition suppressed macrophage infiltration in tumors, and depletion of macrophages from the tumors of Gan mice led to signs of tumor regression. Wnt signaling was suppressed in the tumors of GF-Gan and Gan mice given injections of tumor necrosis factor-α neutralizing antibody. Bacterial infection and PGE(2) signaling are required for gastric tumorigenesis in mice; they cooperate to up-regulate CCL2, which recruits macrophage to gastric tumors. Macrophage-derived tumor necrosis factor-α promotes Wnt signaling in epithelial cells, which contributes to gastric tumorigenesis.

Variation with reproductive status of PGE2 receptor immunoreactivities in the Bostrichthys sinensis olfactory system

The role of PGE(2) as a putative sex pheromone in Chinese black sleeper Bostrichthys sinensis was investigated, using immunocytochemistry and how the immunoreactivities of the prostaglandin E(2) (PGE(2) ) receptor subtypes EP(1) , EP(2) , EP(3) and EP(4) varied with reproductive status in the olfactory system was determined. The results showed that PGE(2) receptors were present in the whole of the olfactory system of B. sinensis, and that the number of receptors was linked to the reproductive status of the fish. The densities of EP(1) immunoreactivity in the olfactory epithelium of mature fish were significantly (P < 0·01) higher than those in immature fish of both sexes, and the densities of EP(2) and EP(3) immunoreactivities in mature fish were higher (but not significantly) than those in immature fish of both sexes. In the olfactory nerve, the density of EP(2) immunoreactivity in mature fish was higher (but not significantly) than that in immature fish in both sexes. In the olfactory bulb, the densities of EP(1-4) immunoreactivities in mature females were significantly (P < 0·05 or <0·01) higher than those in immature females, and the density of EP(4) immunoreactivity in mature males was significantly (P < 0·01) higher than that in immature males. As far as is known, the present study is the first report of the immunoreactivities of PGE(2) receptor subtypes in the olfactory system of a teleost, and offers new findings regarding the role of PGE(2) as sex pheromone and hormone in the reproductive behaviour and pheromonal communication of B. sinensis.

Regulation of Ocular Surface Inflammation by Prostaglandin E Receptor Subtype EP3

We first investigated whether the prostaglandin (PG) E2-PGE receptor subtype EP3 axis regulates the development of murine experimental allergic conjunctivitis because it has been reported that this pathway negatively regulates allergic reactions in a murine allergic asthma model. We observed that EP3 is constitutively expressed in mice conjunctival epithelium. EP3 knockout mice demonstrated significantly increased eosinophil infiltration in conjunctiva after ragweed challenge compared with wild-type mice. Consistently, significantly higher expression of eotaxin-1 messenger RNA was observed in Ptger3-/- mice. Conversely, treatment of wild-type mice with an EP3-selective agonist significantly decreased eosinophil infiltration, which was blunted in Ptger3-/- mice. Expression of cyclooxygenase-2 and PGE synthases was upregulated and PGE2 content increased in the eyelids after ragweed challenge. These data suggest that PGE2 acts on EP3 in the conjunctival epithelium and downregulates the progression of experimental allergic conjunctivitis. We next examined and compared the expression of EP3 in human conjunctival epithelium in various ocular surface diseases. Human conjunctival epithelium expressed EP3-specific messenger RNA and EP3 protein. Although we could clearly find positive signals in the conjunctival epithelium from patients with noninflammatory ocular surface diseases such as conjunctivochalasis and pterygium, we could not find positive signals in that from those with inflammatory disorders such as Stevens-Johnson syndrome and ocular cicatricial pemphigoid. Likewise, expression of the PGE receptor subtype EP4 was clearly found in the conjunctival epithelium from patients with conjunctivochalasis and pterygium but not from patients with Stevens-Johnson syndrome and ocular cicatricial pemphigoid.

A role for COX2-derived PGE2 and PGE2-receptor subtypes in head and neck squamous carcinoma cell proliferation

The overexpression of cyclooxygenase (COX)-2 is a frequent event in squamous cell carcinomas of the head and neck (HNSCC), and non-steroidal anti-inflammatory drugs, which are potent inhibitors of COX-1 and COX-2, exert chemopreventive effects on HNSCC cancer development. COX-2 promotes the release of the pro-inflammatory mediator prostaglandin E2 (PGE2), which acts on its cell surface G protein-coupled receptors EP1, EP2, EP3, and EP4. Here, we investigated the role of PGE2 and its receptors in cellular proliferation in HNSCC. The expression of COX-2 and EP1-4 was examined in immortalized oral epithelial cells and in a representative panel of HNSCC cell lines, and based on these data EP1-EP3 and COX-2 expression were evaluated by immunohistochemistry in a large clinical sample collection using HNSCC tissue microarrays. The ability of selective COX-2 inhibition to block PGE2 secretion was measured by ELISA specific assays. The effects of PGE2 on cell proliferation were evaluated using PGE2, its stable analog, and EP2 and EP3-specific synthetic agonists. The results presented here show that HNSCC tumoral lesions and their derived cell lines constitutively express COX-2 and the EP1, EP2 and EP3 receptors for PGE2. HNSCC cells secrete PGE2, which can be suppressed by low concentrations of COX-2 selective inhibitors, without inhibiting cell proliferation. Exogenously added stable PGE2 and EP3-specific agonists induce DNA synthesis in all HNSCC cell lines tested. Overall, our study supports the emerging notion that PGE2 produced in the tumor microenvironment by the overexpression of COX-2 in tumoral and inflammatory cells may promote the growth of HNSCC cells in an autocrine and paracrine fashion by acting on PGE2 receptors that are widely expressed in most HNSCC cancer cells. In particular, our findings suggest that EP3 receptor may play a more prominent role in HNSCC cell growth promotion, thus providing a rationale for the future evaluation of this PGE2 receptor as a target for HNSCC prevention strategies.

Emerging roles of prostanoids in T cell‐mediated immunity

Three distinct subsets of T helper (Th) cells, Th1, Th2, and Th17, not only contribute to host defense against pathogens, but also cause many types of immune diseases. Differentiation and functions of these T cell subsets are mainly regulated by specific cytokines. Intriguingly, recent studies have revealed that prostanoids including various types of prostaglandins (PGs) and thromboxane (TX) are also involved in these processes. Prostanoids exert their actions by binding to their specific receptors. They include PGD receptor, EP1, EP2, EP3, and EP4 subtypes of PGE receptor, PGF receptor, PGI receptor, and TX receptor. From many in vitro findings, prostanoids, especially PGE(2), were traditionally believed to be an immunosuppressant. However, studies using mice deficient in each type or subtype of prostanoid receptors and their selective agonists and antagonists have revealed that prostanoids collaborate with cytokines, and critically regulate T cell proliferation, differentiation and functions. Recent studies have revealed that PGE(2) facilitates Th1 cell differentiation and Th17 cell expansion in collaboration with IL-12 and IL-23, respectively, and that these PGE(2) actions contribute to development of immune diseases mediated by these Th subsets. Furthermore, studies using the receptor-deficient mice have also revealed that other prostanoids including PGD(2) and PGI(2) contribute to regulation of immune diseases of the Th2 type such as allergic asthma. These findings shed a new light on the roles of prostanoids in T cell-mediated immunity and immune diseases.

Dual roles of PGE 2 -EP4 signaling in mouse experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS). Although prostaglandin (PG) concentrations are increased in cerebrospinal fluid of MS patients, the role of PGs in MS is unknown. We examined this issue by subjecting mice deficient in each PG receptor type or subtype to EAE induction and using agonists or antagonists selective for each of the four PGE receptor (EP) subtypes. Among PG receptor-deficient mice, only EP4(-/-) mice manifested significant suppression of EAE, which was mimicked in wild-type mice and to a greater extent, in EP2(-/-) mice by administration of the EP4 antagonist ONO-AE3-208 during the immunization phase. EP4 antagonism during immunization also suppressed the generation of antigen-specific T helper (Th) 1 and Th17 cells in wild-type mice and to a greater extent, in EP2(-/-) mice. ONO-AE3-208 administration at EAE onset had little effect on disease severity, and its administration throughout the experimental period did not cause significant reduction of the peak of disease, suggesting that, in addition to its facilitative action during the immunization phase, EP4 exerts a preventive action in the elicitation phase. Administration of the EP4 agonist ONO-AE1-329 at EAE onset delayed and suppressed disease progression as well as inhibited the associated increase in permeability of the blood-brain barrier. Thus, PGE(2) exerts dual functions in EAE, facilitating Th1 and Th17 cell generation redundantly through EP4 and EP2 during immunization and attenuating invasion of these cells into the brain by protecting the blood-brain barrier through EP4.

Prostaglandin E2receptor subtype EP2- and EP4-regulated gene expression profiling in human ciliary smooth muscle cells

Prostanoids are an important class of intraocular pressure (IOP)-lowering antiglaucoma agents that act primarily via increased uveo-scleral aqueous humor outflow through the ciliary body. We have developed two novel PGE(2) analogs that are specific agonists for the PGE(2) receptor subtypes EP2 and EP4, respectively. To identify gene regulatory networks and key players that mediate the physiological effects observed in vivo, we performed genomewide expression studies using human ciliary smooth muscle cells. Quantitative real-time RT-PCR confirmed a largely overlapping gene expression profile subsequent to EP2 and EP4 agonist treatment, with 65 significantly regulated genes identified overall, 5 being specific for the EP2 agonist and 6 specific for the EP4 agonist. We found predicted functional cAMP-response elements in promoter regions of a large fraction of the predominantly upregulated genes, which suggests that the cAMP signaling pathway is the most important intracellular signaling pathway for these agonists in these cells. Several target genes were identified that, as part of complex regulatory networks, are implicated in tissue remodeling processes and osmoregulation (e.g., AREG, LOXL3, BMP2, AQP3) and thus may help elucidate the mechanism of action of these IOP-lowering drugs involving the uveo-scleral outflow path.

Effects of prostaglandin E and F receptor agonists in vivo on luteal function in ewes

Loss of progesterone secretion at the end of the estrous cycle is via uterine PGF 2α secretion; however, uterine PGF 2α is not decreased during early pregnancy in ewes to prevent luteolysis. Instead the embryo imparts resistance to PGF 2α-induced luteolysis, which is via the 2-fold increase in prostaglandins E 1 and E 2 (PGE 1, PGE 2; PGE) in the endometrium during early pregnancy. Chronic intrauterine infusion of PGE 1 or PGE 2 prevents spontaneous or an estradiol-17β, IUD, or PGF 2α-induced luteolysis. Four PGE receptor subtypes (EP 1, EP 2, EP 3, and EP 4) and an FP receptor specific for PGF 2α have been identified. The objective of this experiment was to determine the effects of EP 1, EP 2, EP 3, or FP receptor agonists in vivo on luteal mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone in ewes. Ewes received a single treatment of 17-phenyl-tri-Nor-PGE 2 (EP 1, EP 3), butaprost (EP 2), 19-(R)-OH-PGE 2 (EP 2), sulprostone (EP 1, EP 3), or PGF 2α (FP) receptor agonists into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary. 17-Phenlyl-tri-Nor-PGE 2 had no effect ( P ≥ 0.05) on any parameter analyzed. Butaprost and 19-(R)-OH-PGE 2 increased ( P ≤ 0.05) mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone. Both sulprostone and PGF 2α decreased ( P ≤ 0.05) mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone. It is concluded that both EP 3 and FP receptors may be involved in luteolysis. In addition, EP 2 receptors may mediate prevention of luteolysis via regulation of luteal mRNA for LH receptors to prevent loss of occupied and unoccupied LH receptors and therefore to sustaining luteal function.

Prostanoids and inflammation: a new concept arising from receptor knockout mice

Prostanoids including various types of prostaglandins and thromboxanes are arachidonate metabolites produced and released in response to a variety of physiological and pathological stimuli and function to maintain the body homeostasis. Since cyclooxygenase, the enzyme initiating their biosynthesis, is inhibited by aspirin-like antipyretic, anti-inflammatory, and analgesic drugs, contribution of prostanoids to acute inflammation such as fever generation, pain sensitization, and inflammatory swelling has been recognized very early. On the other hand, since aspirin-like drugs generally show little effects on allergy and immunity, it has been believed that prostanoids play little roles in these processes. Prostanoids act on a family of G-protein-coupled receptors designated PGD receptor, PGE receptor subtypes EP1-EP4, PGF receptor, PGI receptor, and TX receptor to elicit their actions. Studies using mice deficient in each of these receptors have revealed that prostanoids indeed function in the above aspirin-sensitive processes. However, these studies have also revealed that prostanoids exert both pro-inflammatory and anti-inflammatory actions not only by acting as mediators of acute inflammation but also by regulating gene expression in mesenchymal and epithelial cells at inflammatory site. Such dual actions of prostanoids are frequently seen in immune and allergic reactions, where different type of prostanoids and their receptors often exert opposite actions in a single process. Thus, a new concept on the role of prostanoids in inflammation has arisen from studies using the receptor knockout mice.

Roles of prostaglandin E receptors in stress responses

Stress is a condition in which the body homeostasis is perturbed by various stimuli such as sickness and psychological stimuli. Stress evokes adaptive responses including febrile, neuroendocrine, and behavioral responses. Prostaglandin (PG) E(2) is a metabolite from arachidonic acid, and exerts its functions through G-protein-coupled receptors called EP1, EP2, EP3, and EP4. Recent studies using knockout mice and selective drugs revealed the roles of these receptors in stress responses. Upon sickness, PGE(2) mediates fever through EP3, while both EP1 and EP3 are involved in ACTH release. In addition, EP1 controls impulsive behaviors under psychological stress through the dopaminergic system. PGE(2) produced by various cell types may integrate multiple stress stimuli through PGE receptor subtypes for adaptive responses.

Phosphodiesterase 3 and 4 Negatively Regulate Receptor Activator of Nuclear Factor-.KAPPA.B Ligand-Mediated Osteoclast Formation by Prostaglandin E2

Prostaglandin E2 (PGE2) stimulates osteoclast formation by increasing receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) mRNA expression via cAMP-protein kinase A (PKA) pathways in osteoblasts. Since phosphodiesterases (PDEs) balance the concentration of intracellular cAMP stimulated by PGE2, we investigated the role of PDEs in PGE2-mediated osteoclast formation using various cAMP-specific PDEs inhibitors. In the presence of PGE(2), PDE3 and 4 inhibitors were shown to dose-dependently increase the osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. In agreement with this finding, they stimulated PGE2-induced cAMP production followed by increased RANKL mRNA expression in osteoblasts, suggesting that PDE3 and 4 negatively regulate PGE2-mediated RANKL expression in osteoblasts. RT-PCR analysis revealed that PDE3A, 3B, 4A, 4B and 4D are expressed in osteoblasts. The PDE8 inhibitor did not increase osteoclast formation, although it stimulated PGE2-induced RANKL mRNA expression in osteoblasts. The four subtypes of PGE receptors are designated EP1, EP2, EP3, and EP4. PDE3 and 4 inhibitors were found to increase EP1/3, EP4 and/or EP2 agonist-stimulated RANKL expression, indicating that PDE3 and PDE4 negatively regulate PGE2-induced RANKL mRNA expression through four EPs. Taken together, these data suggest that PDE3 and PDE4 could have important pharmacological and clinical implications in bone-related diseases.

Essential role of EP3 subtype in prostaglandin E2-induced adhesion of mouse cultured and peritoneal mast cells to the Arg-Gly-Asp-enriched matrix

Accumulating evidence has indicated that mast cells can modulate a wide variety of immune responses. Migration and adhesion play a critical role in regulation of tissue mast cell function, in particular, under inflammatory conditions. We previously demonstrated that prostaglandin (PG) E(2) stimulates adhesion of a mouse mastocytoma cell line, P-815, to the Arg-Gly-Asp (RGD)-enriched matrix through cooperation between two PGE(2) receptor subtypes: EP3 and EP4 (Hatae N, Kita A, Tanaka S, Sugimoto Y, Ichikawa A. J Biol Chem 278: 17977-17981, 2003). We here investigated PGE(2)-induced adhesion of IL-3-dependent bone marrow-derived cultured mast cells (BMMCs). In contrast to the elevated cAMP-dependent adhesion of P-815 cells, EP3-mediated Ca(2+) mobilization plays a pivotal role in PGE(2)-induced adhesion of BMMCs. Adhesion and Ca(2+) mobilization induced by PGE(2) were abolished in the Ptger3(-/-) BMMCs and were significantly suppressed by treatment with pertussis toxin, a phospholipase C inhibitor, U-73122, and a store-operated Ca(2+) channel inhibitor, SKF 36965, indicating the involvement of G(i)-mediated Ca(2+) influx. We then investigated PGE(2)-induced adhesion of peritoneal mast cells to the RGD-enriched matrix. EP3 subtype was found to be the dominant PGE receptor that expresses in mouse peritoneal mast cells. PGE(2) induced adhesion of the peritoneal mast cells of the Ptger3(+/+) mice, but not that of the Ptger3(-/-) mice. In rat peritoneal mast cells, PGE(2) or an EP3 agonist stimulated both Ca(2+) mobilization and adhesion to the RGD-enriched matrix. These results suggested that the EP3 subtype plays a pivotal role in PGE(2)-induced adhesion of murine mast cells to the RGD-enriched matrix through Ca(2+) mobilization.

PGE2-receptor subtype EP4-dependent adherence of mastocytoma P-815 cells to matrix components in subcutaneous tissues overlaying inside surface of air pouch cavity in CDF1 mouse

It remains to be fully clarified how adhesion of mast cells is regulated in vivo. We previously reported that PGE2-receptor EP4 stimulated the adhesion of mouse mastocytoma P-815 cells to plate-bound fibronectin. Our purpose in this study is to evaluate the adhesion using a system, which can mimic the in vivo adhesion. P-815 cells were transplanted in an air pouch produced in the transplantable mice, CDF1. The number of cells that adhere to the subcutaneous tissues overlaying the inside cavity surface was determined. The number of adhered cells was decreased in mice administered with ibuprofen or an EP4 antagonist, ONO AE3-208. A local administration of PGE(2) or a phorbol ester, PMA, increased the number of adhered cells, which was also suppressed in the mice treated with ONO AE3-208. Our results suggest that PGE(2)-mediated adhesion of P-815 cells in the subcutaneous tissues of the air pouch is mediated by the EP4 subtype.