Prostaglandin E2 facilitates subcellular translocation of the EP4 receptor in neuroectodermal NE-4C stem cells

Prostaglandin E2 (PGE₂) is a lipid mediator released from the phospholipid membranes that mediates important physiological functions in the nervous system via activation of four EP receptors (EP1-4). There is growing evidence for the important role of the PGE₂/EP4 signaling in the nervous system. Previous studies in our lab show that the expression of the EP4 receptor is significantly higher during the neurogenesis period in the mouse. We also showed that in mouse neuroblastoma cells, the PGE₂/EP4 receptor signaling pathway plays a role in regulation of intracellular calcium via a phosphoinositide 3-kinase (PI3K)-dependent mechanism. Recent research indicates that the functional importance of the EP4 receptor depends on its subcellular localization. PGE₂-induced EP4 externalization to the plasma membrane of primary sensory neurons has been shown to play a role in the pain pathway. In the present study, we detected a novel PGE₂–dependent subcellular trafficking of the EP4 receptor in neuroectodermal (NE-4C) stem cells and differentiated NE-4C neuronal cells. We show that PGE₂ induces EP4 externalization from the Golgi apparatus to the plasma membrane in NE-4C stem cells. We also show that the EP4 receptors translocate to growth cones of differentiating NE-4C neuronal cells and that a higher level of PGE₂ enhances its growth cone localization. These results demonstrate that the EP4 receptor relocation to the plasma membrane and growth cones in NE-4C cells is PGE₂ dependent. Thus, the functional role of the PGE₂/EP4 pathway in the developing nervous system may depend on the subcellular localization of the EP4 receptor.     

Discovery of AAT-008, a novel,potent, and selective prostaglandin EP4 receptor antagonist

Starting from acylsufonamide HTS hit 2, a novel series of para-N-acylaminomethylbenzoic acids was identified and developed as selective prostaglandin EP4 receptor antagonists. Structural modifications on lead compound 4a were explored with the aim of improving potency, physicochemical properties, and animal PK predictive of QD (once a day) dosing regimen in human. These efforts led to the discovery of the clinical candidate AAT-008 (4j), which exhibited significantly improved pharmacological profiles over grapiprant (1).     

Indomethacin decreases EP2 prostanoid receptor expression in colon cancer cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) can decrease the risk of colorectal cancer; however, it has not been established if this effect is solely through their ability to inhibit cyclooxygenase (COX). In this study the effects of indomethacin, a potent NSAID and nonselective COX inhibitor, was examined in LS174T human colon cancer cells. These cells were found to express EP2 prostanoid receptors, but not the EP1, EP3 or EP4 subtypes. Pretreatment of LS174T cells with indomethacin produced a complete inhibition of prostaglandin E(2) (PGE(2)) stimulated cyclic AMP (cAMP) formation in a dose dependent manner with an IC(50) of 21 microM. Interestingly, the inhibition of PGE(2)-stimulated cAMP formation by indomethacin was accompanied by a decrease in EP2 mRNA expression and by a decrease in the whole cell specific binding of [(3)H]PGE(2). Thus, treatment of LS174T cells with indomethacin causes a down regulation of EP2 prostanoid receptors expression that may be independent of COX inhibition.     

EP2 receptor activation by prostaglandin E2 leads to induction of HO-1 via PKA and PI3K pathways in C6 cells

Recently we proposed that COX-2 induction precedes expression of HO-1 in ischemic preconditioned rat brain. In the current study, we investigated the molecular mechanism by which prostaglandin E₂, one of COX-2 metabolites, induces HO-1 in rat C6 brain cells. We demonstrated that concentration of PGE₂ increased HO-1 expression in C6 cells in vitro. The effects of PGE₂ were mimicked by PGE₂ receptor EP₂ agonists, 11-deoxy PGE₂, and cAMP analog, dibutyl-cAMP. HO-1 expression by PGE₂ was inhibited by LY294002, PI3K inhibitor and H89, PKA inhibitor. The EP₂-specific antagonist, AH8006 also inhibited PGE₂-mediated HO-1 expression in a concentration-dependent manner. Finally, PGE₂ inhibited GOX-induced apoptosis as assayed by FACS analysis or DNA strand breaks assay, and this cell death was reversed by ZnPPIX, HO-1 inhibitor. In addition to HO-1 induction, PGE₂ also increased phosphorylation of Bad by PKA- and PI3K-depednent manner. Taken together, we conclude that PGE₂ induces HO-1 protein expression through PKA and PI3K signaling pathways via EP₂ receptor in C6 cells. The induction of HO-1 along with increase of p-Bad by PGE₂ is responsible for anti-apoptosis against oxidant stress.     

The EP1 subtype of prostaglandin E2 receptor: Role in keratinocyte differentiation and expression in non-melanoma skin cancer

We have previously demonstrated that the EP₁ subtype of PGE₂ receptor is expressed in the differentiated compartment of normal human epidermis and is coupled to intracellular calcium mobilization. We therefore hypothesized that the EP₁ receptor is coupled to keratinocyte differentiation. In in vitro studies, radioligand binding, RT-PCR, immunoblot and receptor agonist-induced second messenger studies demonstrate that the EP₁ receptor is up-regulated by high cell density in human keratinocytes and this up-regulation precedes corneocyte formation. Moreover, two different EP₁ receptor antagonists, SC51322 and AH6809, both inhibited corneocyte formation. SC51322 also inhibited the induction of differentiation-specific proteins, cytokeratin K10 and epidermal transglutaminase. We next examined the immunolocalization of the EP₁ receptor in non-melanoma skin cancer in humans. Well-differentiated SCCs exhibited significantly greater membrane staining, while spindle cell carcinomas and BCCs had significantly decreased membrane staining compared with normal epidermis. This data supports a role for the EP₁ receptor in regulating keratinocyte differentiation.     

Discovery of {4-[4,9-bis(ethyloxy)-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl]-2-fluorophenyl}acetic acid (GSK726701A), a novel EP4 receptor partial agonist for the treatment of pain

A novel series of EP₄ agonists and antagonists have been identified, and then used to validate their potential in the treatment of inflammatory pain. This paper describes these novel ligands and their activity within a number of pre-clinical models of pain, ultimately leading to the identification of the EP₄ partial agonist GSK726701A.     

The mRNA expression of prostaglandin E receptors EP2 and EP4 and the changes in glycosaminoglycans in the sheep cervix during the estrous cycle

Transcervical artificial insemination in sheep is limited by the inability to completely penetrate the cervix with an inseminating pipette. Penetration is partially enhanced at estrus due to a degree of cervical relaxation, which is probably regulated by cervical prostaglandin synthesis and extracellular matrix remodeling. Prostaglandin E₂ acts via prostaglandin E receptors EP₁ to EP₄, and EP₂ and EP₄ stimulate smooth muscle relaxation and glycosaminoglycan synthesis. This study investigated the expression of EP₂ and EP₄ mRNA and glycosaminoglycans in the sheep cervix during the estrous cycle. Sheep cervices were collected prior to, during, and after the luteinizing hormone (LH) surge and during the luteal phase. The mRNA expression of EP₂ and EP₄ was determined by in situ hybridization, glycosaminoglycan composition was assessed by Alcian blue staining, and hyaluronan concentration was investigated by ELISA. The expression of EP₂ mRNA was greatest prior to the LH surge (P = 0.02), although EP₂ and EP₄ were expressed throughout the estrous cycle. Hyaluronan was the predominant glycosaminoglycan, and hyaluronan content increased prior to the LH surge (P < 0.05). Cervical EP₂ mRNA expression changed throughout the estrous cycle and was greatest prior to the LH surge. We propose that prostaglandin E₂ binds to EP₂ and EP₄ stimulating hyaluronan synthesis, which may cause remodeling of the cervical extracellular matrix, culminating in cervical relaxation.     

EP2 receptor mediated cAMP release is augmented by PGF2α activation of the FP receptor via the calcium-calmodulin pathway

Prostaglandins exert their effects on target cells by coupling to specific G protein-coupled receptors (GPCRs) that are often co-expressed in the same cells and use alternate and in some cases opposing intracellular signaling pathways. This study investigated the cross-talk that influences intracellular signaling and gene expression profiling in response to co-activation of the EP2 and FP prostanoid receptors in Ishikawa cells stably expressing both receptors (FPEP2 cells). In this study we show that in FPEP2 cells, PGF alone does not alter adenosine 3′,5′-cyclic monophosphate (cAMP) production, but in combination with Butaprost enhances EP2 receptor mediated cAMP release compared to treatment with Butaprost alone. PGF-mediated potentiation of cAMP release was abolished by antagonism of the FP receptor, inhibition of phospholipase C (PLC) and inositol phosphate receptor (IP3R) whereas inhibition of protein kinase C (PKC) had no effect. Moreover, inhibition of calcium effectors using calmodulin antagonist (W7) or Ca²⁺/calmodulin-dependent kinase II (CaMK-II) inhibitor (KN-93) abolished PGF potentiation of Butaprost-mediated cAMP release. Using siRNA molecules targeted against the adenylyl cyclase 3 (AC3) isoform, we show that AC3 is responsible for the cross-talk between the FP and EP2 receptors. Using gene array studies we have identified a candidate gene, Spermidine/N1-acetyltransferase (SAT1), which is regulated by this cAMP mediated cross-talk. In conclusion, this study demonstrates that co-activation of the FP and EP2 receptors results in enhanced release of cAMP via FP receptor-Gα_q-Ca²⁺-calmodulin pathway by activating calcium sensitive AC3 isoform.     

Nuclear factor-κB activation in human monocytes stimulated with lipopolysaccharide is inhibited by fibroblast conditioned medium and exogenous PGE2

The nuclear factor κB (NF-κB) is thought to be crucially involved in the gene activation of several cytokines, including tumor necrosis factor α (TNF). Previously, we showed that fibroblast conditioned medium (FCM) is able to inhibit both TNF mRNA accumulation and protein release in peripheral blood-derived human monocytes (PBM) stimulated with lipopolysaccharide (LPS). In this study we have investigated the effect of FCM on the LPS-induced DNA-binding activity of NF-κB, by means of electrophoretic shift assay (EMSA). We provide evidence that FCM strongly inhibits the LPS-induced NFκB activation in PBM. Furthermore, we show that exogenous PGE₂ mimics the NFκB inhibitory effect of FCM. On the other hand, FCM produced in the presence of indomethacin does not inhibit NF-κB activation by LPS. Our results lend further support to the hypothesis that inflammatory and immune responses of monocytes/macrophages may be modulated at the molecular level by signals originating from tissue structural cells such as fibroblasts.     

Exogenous arachidonic acid mediates permeability of human brain microvessel endothelial cells through prostaglandin E2 activation of EP3 and EP4 receptors

The blood–brain barrier, formed by microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. Arachidonic acid (ARA; 5,8,11,14‐cis‐eicosatetraenoic acid) is a conditionally essential polyunsaturated fatty acid [20:4(n ? 6)] and is a major constituent of brain lipids. The current study examined the transport processes for ARA in confluent monolayers of human brain microvascular endothelial cells (HBMEC). Addition of radioactive ARA to the apical compartment of HBMEC cultured on Transwell^® inserts resulted in rapid incorporation of radioactivity into the basolateral medium. Knock down of fatty acid transport proteins did not alter ARA passage into the basolateral medium as a result of the rapid generation of prostaglandin E₂ (PGE₂), an eicosanoid known to facilitate opening of the blood–brain barrier. Permeability following ARA or PGE₂ exposure was confirmed by an increased movement of fluorescein‐labeled dextran from apical to basolateral medium. ARA‐mediated permeability was attenuated by specific cyclooxygenase‐2 inhibitors. EP₃ and EP₄ receptor antagonists attenuated the ARA‐mediated permeability of HBMEC. The results indicate that ARA increases permeability of HBMEC monolayers likely via increased production of PGE₂ which acts upon EP₃ and EP₄ receptors to mediate permeability. These observations may explain the rapid influx of ARA into the brain previously observed upon plasma infusion with ARA.

     

The effects of 11-methyl 16,16 dimethyl prostaglandin E2 on gastric acid secretion in man

11-Methyl 16,16 Dimethyl Prostaglandin E₂ (TM-PGE) was administered orally to man in dosages of 2.5, 5, 7.5 and 10 μg/kg. Maximal inhibition of basal secretion was 52 and 78% and submaximal histamine-stimulated secretion 45 and 70% for volume and acid output, respectively. Secretory inhibition was observed for approximately two hours after ingestion of the drug. No effect was observed on serum gastrin levels. Side effects occurred with equal frequency in the placebo and drug groups. TM-PGE is well tolerated and inhibits both basal and submaximal histamine-stimulated acid secretion in man. Further evaluation may prove it to be helpful in the clinical treatment of acid hypersecretory states and peptic ulcer disease.     

Role of phospholipase A2 and prostaglandin E in growth and differentiation of myeloid leukemic cells

Phospholipase A₂ activity and prostaglandin E synthesis have been studied in different clones of myeloid leukemic cells, which differ in their competence to be induced to differentiate by the macrophage and granulocyte differentiation-inducing protein or the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). Clones that could be induced to differentiate by this protein showed a higher basal phospholipase A₂ activity than clones that could not be induced to differentiate by this protein inducer. Cell competence to be induced to differentiate by TPA did not show this correlation, and the clone with the least ability to respond to TPA showed the lowest number of binding sites for [20-³H]phorbol 12,13-dibutyrate. Differentiation induced by the protein was accompanied by a 7–14-fold increase in prostaglandin E synthesis, whereas differentiation induced by TPA did not show this increase. Externally added prostaglandin E₁ did not induce differentiation but inhibited cell proliferation and the degree of inhibition in the different clones was related to the basal phospholipase A₂ activity. The results indicate that increase of prostaglandin E synthesis was not an essential pre-requisite for differentiation, that prostaglandin E seems to be involved in the inhibition of cell proliferation in association with phospholipase A₂, and that the differentiation-inducing protein and TPA can induce differentiation by different pathways. The amount of basal phospholipase A₂ activity was also related to previously found differences in the ability of the clones to develop desensitization to β-adrenergic hormones or prostaglandin E₁.     

N-3 but not N-6 fatty acids reduce the expression of the combined adhesion and scavenger receptor CD36 in human monocytic cells

CD36, a multifunctional adhesion receptor e.g. for thrombospondin and collagen, as well as a scavenger receptor for oxidized low density lipoprotein, is expressed e.g. on platelets and monocytes. By this dual role it might be involved in early steps of atherosclerosis like the recruitment of monocytes and formation of foam cells. We therefore studied the effects of n-3 fatty acids on CD36 expression in human monocytic cells. Incorporation of eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) into cellular phospholipids resulted in a significant reduction of CD36 expression at the mRNA and protein level, whereas arachidonic acid (AA, C20: 4n-6) and linoleic acid (LA, C18:2n-6) tended to increase CD36 expression compared to the control. This specific down-regulation of CD36 by n-3 fatty acids in cells involved in the initiation and progression of atherogenesis and inflammation, represents a further mechanism that may contribute to the beneficial effects of n-3 polyunsaturated fatty acids (PUFA) in these disorders.     

Expression of prostaglandin E2 receptor subtypes in the canine lower urinary tract varies according to the gonadal status and gender

Locally-synthesised prostaglandin E₂ (PGE₂) is pivotal for the function of the lower urinary tract (LUT). This study aimed at investigating the expression and distribution pattern of the four PGE₂ receptor (EP) subtypes in the LUT of intact and gonadectomised male and female dogs. Expression for EP1, EP2, EP3, and EP4 and their mRNA (EP2, EP3, and EP4) was investigated. Twenty clinically healthy dogs were allotted into 4 groups based on their gonadal status and gender including 5 intact males, 5 anoestrous females, 4 castrated males, and 6 spayed females. In situ hybridization and immunohistochemistry showed variation in the expression of mRNA and protein for the EP subtypes among tissue layers (epithelium, sub-epithelial stroma, and muscle), regions (body and neck of the bladder as well as proximal and distal urethra) and between gonadal statuses and genders. The expression for the four EPs was intense in the luminal epithelium, intermediate to low in the muscle and the sub-epithelial stroma regardless of gonadal status or gender. Higher expression of all EPs and their mRNAs was observed in the proximal urethra compared to other regions in intact dogs. However, in gonadectomised dogs, the expression did not differ among different regions and was generally lower than in intact dogs particularly in the proximal urethra. Differences in the expression between genders were found and depended on EP subtypes. In conclusion, the results have shown that four subtypes of EP receptors and their mRNAs are present in the canine LUT and their expression was affected by the gonadal status and the gender. The results lead to suggest that an impaired LUT function post-neutering may partly be associated with differences in PGE₂ receptor expression between intact and gonadectomised dogs.     

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.     

The pyrimidinergic P2Y6 receptor mediates a novel release of proinflammatory cytokines and chemokines in monocytic cells stimulated with UDP

The human P2Y6 receptor (hP2Y6) is a member of the G protein-coupled pyrimidinergic P2 receptor family that responds specifically to the extracellular nucleotide uridine diphosphate (UDP). Recently, the hP2Y6 receptor has been reported to mediate monocyte IL-8 production in response to UDP or lipopolysaccharide (LPS), but the role of hP2Y6 in regulating other pro-inflammatory cytokines or mediators is largely unknown. We demonstrate here that UDP specifically induces soluble TNF-alpha and IL-8 production in a promonocytic U937 cell line stably transfected with hP2Y6. However, we did not detect IL-1alpha, IL-1beta, IL-6, IL-10, IL-18, and PGE2 in the conditioned media from the same cell line. These results distinguish UDP/P2Y6 signaling from LPS signaling. Interestingly, UDP induces the production of IL-8, but not TNF-alpha, in human astrocytoma 1321N1 cell lines stably transfected with hP2Y6. Therefore, the immune effect of UDP/P2Y6 signaling on the production of proinflammatory cytokines is selective and dependent on cell types. We further identify that UDP can also induce the production of proinflammatory chemokines MCP-1 and IP-10 in hP2Y6 transfected promonocytic U937 cell lines, but not astrocytoma 1321N1 cell lines stably transfected with hP2Y6. From the Taqman analysis, UDP stimulation significantly upregulates the mRNA levels of IL-8, IP-10, and IL-1beta, but not TNF-alpha. Taken together, these new findings expand the pro-inflammatory biology of UDP mediated by the P2Y6 receptor.     

Biphasic effect of sodium fluoride and guanyl nucleotides on binding to prostaglandin E2 receptors in rat epididymal adipocyte membranes

Both NaCl and NaF promoted PGE₂ binding to epididymal adipocyte membranes by apparent increase in the binding affinity. In order to distinguish between the effect of fluoride and the ‘salt effect’ of sodium on PGE₂ binding, the effects of Mg²⁺ and guanyl nucleotides on PGE₂ binding in the presence of NaCl or NaF were compared. Mg²⁺ decreased PGE₂ binding; high NaF concentration abolished this inhibition, while increased NaCl concentratipns did not affect the Mg²⁺ inhibition. In the presence of Mg²⁺ the effects of NaCl and NaF were additive. The enhancement of PGE₂ binding by fluoride, unlike sodium, was dependent on the presence of Mg²⁺. Induction of the membranes with GDPβS, Gpp(NH)p, GTP or GTPγS increased PGE, binding. Gradual increase in NaF concentrations in the presence of guanyl nucleotides resulted in stimulation of PGE₂ binding at low NaF concentrations and inhibition of PGE₂ binding at higjh NaF concentrations. No changes in the stimulatory action of NaCl on PGE₂ binding were observed in the simulatenous presence of NaCl and guanyl nucleotides. A biphasic effect on PGE₂ binding was observed with a wide concentration range of guanyl nucleotides. Treatment of the isolated membranes with cholera or pertussis toxins stimulated the adenylyl cyclase activity of the membranes, but failed to influence PGE₂ binding. The implications of these findings are discussed.