EP4 Agonist Research Articles

Prostaglandin E2 down-regulates the expression of CD25 on bovine T cells, and this effect is mediated through the EP4 receptor

A crucial event in the initiation of an immune response is the activation of T cells, which requires IL-2 binding to its high-affinity IL-2 receptor for optimal signaling. The IL-2 receptor α-chain (CD25) is needed for the high affinity binding of IL-2 to effector cells and is potently induced after T cell activation. The aim of this research has been to determine whether prostaglandin E2 (PGE2) affects the CD25 expression on bovine T cells, and if it does, then which of the PGE2 receptor (EP) subtype(s) mediate(s) this effect. Herein, we report that exposure of peripheral blood mononuclear cells (PBMC) to PGE2 considerably reduces the percentage and absolute counts of CD25+CD4+, CD25+CD8+ and CD25+WC1+ T cells, significantly increases the value of these parameters with respect of CD25−CD4+, CD25−CD8+ and CD25−WC1+ T cells, and does not affect counts of the total populations of CD4+, CD8+ and WC1+ T cells. These results indicate that PGE2 down-regulates the CD25 expression on bovine T cells. Moreover, we show that the selective blockade of EP4 receptor, but not EP1 and EP3 receptors, prevents this effect. Interestingly, the exposure of PBMC to a selective EP2 receptor agonist leads to a substantial increase in the percentage and absolute number of CD25+CD4+, CD25+CD8+ and CD25+WC1+ T cells. In conclusions, the PGE2-induced down-regulation of CD25 expression on bovine CD4+, CD8+ and WC1+ T cells should be considered as immunosuppressive and anti-inflammatory action, because these lymphocytes primarily represent effector cells and adequate CD25 expression is essential for their correct functioning. The PGE2-mediated down-regulation of the CD25 expression on bovine T cells is mediated via the EP4 receptor, although selective activation of the EP2 receptor up-regulates the CD25 expression on these cells. Thus, with respect to the effect of PGE2 on the CD25 expression on bovine T cells, EP4 receptor serves as an inhibitory receptor, whereas EP2 receptor functions as a stimulatory receptor. The fact that non-selective stimulation of EP receptors, i.e. triggered by PGE2, leads to weaker CD25 expression proves that inhibitory actions prevail over stimulatory ones. These results indicate the possibility of pharmacological manipulation of the CD25 expression on T cells via selective antagonists and agonists of EP2 and EP4 receptors.

Investigation of Ocular Events Associated with Taprenepag Isopropyl, a Topical EP2 Agonist in Development for Treatment of Glaucoma

Taprenepag isopropyl is an EP2 receptor agonist that is in development for the treatment of glaucoma. Iritis, photophobia, and increased corneal thickness observed in a Phase 2 clinical trial with taprenepag isopropyl were not previously observed in topical ocular toxicity studies in rabbits and dogs. In vivo studies using cynomolgus monkeys and in vitro models were used to elucidate the mechanisms underlying these ocular events. Monkeys were dosed daily for 28 days in 1 eye with taprenepag and in the other with vehicle control. Complete ophthalmic examinations were performed at baseline and weekly thereafter. Serial sections of eyes were examined histopathologically at the end of the study. Recovery after the discontinuation of taprenepag was assessed for 28 days in the monkeys in the high-dose group. In vitro studies evaluated cell viability, paracellular permeability, and cytokine induction with human corneal epithelial or endothelial cell cultures. Monkeys demonstrated a dose-related incidence of iritis and increased corneal thickness that resolved within 28 days of discontinuing taprenepag. There was no evidence in vivo of taprenepag toxicity to the corneal endothelium or epithelium. Cell viability of stratified epithelial cells was primarily affected by excipients and was similar to Xalatan(®). The viability of HCEC-12 cells was not affected by taprenepag at concentrations up to 100 μM. The lack of in vivo or in vitro endothelial cytotoxicity and the reversibility of the increase in corneal thickness and iritis in the monkey provide confidence to permit further clinical development of taprenepag.

Prostaglandin E2 receptor EP4 signaling in vascular smooth muscle cells decreases aortic elasticity (1123.2)

Arterial elasticity was impaired in advancing aortic aneurysm (AA). Since we have reported that the PGE2 receptor EP4 signaling was involved in progression of AA, we further examined whether EP4 signaling in smooth muscle cells (SMCs) decreases aortic elasticity. We generated mice (EP4TG) with vascular smooth muscle‐specific overexpression of human EP4 (hEP4). hEP4 mRNA was over‐expressed in the aorta of EP4TG (n=8). Using a wire myograph, we found that vasodilative effect of EP4 agonist (10 µM) was greater in the aortae of EP4TG than those of non‐transgenic mice (non‐TG) (25.0±2.9 vs. 9.6±2.0% of phenylephrine‐induced contraction, p<0.01, n=4‐6). Under basal conditions, aortic diameter and elasticity were not different. However, pressure‐induced dilation (20 to 220mmHg) was decreased in EP4TG than in non‐TG (0.84±0.04‐fold, n=5‐6, p<0.05). After angiotensin II (ATII) infusion (3.0µg/kg/min for 28 days), the aortae of EP4TG were enlarged to a greater degree than those of non‐TG (1.1±0.02‐fold, n=5‐6, p<0.05). A zelatin zymography revealed that MMP‐2 activity is higher in the aortae of EP4TG than in that of non‐TG (1.7±0.07‐fold, n=5, p<0.05). After ATII treatment, enhancement of MMP‐9 activation was greater in EP4TG than in non‐TG (1.6±0.19‐fold, n=5, p<0.05). These data suggest that EP4 signaling in SMCs decreases aortic elasticity by increasing MMP activation.

Suppression of polyi:c-inducible gene expression by EP3 in murine conjunctival epithelium

We previously reported that EP3, a subtype of prostaglandin E2 receptors, negatively regulates eosinophilic infiltration in murine experimental allergic conjunctivitis induced by TLR3, which causes reduced eosinophilic conjunctival inflammation in TLR3/EP3 double knock-out (DKO) mice although in EP3-KO mice eosinophilic conjunctival inflammation is pronounced. We also documented that in human conjunctival epithelial cells, the EP3 agonist suppressed the production of cytokines such as CXCL10, CXCL11, IL6, CCL5, TSLP, and MCP-1 induced by polyI:C, a TLR3 ligand. EP3 was dominantly expressed in conjunctival epithelial cells, airway epithelial cells, and keratinocytes. To examine the effects of EP3 against polyI:C-inducible gene expression in conjunctival epithelium we performed gene expression analysis of the polyI:C-stimulated conjunctival epithelium in wild-type, EP3-KO-, and EP3/TLR3 DKO mice. Using GeneChip® we first examined the comprehensive effects of gene expression in polyI:C-stimulated conjunctival epithelium of wild-type mice. We found that after 6-hr stimulation, 31 transcripts were up-regulated more than 10-fold. Quantitative RT-PCR confirmed that 21 of the 31 transcripts were significantly (> 3-fold) up-regulated. Next, to identify the transcripts regulated by EP3 we compared the gene expression of these 21 transcripts in polyI:C stimulated conjunctival epithelium of wild-type and EP3-KO mice by quantitative RT-PCR. We found that all 21 transcripts were expressed significantly stronger in polyI:C stimulated conjunctival epithelium of EP3-KO mice. We also confirmed that the mRNA expression of these 21 transcripts was significantly reduced in polyI:C stimulated conjunctival epithelium of EP3/TLR3 DKO- compared to EP3- KO mice. GeneChip® analysis also showed that the number of 4 transcripts was more than 5 times greater in polyI:C stimulated conjunctival epithelium of EP3-KO- than wild-type mice although in wild-type mice these 4 transcripts were not significantly up-regulated after 6-hr polyI:C stimulation. Quantitative RT-PCR confirmed that the number of 2 of the 4 transcripts was more than 100-fold higher in polyI:C stimulated EP3 KO- than wild-type mice. In summary, we found that EP3 suppressed polyI:C, a TLR3 ligand, inducible genes in polyI:C stimulated murine conjunctival epithelium. Our findings suggest that EP3 and TLR3 in conjunctival epithelium play a critical role in regulating ocular surface inflammation.

Prostaglandin E2 upregulates β1 integrin expression via the E prostanoid 1 receptor/nuclear factor κ-light-chain-enhancer of activated B cells pathway in non-small-cell lung cancer cells

The prostaglandin E2 (PGE2) E prostanoid (EP)1 receptor shown to be associated with lung cancer cell invasion. However, the mechanism of EP1 receptor-mediated cell migration remains to be elucidated. β1 integrin is an essential regulator of the tumorigenic properties of non-small-cell lung carcinoma (NSCLC) cells. To date, little is known regarding the association between the EP1 receptor and β1 integrin expression. The present study investigated the effect of EP1 receptor activation on β1 integrin expression and cell migration in NSCLC cells. A total of 34 patients with clinical diagnosis of NSCLC and 10 patients with benign disease were recruited for the present study. The expression levels of the EP1 receptor and β1 integrin expression were studied in resected lung tissue using immunohistochemistry. A statistical analysis was performed using Stata se12.0 software. The effects of PGE2, EP1 agonist 17-phenyl trinor-PGE2 (17-PT-PGE2) and the nuclear factor κ-B (NF-κB) inhibitor on β1 integrin expression were investigated on A549 cells. The expression of β1 integrin and the phosphorylation of NF-κB‑p65 Ser536 was investigated by western blot analysis. Cell migration was assessed by a transwell assay. The results demonstrated that β1 integrin and EP1 receptor expression exhibited a positive correlation of evident significance in the 44 samples. The in vitro migration assay revealed that cell migration was increased by 30% when the cells were treated with 5 µM 17-PT-PGE2 and that the pre-treatment of β1 integrin monoclonal antibody inhibited 17-PT-PGE2‑mediated cell migration completely. PGE2 and 17-PT-PGE2 treatment increased β1 integrin expression. RNA interference against the EP1 receptor blocked the PGE2-mediated β1 integrin expression in A549 cells. Treatment with 17-PT-PGE2 induced NF-κB activation, and the selective NF-κB inhibitor pyrrolidinedithiocarbamate inhibited 17-PT-PGE2-mediated β1 integrin expression. In conclusion, the present study indicated that the PGE2 EP1 receptor regulates β1 integrin expression and cell migration in NSCLC cells by activating the NF-κB signaling pathway. Targeting the PGE2/EP1/β1 integrin signaling pathway may aid in the development of new therapeutic strategies for the prevention and treatment of this type of cancer.

Prostaglandin E2 (EP) Receptors Mediate PGE2-Specific Events in Ovulation and Luteinization Within Primate Ovarian Follicles

Prostaglandin E2 (PGE2) is a key mediator of ovulation. All 4 PGE2 receptors (EP receptors) are expressed in the primate follicle, but the specific role of each EP receptor in ovulatory events is poorly understood. To examine the ovulatory events mediated via these EP receptors, preovulatory monkey follicles were injected with vehicle, the PG synthesis inhibitor indomethacin, or indomethacin plus PGE2. An ovulatory dose of human chorionic gonadotropin was administered; the injected ovary was collected 48 hours later and serially sectioned. Vehicle-injected follicles showed normal ovulatory events, including follicle rupture, absence of an oocyte, and thickening of the granulosa cell layer. Indomethacin-injected follicles did not rupture and contained oocytes surrounded by unexpanded cumulus; granulosa cell hypertrophy did not occur. Follicles injected with indomethacin plus PGE2 were similar to vehicle-injected ovaries, indicating that PGE2 restored the ovulatory changes inhibited by indomethacin. Additional follicles were injected with indomethacin plus an agonist for each EP receptor. EP1, EP2, and EP4 agonists each promoted aspects of follicle rupture, but no single EP agonist recapitulated normal follicle rupture as seen in follicles injected with either vehicle or indomethacin plus PGE2. Although EP4 agonist-injected follicles contained oocytes in unexpanded cumulus, the absence of oocytes in EP1 agonist- and EP2 agonist-injected follicles suggests that these EP receptors promote cumulus expansion. Surprisingly, the EP3 agonist did not stimulate any of these ovulatory changes, despite the high level of EP3 receptor expression in the monkey follicle. Therefore, agonists and antagonists selective for EP1 and EP2 receptors hold the most promise for control of ovulatory events in women.

Prostacyclin Regulates Spinal Nociceptive Processing through Cyclic Adenosine Monophosphate–induced Translocation of Glutamate Receptors

Prostacyclin (PGI2) is known to be an important mediator of peripheral pain sensation (nociception) whereas little is known about its role in central sensitization. The levels of the stable PGI2-metabolite 6-keto-prostaglandin F1α (6-keto-PGF1α) and of prostaglandin E2 (PGE2) were measured in the dorsal horn with the use of mass spectrometry after peripheral inflammation. Expression of the prostanoid receptors was determined by immunohistology. Effects of prostacyclin receptor (IP) activation on spinal neurons were investigated with biochemical assays (cyclic adenosine monophosphate-, glutamate release-measurement, Western blot analysis) in embryonic cultures and adult spinal cord. The specific IP antagonist Cay10441 was applied intrathecally after zymosan-induced mechanical hyperalgesia in vivo. Peripheral inflammation caused a significant increase of the stable PGI2 metabolite 6-keto-PGF1α in the dorsal horn of wild-type mice (n = 5). IP was located on spinal neurons and did not colocalize with the prostaglandin E2 receptors EP2 or EP4. The selective IP-agonist cicaprost increased cyclic adenosine monophosphate synthesis in spinal cultures from wild-type but not from IP-deficient mice (n = 5-10). The combination of fluorescence-resonance-energy transfer-based cyclic adenosine monophosphate imaging and calcium imaging showed a cicaprost-induced cyclic adenosine monophosphate synthesis in spinal cord neurons (n = 5-6). Fittingly, IP activation increased glutamate release from acute spinal cord sections of adult mice (n = 13-58). Cicaprost, but not agonists for EP2 and EP4, induced protein kinase A-dependent phosphorylation of the GluR1 subunit and its translocation to the membrane. Accordingly, intrathecal administration of the IP receptor antagonist Cay10441 had an antinociceptive effect (n = 8-11). Spinal prostacyclin synthesis during early inflammation causes the recruitment of GluR1 receptors to membrane fractions, thereby augmenting the onset of central sensitization.

Prostaglandin E2 Induces IL-6 and IL-8 Production by the EP Receptors/Akt/NF-κB Pathways in Nasal Polyp-Derived Fibroblasts.

PurposeInterleukin 6 (IL-6) and IL-8 participate in the pathogenesis of chronic rhinosinusitis with nasal polyps, and their levels are increased by prostaglandin E2 (PGE2) in different cell types. The purposes of this study were to determine whether PGE2 has any effect on the increase in the levels of IL-6 and IL-8 in nasal polyp-derived fibroblasts (NPDFs) and subsequently investigate the possible mechanism of this effect.MethodsDifferent concentrations of PGE2 were used to stimulate NPDFs at different time intervals. NPDFs were treated with agonists and antagonists of E prostanoid (EP) receptors. To determine the signaling pathway for the expression of PGE2-induced IL-6 and IL-8, PGE2 was treated with Akt and NF-κB inhibitors in NPDFs. Reverse transcription-polymerase chain reaction for IL-6 and IL-8 mRNAs was performed. IL-6 and IL-8 levels were measured byenzyme-linked immunosorbent assay (ELISA). The activation of Akt and NF-κB was evaluated by western blot analysis.ResultsPGE2 significantly increased the mRNA and protein expression levels of IL-6 and IL-8 in NPDFs. The EP2 and EP4 agonists and antagonists induced and inhibited IL-6 expression. However, the EP4 agonist and antagonist were only observed to induce and inhibit IL-8 expression level. The Akt and NF-κB inhibitors significantly blocked PGE2-induced expression of IL-6 and IL-8.ConclusionsPGE2 increases IL-6 expression via EP2 and EP4 receptors, and IL-8 expression via the EP4 receptor in NPDFs. It also activates the Akt and NF-κB signal pathways for the production of IL-6 and IL-8 in NPDFs. These results suggest that signaling pathway for IL-6 and IL-8 expression induced by PGE2 might be a useful therapeutic target for the treatment of nasal polyposis.

Role of PGE2 in the colonic motility: PGE2 generates and enhances spontaneous contractions of longitudinal smooth muscle in the rat colon

The aim of this study was to determine which PGE2 receptors (EP1-4 receptors) influence colonic motility. Mucosa-free longitudinal smooth muscle strips of the rat middle colon spontaneously induced frequent phasic contractions (giant contractions, GCs) in vitro, and the GCs were almost completely abolished by a cyclooxygenase inhibitor, piroxicam, and by an EP3 receptor antagonist, ONO-AE3-240, but enhanced by tetrodotoxin (TTX). In the presence of piroxicam, exogenous PGE2, both ONO-AE-248 (EP3 agonist), and ONO-DI-004 (EP1 agonist) induced GC-like contractions, and increased the frequency and amplitude. These effects of EP receptor agonists were insensitive to TTX and ω-conotoxins. In immunohistochemistry, the EP1 and EP3 receptors were expressed in the longitudinal smooth muscle cells. These results suggest that the endogenous PGE2 spontaneously generates and enhances the frequent phasic contractions directly activating the EP1 and EP3 receptors expressed on longitudinal smooth muscle cells in the rat middle colon.

Silymarin suppresses the PGE2‐induced cell migration through inhibition of EP2 activation; G protein‐dependent PKA‐CREB and G protein‐independent Src‐STAT3 signal pathways

Silymarin has been known as a chemopreventive agent, and possesses multiple anti-cancer activities including induction of apoptosis, inhibition of proliferation and growth, and blockade of migration and invasion. However, whether silymarin could inhibit prostaglandin (PG) E2 -induced renal cell carcinoma (RCC) migration and what are the underlying mechanisms are not well elucidated. Here, we found that silymarin markedly inhibited PGE2 -stimulated migration. PGE2 induced G protein-dependent CREB phosphorylation via protein kinase A (PKA) signaling, and PKA inhibitor (H89) inhibited PGE2 -mediated migration. Silymarin reduced PGE2 -induced CREB phosphorylation and CRE-promoter activity. PGE2 also activated G protien-independent signaling pathways (Src and STAT3) and silymarin reduced PGE2 -induced phosphorylation of Src and STAT3. Inhibitor of Src (Saracatinib) markedly reduced PGE2 -mediated migration. We found that EP2, a PGE2 receptor, is involved in PGE2 -mediated cell migration. Down regulation of EP2 by EP2 siRNA and EP2 antagonist (AH6809) reduced PGE2 -inudced migration. In contrast, EP2 agonist (Butaprost) increased cell migration and silymarin effectively reduced butaprost-mediated cell migration. Moreover, PGE2 increased EP2 expression through activation of positive feedback mechanism, and PGE2 -induced EP2 expression, as well as basal EP2 levels, were reduced in silymarin-treated cells. Taken together, our study demonstrates that silymarin inhibited PGE2 -induced cell migration through inhibition of EP2 signaling pathways (G protein dependent PKA-CREB and G protein-independent Src-STAT3).

TGF-β1 Downregulates COX-2 Expression Leading to Decrease of PGE2 Production in Human Lung Cancer A549 Cells, Which Is Involved in Fibrotic Response to TGF-β1

Transforming growth factor-ß1 (TGF-β1) is a multifunctional cytokine that is involved in various pathophysiological processes, including cancer progression and fibrotic disorders. Here, we show that treatment with TGF-β1 (5 ng/mL) induced downregulation of cyclooxygenase-2 (COX-2), leading to reduced synthesis of prostaglandin E2 (PGE2), in human lung cancer A549 cells. Treatment of cells with specific inhibitors of COX-2 or PGE2 receptor resulted in growth inhibition, indicating that the COX-2/PGE2 pathway contributes to proliferation in an autocrine manner. TGF-β1 treatment induced growth inhibition, which was attenuated by exogenous PGE2. TGF-β1 is also a potent inducer of epithelial mesenchymal transition (EMT), a phenotype change in which epithelial cells differentiate into fibroblastoid cells. Supplementation with PGE2 or PGE2 receptor EP4 agonist PGE1-alcohol, as compared with EP1/3 agonist sulprostone, inhibited TGF-β1-induced expression of fibronectin and collagen I (extracellular matrix components). Exogenous PGE2 or PGE2 receptor agonists also suppressed actin remodeling induced by TGF-β1. These results suggest that PGE2 has an anti-fibrotic effect. We conclude that TGF-β1-induced downregulation of COX-2/PGE2 signaling is involved in facilitation of fibrotic EMT response in A549 cells.

Phosphodiesterase-4 Inhibition Augments Human Lung Fibroblast Vascular Endothelial Growth Factor Production Induced by Prostaglandin E2

Lung fibroblasts are believed to be a major source of vascular endothelial growth factor (VEGF), which supports the survival of lung endothelial cells and modulates the maintenance of the pulmonary microvasculature. VEGF has been related to the pathogenesis of lung diseases, including chronic obstructive pulmonary disease (COPD). Prostaglandin E2 (PGE2) stimulates VEGF production from lung fibroblasts via the E-prostanoid (EP)-2 receptor. The EP2 signaling pathway uses cyclic adenosine monophosphate (cAMP) as a second messenger, and cAMP is degraded by phosphodiesterases (PDEs). This study investigates whether phosphodiesterase inhibition modulates the human lung fibroblast VEGF production induced by PGE2. Human fetal lung fibroblasts were cultured with PGE2 and PDE inhibitors. The PDE4 inhibitors roflumilast, roflumilast N-oxide, and rolipram with PGE2 increased VEGF release, as quantified in supernatant media by ELISA. In contrast, PDE3, PDE5, and PDE7 inhibitors did not affect VEGF release. Roflumilast increased VEGF release with either an EP2 or an EP4 agonist. Roflumilast augmented the cytosolic cAMP levels induced by PGE2 and VEGF release with other agents that use the cAMP signaling pathway. Roflumilast-augmented VEGF release was completely inhibited by a protein kinase A (PKA) inhibitor. Roflumilast with PGE2 increased VEGF mRNA levels, and the blockade of mRNA synthesis inhibited the augmented VEGF release. The stimulatory effect of roflumilast on VEGF release was replicated using primary healthy and COPD lung fibroblasts. These findings demonstrate that PDE4 inhibition can modulate human lung fibroblast VEGF release by PGE2 acting through the EP2 and EP4 receptor-cAMP/PKA signaling pathway. Through this action, PDE4 inhibitors such as roflumilast could contribute to the survival of lung endothelial cells.

Prostaglandin E2deficiency causes a phenotype of aspirin sensitivity that depends on platelets and cysteinyl leukotrienes

Aspirin-exacerbated respiratory disease (AERD) is characterized by asthma, tissue eosinophilia, overproduction of cysteinyl leukotrienes (cysLTs), and respiratory reactions to nonselective cyclooxygenase (COX) inhibitors. Ex vivo studies suggest that functional abnormalities of the COX-2/microsomal prostaglandin (PG)E2 synthase-1 system may underlie AERD. We demonstrate that microsomal PGE2 synthase-1 null mice develop a remarkably AERD-like phenotype in a model of eosinophilic pulmonary inflammation. Lysine aspirin (Lys-ASA)-challenged PGE2 synthase-1 null mice exhibit sustained increases in airway resistance, along with lung mast cell (MC) activation and cysLT overproduction. A stable PGE2 analog and a selective E prostanoid (EP)2 receptor agonist blocked the responses to Lys-ASA by ∼90%; EP3 and EP4 agonists were also active. The increases in airway resistance and MC products were blocked by antagonists of the type 1 cysLT receptor or 5-lipoxygenase, implying that bronchoconstriction and MC activation were both cysLT dependent. Lys-ASA-induced cysLT generation and MC activation depended on platelet-adherent granulocytes and T-prostanoid (TP) receptors. Thus, lesions that impair the inducible generation of PGE2 remove control of platelet/granulocyte interactions and TP-receptor-dependent cysLT production, permitting MC activation in response to COX-1 inhibition. The findings suggest applications of antiplatelet drugs or TP receptor antagonists for the treatment of AERD.

H2O2 Stimulates Cystic Fibrosis Transmembrane Conductance Regulator through an Autocrine Prostaglandin Pathway, Using Multidrug-Resistant Protein–4

Cystic fibrosis transmembrane conductance regulator (CFTR) activity is essential for the maintenance of airway surface liquid depth, and therefore mucociliary clearance. Reactive oxygen species, increased during inflammatory airway diseases, alter CFTR activity. Here, H2O2 levels in the surface liquid of normal human bronchial epithelial cultures differentiated at the air-liquid interface were estimated, and H2O2-mediated changes in CFTR activity were examined. In Ussing chambers, H2O2-induced anion currents were sensitive to the CFTR inhibitors CFTRinh172 and GlyH-101. These currents were absent in cells from patients with cystic fibrosis. Responses to greater than 500 μM H2O2 were transient. Cyclooxygenase inhibitors blocked the H2O2 response, as did EP1 and EP4 receptor antagonists. A multidrug-resistant protein (MRP) inhibitor and short hairpin RNA directed against MRP4 blocked H2O2 responses. EP1 and EP4 agonists mimicked H2O2 in both control and MRP4 knockdown cells. Thus, H2O2 activates the synthesis, export, and binding of prostanoids via EP4 and, interestingly, EP1 receptors in normal, differentiated human airway epithelial cells to activate cyclic adenosine monophosphate pathways that in turn activate CFTR channels in the apical membrane.

EP3 receptor isoforms are differentially expressed in subpopulations of primate granulosa cells and couple to unique G-proteins

Prostaglandin E2 (PGE2) produced within the ovarian follicle is necessary for ovulation. PGE2 is recognized by four distinct G-protein-coupled receptors. Among them, PTGER3 (also known as EP3) is unique in that mRNA splicing generates multiple isoforms. Each isoform has a distinct amino acid composition in the C-terminal region, which is involved in G-protein coupling. To determine whether monkey EP3 isoforms couple to different G-proteins, each EP3 isoform was expressed in Chinese hamster ovary cells, and intracellular signals were examined after stimulation with the EP3 agonist sulprostone. Stimulation of EP3 isoform 5 (EP3-5) reduced cAMP in a pertussis toxin (PTX)-sensitive manner, indicating involvement of Gαi. Stimulation of EP3-9 increased cAMP, which was reduced by the general G-protein inhibitor GDP-β-S, and also increased intracellular calcium, which was reduced by PTX and GDP-β-S. So, EP3-9 likely couples to both Gαs and a PTX-sensitive G-protein to regulate intracellular signals. Stimulation of EP3-14 increased cAMP, which was further increased by PTX, so EP3-14 likely regulates cAMP via multiple G-proteins. Granulosa cell expression of all EP3 isoforms increased in response to an ovulatory dose of human chorionic gonadotropin. Two EP3 isoforms were differentially expressed in functional subpopulations of granulosa cells. EP3-5 was low in granulosa cells at the follicle apex while EP3-9 was high in cumulus granulosa cells. Differential expression of EP3 isoforms may yield different intracellular responses to PGE2 in granulosa cell subpopulations, contributing to the different roles played by granulosa cell subpopulations in the process of ovulation.

The Role of Prostaglandin E<sub>2</sub> in Stroke-Reperfusion Injury

Although augmented prostaglandin E2 (PGE2) accumulation has been demonstrated at the lesion sites of rodent ischemia models, the role of postischemic PGE2 in neuronal survival has remained obscure. We recently identified the microsomal prostaglandin E synthase-1 (mPGES-1), an inducible terminal enzyme for prostaglandin E2 synthesis, as a critical factor in stroke-reperfusion injury. Co-induction of mPGES-1 and cyclooxygenase (COX)-2, an upstream enzyme for PGE2 production, was observed after brain ischemia. In mPGES-1 knockout (KO) mice, in which the postischemic PGE2 production in the cortex was completely absent, the ischemic injuries were less severe compared to those in wild-type (WT) mice. The ameliorated symptoms observed in KO mice after ischemia were reversed to almost the same severity as in the WT mice by intracerebroventricular injection of PGE2 into KO mice. The induction of mPGES-1 was also observed after glutamate exposure in cultured hippocampal slices. In mPGES-1 KO slices, glutamate-induced excitotoxicity was less severe compared to that in WT slices. Among the EP1-4 antagonists and agonists, only the EP3 antagonist attenuated and only the EP3 agonist augmented the glutamate-induced excitotoxicity. Furthermore, intraperitoneal injection of COX-2 inhibitor or EP3 antagonist reduced the ischemic injuries in WT mice, but not in mPGES-1 KO mice. In EP3 KO mice, the ischemic injuries were less severe compared to those in WT mice. These results suggest that mPGES-1 and COX-2 are co-induced by excessive glutamate in the ischemic brain and act together to exacerbate stroke injury through PGE2 production followed by activation of EP3 receptors.

Chronic administration of EP4-selective agonist exacerbates albuminuria and fibrosis of the kidney in streptozotocin-induced diabetic mice through IL-6

Diabetic nephropathy is currently the most common cause of end-stage renal disease in the western world. Exacerbated inflammation of the kidney is known to contribute acceleration of nephropathy. Despite increased COX-2-mediated production of prostanoid metabolite PGE2, knowledge on its involvement in the progression of diabetic kidney disease is not complete. Here, we show the cross talk of the PGE2–EP4 pathways and IL-6 in inducing albuminuria and fibrosis in an animal model of type 1 diabetes. Hyperglycemia causes enhanced COX-2 expression and PGE2 production. Administration of PGE2 receptor EP4-selective agonist ONO-AE1-329 for 12 weeks exacerbated fibrosis and albuminuria. Diabetes-induced expression of inflammatory cytokines TNFα and TGFβ1 was enhanced in EP4 agonist-treated mice kidney. In addition, urinary excretion of cytokines (TNFα and IL-6) and chemokines (MCP-1 and IP-10) were significantly more in EP4-treated mice than vehicle-treated diabetes. Diabetes-induced collagen I and CTGF expression were also significantly higher in EP4-treated mice. However, EP4 agonist did not alter macrophage infiltration but increased cytokine and chemokine production in RAW264.7 cells. Interestingly, EP4-induced IL-6 expression in the kidney was localized in proximal and distal tubular epithelial cells. To confirm further whether EP4 agonist increases fibrosis and albuminuria through an increase in IL-6 expression, IL-6-knockout mice were administered with EP4 agonist. IL-6-knockout mice were resistant to EP4-induced exacerbation of albuminuria and diabetes and EP4-induced fibrosis. Our data suggest that EP4 agonist through IL-6 induces glomerulosclerosis and interstitial fibrosis, and IL-6 represents a new factor in the EP4 pathway.

Enhancement of PLGF production by 15-(S)-HETE via PI3K-Akt, NF-κB and COX-2 pathways in rheumatoid arthritis synovial fibroblast

Metabolites from arachidonic acids play the pivotal roles in inflammatory arthritis. Arachidonic acid could be metabolized by cyclooxygenase (COX) and lipoxygenase (LOX) to produce the bioactive eicosanoids. Although the down-stream products of COX including prostaglandin E2 are well-known inflammatory stimulators, the role of LOX products in inflammatory arthritis is still unclear. Here we found that the downstream product of 15-LOX, 15-S-hydroxyeicosatetraenoic acid (15-(S)–HETE), can enhance the expression of placenta growth factor (PLGF), which is recently considered to play an important role in rheumatoid arthritis. 15-(S)-HETE increased the expression of PLGF in human rheumatoid arthritis synovial fibroblasts in a time-dependent and concentration-dependent manner. PI3K-Akt, NF-κB signaling pathways were involved in the potentiation effects of 15-(S)–HETE. In addition, COX-2 was up-regulated by the treatment of 15-(S)-HETE and the increase of COX-2 expression participated in 15-(S)-HETE-induced PLGF expression, which was confirmed by COX-2 shRNA or pharmacological COX-2 inhibitor. Moreover, it was found that treatment of prostaglandin E2 (PGE2), which was the main down-stream metabolite of COX-2, increased the expression of PLGF. EP1, EP2, EP3 and EP4 agonists could up-regulate PLGF as well. In animal studies, we found that the adjuvant-induced expression of PLGF and COX-2 was inhibited in 15-LOX knockout mice. These results indicated that PLGF up-regulation by 15-LOX downstream product may be involved in inflammatory arthritis.

Differential Effects of Prostaglandin E2-Sensitive Receptors on Contractility of Human Ocular Cells That Regulate Conventional Outflow

The goal of this study was to functionally compare prostaglandin E2 (PGE2)-sensitive receptors in human primary cells involved in conventional outflow. The expression profile of prostaglandin (PG) receptors in primary cultures of human trabecular meshwork (TM) and Schlemm's canal (SC) cells were determined by quantitative-PCR. The functional activities of endogenous PGE2-sensitive receptors were evaluated using subtype-selective agonists and antagonists with cell impedance technology. Agonist-sensitive EP1, EP2, and EP4 receptors were present in TM cells, all increasing cell stiffness (or contractility) in a dose-dependent manner. Rank order of efficacy (Emax) for agonists in TM cells were EP1 greater than EP2 greater than EP4 with EC50 1.1 μM, 0.56 μM, and 0.1 μM, respectively, and no functional EP3 receptors were found. Of the four EP receptor subtypes active in SC cells, EP1 and EP3 receptor activation increased cell stiffness, while EP2 and EP4 agonists dose-dependently decreased cell stiffness 47% and 23% with EC50 values of 170 nM and 69 nM, respectively. Consistent with these observations, the Rho kinase inhibitor Y-27632 decreased cell impedance (stiffness) of TM and SC cells (∼60%), while Rho GTPase activator thrombin caused cell impedance to increase in both cell types (168%-190%). Cell impedance positively correlates with cellular stiffness/contractility. Because EP2/4 receptors caused decreased cell stiffness in SC, but not in TM cells, both receptors appear to mediate IOP lowering via changes in SC cell stiffness in the conventional outflow pathway.

Calcium-mediated increased expression of fibroblast growth factor-2 acts through NF-κB and PGE2/EP4 receptor signaling pathways in cementoblasts

We reported previously that cementoblasts are provided with sensing mechanisms for extracellular Ca2+ and that elevated extracellular Ca2+ increases fibroblast growth factor-2 (FGF-2) gene and protein expression levels via a cyclic AMP/protein kinase A (PKA) dependent pathway. In the present study, we found that stimulation of murine cementoblasts with 10mM CaCl2 induced cyclooxygenase-2 (COX-2) gene expression and prostaglandin E2 (PGE2) biosynthesis. NS-398, a COX-2 inhibitor, significantly reduced CaCl2-induced increase in Fgf-2 gene expression, indicating that PGE2 synthesized by COX-2 may be involved in FGF-2 induction. The inhibitory effect of NS-398 was restored completely by the addition of PGE2 receptor 4 (E-prostanoid receptor 4, called EP4) agonist, but not agonists for EP1, EP2, and EP3. Furthermore, EP4 antagonist significantly reduced CaCl2-induced Fgf-2 induction, suggesting that it is mediated by EP4 activation. However, stimulation with EP4 agonist alone in the absence of CaCl2 had no effect on the Fgf-2 induction, indicating that EP4 signaling alone is not sufficient. CaCl2 also upregulated gene expression levels of Ep4 and Cox-2, as well as Fgf-2 and induction of these genes was abolished by pretreatment with BMS-345541, a nuclear factor-κB (NF-κB) inhibitor, indicating that NF-κB signaling triggered by CaCl2 is indispensable for FGF-2 induction. Furthermore, CaCl2-induced Fgf-2 induction was synergistically enhanced by the addition of EP4 agonist. This indicates that the signaling triggered via CaCl2 and its combination with EP4 agonist may be useful as a novel strategy for periodontal regeneration.