PGD₂ has long been implicated in allergic diseases. Recent cloning of a second PGD₂ receptor, DP2 (also known as CRTh2), led to a greater understanding of the physiological and pathophysiological implications of PGD₂. PGD₂ signaling through DP1 and DP2 mediates different and often opposite effects in many cell types of the immune system. Although mast cells (MC) are the largest source of PGD₂ in the body, there is little information about their potential expression of DP2 and its functional significance. In this study, we show that tissue MC in human nasal polyps express DP2 protein, and that human MC lines and primary cultured human MC express mRNA as well as protein of DP2. By immunohistochemistry, we detected that 34% of MC in human nasal polyps expressed DP2. In addition, flow cytometry showed that 87% of the LAD2 human MC line and 98% of primary cultured human MC contained intracellular DP2. However, we could not detect surface expression of DP2 on human MC by single cell analysis using imaging flow cytometry. Blocking of endogenous PGD2 production with aspirin did not induce surface expression of DP2 in human MC. Two DP2 selective agonists, DK-PGD₂ and 15R-15-methyl PGD₂ induced dose-dependent intracellular calcium mobilization that was abrogated by pertussis toxin, but not by three DP2 selective antagonists. MC mediator release including degranulation was not affected by DP2 selective agonists. Thus, human MC express DP2 intracellularly rather than on their surface, and the function of DP2 in human MC is different than in other immune cells such as Th2 cells, eosinophils and basophils where it is expressed on the cell surface and induces Th2 cytokine and/or granule associated mediator release. Further studies to elucidate the role of intracellular DP2 in human MC may expand our understanding of this molecule and provide novel therapeutic opportunities.

Recently, certain studies have demonstrated in vitro that prostaglandin E2 (PGE2) promotes human cluster of differentiation (CD)34+ cell homing. However, the sub‑type receptors activated by PGE2 are unknown, as the PGE2 receptor EP1-4 subtypes (EP1-4) are expressed on the membrane of human CD34+ cells. Based on the above, the present study aimed to screen the receptor subtype activity by PGE2 to promote human CD34+ cell homing. It was observed that human CD34+ cells expressed the four PGE2 sub‑receptors, particularly EP2 and 4. PGE2 increased EP2 and 4 mRNA expression significantly, while EP1 and 3 mRNA exhibited no significant alteration. PGE2, EP2 agonist (EP2A), and EP4A upregulated C‑X‑C chemokine receptor 4 mRNA and protein expression in human CD34+ cells, and promoted stromal cell‑derived factor 1α (SDF‑1α) expression in bone marrow mesenchymal stem cells (BMMSCs). These phenomena were inhibited by the associated receptor antagonists. PGE2, EP2A, and EP4A facilitated human CD34+ cell migration towards SDF‑1α and BMMSCs. The results of the present study suggested that PGE2 promoted human CD34+ cell homing through EP2 and 4 receptors in vitro.

Non-hormonal therapeutic strategies for endometriosis are needed. The aim of this study was to characterize the effects of prostaglandin (PG)E2 receptor inhibitors to explore their potential as novel therapeutic strategies for endometriosis. The expression of PGE2 receptors (EP2 and EP4) in donated tissues from human ovarian endometriosis, adenomyosis and peritoneal endometriosis was examined using immunohistochemistry. Human endometriotic stromal cells (ESC) isolated from ovarian endometriotic tissue and peritoneal macrophages were treated with EP2 and EP4 antagonists. cAMP accumulation and the effect of EP antagonists were measured using cAMP assays. DNA synthesis in ESC was detected using bromodeoxyuridine incorporation analysis. Interleukin (IL)-6 and IL-8 protein levels in ESC supernatants were measured using ELISAs. mRNA expression level for aromatase by ESC, and selected cytokines by peritoneal macrophages was measured using RT-PCR. EP2 and EP4 receptors were expressed in cells derived from control and diseased tissue, ovarian endometriotic, adenomyotic and peritoneal lesions. A selective EP2 antagonist reduced DNA synthesis, cAMP accumulation and IL-1β-induced proinflammatory cytokine secretion and aromatase expression. A selective EP4 antagonist negated IL-1β-induced IL-6 secretion and aromatase expression. In peritoneal macrophages, EP expression was elevated in endometriosis samples but the EP4 antagonist reduced cAMP levels and expression of vascular endothelial growth factor, chemokine ligand 2 and chemokine ligand 3 mRNA. EP2 and EP4 are functioning in endometriosis lesions and peritoneal macrophages, and their selective antagonists can reduce EP-mediated actions, therefore, the EP antagonists are potential therapeutic agents for controlling endometriosis.

To investigate the role of excitatory amino acid neurotransmission within the rostral raphe pallidus area (RPa) in thermogenic and cardiovascular responses, changes in sympathetic nerve activity to brown adipose tissue (BAT), BAT temperature, expired CO(2), arterial pressure, and heart rate were recorded after microinjection of excitatory amino acid (EAA) receptor agonists into the RPa in urethan-chloralose-anesthetized, ventilated rats. To determine whether EAA neurotransmission within the RPa is necessary for the responses evoked by disinhibition of the RPa or by prostaglandin E(2) acting within the medial preoptic area, BAT sympathetic nerve activity, BAT temperature, expired CO(2), arterial pressure, and heart rate were measured during these treatments both before and after blockade of EAA receptors within the RPa. Microinjection of EAA receptor agonists into the RPa resulted in significant increases in all measured variables; these increases were attenuated by prior microinjection of the respective EAA receptor antagonists into the RPa. Microinjection of prostaglandin E(2) into the medial preoptic area or microinjection of bicuculline into the RPa resulted in respective significant increases in BAT sympathetic nerve activity (+approximately 190% and +approximately 235% of resting levels), in BAT temperature (approximately 1.8 degrees C and approximately 2 degrees C), in expired CO(2) (approximately 1.1% and approximately 1.1%), and in heart rate (approximately 97 beats per minute (bpm) and approximately 100 bpm). Blockade of ionotropic EAA receptors within the RPa by microinjection of kynurenate completely reversed the prostaglandin E(2) or bicuculline-evoked increases in all of the measured variables. Blockade of either N-methyl-D-aspartate (NMDA) receptors or non-NMDA receptors alone resulted in marked attenuations of the prostaglandin E(2)-evoked effects on all of the measured variables. These data demonstrate that activation of an EAA input to the RPa is necessary for the BAT thermogenic and the cardiovascular effects resulting from the actions of prostaglandin E(2) within the medial preoptic area or from the disinhibition of local neurons in the RPa.

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)E(2) 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 PGE(2) and the characterization of the PGE(2) receptor subtypes (EP(1), EP(2), EP(3) or EP(4)) 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. PGE(2) induced the contraction of HICA (E(max)=7.28 ± 0.16 g; pEC(50) value=0.79 ± 0.18; n=17); contractions were also observed with the EP(3) receptor agonists, sulprostone, 17-phenyl-PGE(2), misoprostol or ONO-AE-248. In conclusion, PGE(2) induced vasoconstriction of HICA via EP(3) 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 EP(3) mRNA. These observations suggest that in the context of perioperative inflammation, increased PGE(2) concentrations could trigger vasoconstriction of HICA and possibly alter spinal vascularization.

Aberrant upregulation of COX-2 enzyme resulting in accumulation of PGE2 in a cancer cell environment is a marker for progression of many cancers, including breast cancer. Four subtypes of cell surface receptors (EP1, EP2, EP3, and EP4), which are coupled with different G-proteins, mediate PGE2 actions. Since migration is an essential step in invasion and metastasis, in the present study we defined the expression of EP receptors and their roles in migratory function of breast cancer cells of murine (C3L5) and human (MDA-MB-231 and MCF-7) origin. Highly metastatic C3L5 and MDA-MB-231 cells, found to be highly migratory in a Transwell migration assay, were shown to accumulate much higher levels of PGE2 in culture media in comparison with nonmetastatic and poorly migrating MCF-7 cells; the levels of PGF2alpha and 6-keto-PGF1alpha were low in all cases. The elevated PGE2 production by metastatic cancer cells was due to COX-2 activity since dual COX-1/2 inhibitor indomethacin and selective COX-2 inhibitor NS-398 equally suppressed both basal and inducible (by IFN-gamma/LPS or Ca2+-ionophores) PGE2 accumulation. RT-PCR analysis revealed that murine C3L5 cells expressed mRNA of EP1, EP3, and EP4 but not EP2 receptors. On the other hand, human MDA-MB-231 and MCF-7 cells expressed all the above receptors. High levels of expression of functional EP4 receptors coupled with Gs-protein was confirmed in C3L5 cells by biochemical assay showing a dose-dependent increase of intracellular cAMP synthesis in response to PGE2. EP receptor antagonists SC-19220, AH-6809, and AH-23848B, having highest affinity for EP1, EP1/EP2/DP, and EP4 receptors, respectively, variably inhibited migration of metastatic breast cancer cells. An autocrine PGE2-mediated migratory activity of these cells appeared to be associated predominantly with EP4 receptor-mediated signaling pathway, which uses cAMP as a second messenger. This conclusion is based on several observations: (1) selective EP4 antagonist AH-23848B effectively inhibited migration of both C3L5 and MDA-MB-231 cells in a dose-dependent manner; (2) exogenous PGE2 and EP4 agonist PGE1 alcohol increased migration of C3L5 cells; (3) forskolin, a potent activator of adenylate cyclase, as well as membrane-permeable analogues of cAMP (8-bromo-cAMP, dibutyryl-cAMP) stimulated migration of C3L5 cells; and (4) Rp-cAMPS, a selective protein kinase A inhibitor, reduced migration of C3L5 cells. Migration of poorly migratory MCF-7 cells remained unaffected with either PGE2 or EP4 antagonist. These findings are relevant for designing therapeutic strategies against breast cancer metastasis.

Prostaglandin E2 (PGE2) is a prostanoid with diverse actions in health and disease. In chronic respiratory diseases driven by inflammation, PGE2 has both positive and negative effects. An enhanced understanding of the receptor-mediated cellular signalling pathways induced by PGE2 may help us separate the beneficial properties from unwanted actions of this important prostaglandin. PGE2 is known to exert anti-inflammatory and bronchoprotective actions in human airways. To date however, whether PGE2 increases production of the anti-inflammatory protein MAPK phosphatase 1 (MKP-1) was unknown. We address this herein and use primary cultures of human airway smooth muscle (ASM) cells to show that PGE2 increases MKP-1 mRNA and protein upregulation in a concentration-dependent manner. We explore the signalling pathways responsible and show that PGE2-induces CREB phosphorylation, not p38 MAPK activation, in ASM cells. Moreover, we utilize selective antagonists of EP2 (PF-04418948) and EP4 receptors (GW 627368X) to begin to identify EP-mediated functional outcomes in ASM cells in vitro. Taken together with earlier studies, our data suggest that PGE2 increases production of the anti-inflammatory protein MKP-1 via cAMP/CREB-mediated cellular signalling in ASM cells and demonstrates that EP2 may, in part, be involved.

In a recent study we have shown that prostaglandin D2 (PGD2) induces human osteoclast (OC) apoptosis through the activation of the chemoattractant receptor homologous molecule expressed on T-helper type 2 cell (CRTH2) receptor and the intrinsic apoptotic pathway. However, the molecular mechanisms underlying this response remain elusive. The objective of this study is to investigate the intracellular signaling pathways mediating PGD2-induced OC apoptosis. OCs were generated by in vitro differentiation of human peripheral blood mononuclear cells (PBMCs), and then treated with or without the selective inhibitors of mitogen-activated protein kinase-extracellular signal-regulated kinase (ERK) kinase, (MEK)-1/2, phosphatidylinositol3-kinase (PI3K) and NF-κB/IκB kinase-2 (IKK2) prior to the treatments of PGD2 as well as its agonists and antagonists. Fluorogenic substrate assay and immunoblotting were performed to determine the caspase-3 activity and key proteins involved in Akt, ERK1/2 and NF-κB signaling pathways. Treatments with both PGD2 and a CRTH2 agonist decreased ERK1/2 (Thr202/Tyr204) and Akt (Ser473) phosphorylation, whereas both treatments increased β-arrestin-1 phosphorylation (Ser412) in the presence of naproxen, which was used to eliminate endogenous prostaglandin production. In the absence of naproxen, treatment with a CRTH2 antagonist increased both ERK1/2 and Akt phosphorylations, and reduced the phosphorylation of β-arrestin-1. Treatment of OCs with a selective MEK-1/2 inhibitor increased caspase-3 activity and OC apoptosis induced by both PGD2 and a CRTH2 agonist. Moreover, a CRTH2 antagonist diminished the selective MEK-1/2 inhibitor-induced increase in caspase-3 activity in the presence of endogenous prostaglandins. In addition, treatment of OCs with a selective PI3K inhibitor decreased ERK1/2 (Thr202/Tyr204) phosphorylation caused by PGD2, whereas increased ERK1/2 (Thr202/Tyr204) phosphorylation by a CRTH2 antagonist was attenuated with a PI3K inhibitor treatment. The DP receptor was not implicated in any of the parameters evaluated. Treatment of OCs with PGD2 as well as its receptor agonists and antagonists did not alter the phosphorylation of RelA/p65 (Ser536). Moreover, the caspase-3 activity was not altered in OCs treated with a selective IKK2/NF-κB inhibitor. In conclusion, endogenous or exogenous PGD2 induces CRTH2-dependent apoptosis in human differentiated OCs; β-arrestin-1, ERK1/2, and Akt, but not IKK2/NF-κB are probably implicated in the signaling pathways of this receptor in the model studied.

In the inflamed uterus, the production and secretion of prostaglandins (PGs) and noradrenergic innervation pattern are changed. Receptor-based control of prostaglandin E2 (PGE2) production and secretion by noradrenaline during uterine inflammation is unknown. The aim of this study was to determine the role of α1-, α2- and β-adrenoreceptors (ARs) in noradrenaline-influenced PG-endoperoxidase synthase-2 (PTGS-2) and microsomal PTGE synthase-1 (mPTGES-1) protein levels in the inflamed pig endometrium, and in the secretion of PGE2 from this tissue. E. coli suspension (E. coli group) or saline (CON group) was injected into the uterine horns. Eight days later, severe acute endometritis developed in the E. coli group. Endometrial explants were incubated with noradrenaline and/or α1-, α2- and β-AR antagonists. In the CON group, noradrenaline did not significantly change PTGS-2 and mPTGES-1 protein expression and increased PGE2 secretion compared to the control values (untreated tissue). In the E. coli group, both enzyme expression and PGE2 release were stimulated by noradrenaline, and these values were higher versus the CON group. The antagonists of α1- and α2-AR isoforms and β-AR subtypes do not significantly alter the noradrenaline effect on PTGS-2 and mPTGES-1 protein levels in the CON group, compared to noradrenaline action alone. In this group, α1A-, α2B- and β2-AR antagonists partly eliminated noradrenaline-stimulated PGE2 release. Compared to the noradrenaline effect alone, α1A-, α1B-, α2A-, α2B-, β1-, β2- and β3-AR antagonists together with noradrenaline reduced PTGS-2 protein expression in the E. coli group. Such effects were also exerted in this group by α1A-, α1D-, α2A-, β2- and β3-AR antagonists with noradrenaline on mPTGES-1 protein levels. In the E. coli group, the antagonists of all isoforms of α1-ARs and subtypes of β-ARs as well as α2A-ARs together with noradrenaline decreased PGE2 secretion versus noradrenaline action alone. Summarizing, in the inflamed pig endometrium, α1(A, B)-, α2(A, B)- and β(1, 2, 3)-ARs mediate the noradrenaline stimulatory effect on PTGE-2 protein expression, while noradrenaline via α1(A, D)-, α2A- and β(2, 3)-ARs increases mPTGES-1 protein expression and α1(A, B, D)-, α2A- and β(1, 2, 3)-ARs are involved in PGE2 release. Data suggest that noradrenaline may indirectly affect the processes regulated by PGE2 by influencing its production. Pharmacological modulation of particular AR isoforms/subtypes can be used to change PGE2 synthesis/secretion to alleviate inflammation and improve uterine function.

The aim of this study was to evaluate the gastroprotective activity of ethanolic extract of geopropolis (EEGP) from Melipona scutellaris and to investigate the possible mechanisms of action. The gastroprotective activity of the EEGP was evaluated using model ulcer induced by ethanol. To elucidate the possible mechanisms of action, we investigated the involvement of the nonprotein sulfhydryl (NP-SH) groups, nitric oxide and prostaglandins. In addition, the antisecretory activity of EEGP was also evaluated by pylorus ligated model. The EEGP orally administrated (300 mg/kg) reduced the ulcerative lesions induced by the ethanol (P < 0.05). Regarding the mechanism of action, the prior administration of nitric oxide and prostaglandins antagonists suppressed the activity of gastroprotective EEGP (P < 0.05). On the other hand the gastroprotective activity of EEGP was kept in the group pretreated with the antagonist of the NP-SH groups; furthermore the antisecretory activity was not significant (P > 0.05). These results support the alternative medicine use of geopropolis as gastroprotective and the activities observed show to be related to nitric oxide and prostaglandins production.

Prostaglandin E2 (PGE2) is an inflammatory mediator synthesized by the brain constitutive cyclooxygenase enzyme. PGE2 binds to G protein-coupled EP1-4 receptors (EP1 to Gq, EP2,4 to Gs, and EP3 to Gi/o). EP2, EP3 and EP4 receptors are expressed in the locus coeruleus (LC), the main noradrenergic nucleus in the brain. EP3 receptors have been explored in the central nervous system, although its role regulating the locus coeruleus neuron activity has not been pharmacologically defined. Our aim was to characterize the function of EP3 receptors in neurons of the LC. Thus, we studied the effect of EP3 receptor agonists on the firing activity of LC cells in rat brain slices by single-unit extracellular electrophysiological techniques. The EP3 receptor agonist sulprostone (0.15 nM-1.28 µM), PGE2 (0.31 nM-10.2 µM) and the PGE1 analogue misoprostol (0.31 nM-2.56 µM) inhibited the firing rate of LC neurons in a concentration-dependent manner (EC50 = 15 nM, 110 nM, and 51 nM, respectively). The EP3 receptor antagonist L-798,106 (3-10 µM), but not the EP2 (PF-04418948, 3-10 µM) or EP4 (L-161,982, 3-10 µM) receptor antagonists, caused rightward shifts in the concentration-effect curves for the EP3 receptor agonists. Sulprostone-induced effect was attenuated by the Gi/o protein blocker pertussis toxin (pertussis toxin, 500 ng ml-1) and the inhibitors of inwardly rectifying potassium channels (GIRK) BaCl2 (300 µM) and SCH-23390 (15 µM). In conclusion, LC neuron firing activity is regulated by EP3 receptors, presumably by an inhibitory Gi/o protein- and GIRK-mediated mechanism.

Dysfunctional regulation of inflammation may contribute to the progression of neurodegenerative diseases. The results of this study revealed that DJ-1, a Parkinson's disease (PD) gene, regulated expression of prostaglandin D2 synthase (PTGDS) and production of prostaglandin D2 (PGD2), by which DJ-1 enhanced anti-inflammatory function of astrocytes. In injured DJ-1 knockout (KO) brain, expression of tumor necrosis factor-alpha (TNF-α) was more increased, but that of anti-inflammatory heme oxygenase-1 (HO-1) was less increased compared with that in injured wild-type (WT) brain. Similarly, astrocyte-conditioned media (ACM) prepared from DJ-1-KO astrocytes less induced HO-1 expression and less inhibited expression of inflammatory mediators in microglia. With respect to the underlying mechanism, we found that PTGDS that induced expression of HO-1 was lower in DJ-1 KO astrocytes and brains compared with their WT counterparts. In addition, PTGDS levels increased in the injured brain of WT mice, but barely in that of KO mice. We also found that DJ-1 regulated PTGDS expression through Sox9. Thus, Sox9 siRNAs reduced PTGDS expression in WT astrocytes, and Sox9 overexpression rescued PTGDS expression in DJ-1 KO astrocytes. In agreement with these results, ACM from Sox9 siRNA-treated astrocytes and that from Sox9-overexpression astrocytes exerted opposite effects on HO-1 expression and anti-inflammation. These findings suggest that DJ-1 positively regulates anti-inflammatory functions of astrocytes, and that DJ-1 dysfunction contributes to the excessive inflammatory response in PD development.

Accumulating evidence indicates that inflammation plays a critical role in cancer development; however, mechanisms of immunosuppression hinder productive anti-tumor immunity to limit immunopathology. Tumor-specific cytotoxic T lymphocyte (CTL) dysfunction or exhaustion by upregulating inhibitory receptors such as programmed cell death 1 (PD-1) in tumor-bearing hosts is one such mechanism. Identification and blockade of the pathways that induce CTL dysfunction has been shown to partially restore CTL function in tumor-bearing hosts. Cyclooxygenase-2 (COX-2) is a rate-limiting enzyme for prostanoid biosynthesis, including prostaglandin E2 (PGE2), and plays a key role in both inflammation and cancer. The disruption of COX2/PGE2 signaling using COX2 inhibitors or PGE2 receptors EP2 and EP4 antagonists, combined with anti-PD-1 blockade was therapeutic in terms of improving eradication of tumors and augmenting the numbers of functional tumor-specific CTLs. Thus, COX2/PGE2 axis inhibition is a promising adjunct therapy to PD-1 blockade for immune-based therapies in cancer.

Isolated cells from adult rat dorsal root ganglia (DRG) are frequently used as a model system to study responses of primary sensory neurons to nociceptor sensitizing agents such as prostaglandin E(2) and prostacyclin, which are presumed to act only on the neurons in typical mixed cell cultures. In the present study, we evaluated the expression of prostaglandin E(2) (EP(4)) and prostacyclin (IP) receptors in cultures of mixed DRG cells and in purified DRG glia. We show here that EP(4) and IP receptor agonists stimulated adenylyl cyclase activity in both mixed DRG cells and in purified DRG glia, and that these responses were specifically inhibited by EP(4) and IP receptor antagonists, respectively. The presence of EP(4) and IP receptors in DRG glia was further confirmed by the expression of EP(4) and IP receptor immunoreactivity and mRNA. With the increasing awareness of neuron-glial interactions within intact DRG and the use of isolated DRG cells in the study of mechanisms underlying nociception, it will be essential to consider the role played by EP(4) and IP receptor-expressing glial cells when evaluating prostanoid-induced sensitization of DRG neurons.

In the present studies we investigated the mechanism of action of prostaglandin E2 (1 mg/kg, i.p.) to induce emesis and defecation and/or tenesmus in the ferret. The emesis was antagonized significantly (P<0.05) by ondansetron (0.3 and 1 mg/kg, i.p.) and (+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenlypiperidine hydrochloride (CP-99,994; 10 mg/kg, i.p.), but neither compound reduced defecations and/or tenesmus, with ondansetron (0.3 mg/kg) actually producing a slight increase (P<0.05). Droperidol (1 and 3 mg/kg), metoclopramide (0.3 and 3 mg/kg), domperidone (0.3 and 3 mg/kg), promethazine (0.3 and 3 mg/kg) and scopolamine (0.3 and 3 mg/kg) failed to reduce prostaglandin E2 induced emesis. However, droperidol (1 and 3 mg/kg) and scopolamine (0.3 and 3 mg/kg) reduced significantly the defecatory and/or tenesmus response (P<0.05). Bilateral abdominal vagotomy was ineffective to reduce emesis and defecations and/or tenesmus. The data suggests that 5-HT3 receptor and NK1 tachykinin receptor antagonists could be useful in the clinic to prevent emesis but not defecations induced by prostaglandin E2.

Although deletion of EP3 receptors is known to ameliorate stroke injury in experimental stroke models, the underlying mechanisms and the effects of EP3-specific antagonists remain poorly understood. Here we demonstrate the protective effect of postischemic treatment with an EP3 antagonist, ONO-AE3-240, through anti-inflammatory and anti-apoptotic effects. In transient focal ischemia models, peritoneal injection of an EP3 antagonist after occlusion-reperfusion reduced infarction, edema and neurological dysfunctions to almost the same levels of those in EP3 knockout (KO) mice. Furthermore, neuronal apoptosis in the ischemic cortex investigated by terminal dUTP nick-end labeling (TUNEL) and caspase-3 immunostaining were ameliorated in EP3 antagonist-treated mice or EP3 KO mice as compared with vehicle-treated mice or wild-type (WT) mice, respectively. There were no significant differences between ONO-AE3-240-injected or EP3 KO mice and vehicle-injected or WT mice, respectively, in mean arterial blood pressure, cerebral blood flow or body temperature. The double-immunostaining showed that EP3 receptor-positive cells were also positive for CD-11b and partially for Neu-N, the marker for microglia and neurons. Deletion of EP3 receptors also reduced damage of the blood-brain barrier, activation of microglia and infiltration of neutrophils into the ischemic cortex. These results suggest that EP3 receptors are involved in stroke injury through the enhancement of inflammatory and apoptotic reactions in the ischemic cortex. Thus, EP3 antagonists may be valuable for the treatment of human stroke.

Cyclooxygenase-2 (COX-2) triggers pro-inflammatory processes that can aggravate neuronal degeneration and functional impairments in many neurological conditions, mainly via producing prostaglandin E2 (PGE2) that activates four membrane receptors, EP1-EP4. However, which EP receptor is the culprit of COX-2/PGE2-mediated neuronal inflammation and degeneration remains largely unclear and presumably depends on the insult types and responding components. Herein, we demonstrated that COX-2 was induced and showed nuclear translocation in two neuronal cell lines - mouse Neuro-2a and human SH-SY5Y - after treatment with neurotoxin 6-hydroxydopamine (6-OHDA), leading to the biosynthesis of PGE2 and upregulation of pro-inflammatory cytokine interleukin-1β. Inhibiting COX-2 or microsomal prostaglandin E synthase-1 suppressed the 6-OHDA-triggered PGE2 production in these cells. Treatment with PGE2 or EP2 selective agonist butaprost, but not EP4 agonist CAY10598, increased cAMP response in both cell lines. PGE2-initiated cAMP production in these cells was blocked by our recently developed novel selective EP2 antagonists - TG4-155 and TG6-10-1, but not by EP4 selective antagonist GW627368X. The 6-OHDA-promoted cytotoxicity was largely blocked by TG4-155, TG6-10-1 or COX-2 selective inhibitor celecoxib, but not by GW627368X. Our results suggest that PGE2 receptor EP2 is a key mediator of COX-2 activity-initiated cAMP signaling in Neuro-2a and SH-SY5Y cells following 6-OHDA treatment, and contributes to oxidopamine-mediated neurotoxicity.

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.

The formation of new blood (angiogenesis) and lymphatic (lymphangiogenesis) vessels are major events associated with most epithelial malignancies, including breast cancer. Angiogenesis is essential for cancer cell survival. Lymphangiogenesis is critical in maintaining tumoral interstitial fluid balance and importing tumor-facilitatory immune cells. Both vascular routes also serve as conduits for cancer metastasis. Intratumoral hypoxia promotes both events by stimulating multiple angiogenic/lymphangiogenic growth factors. Studies on tumor-associated lymphangiogenesis and its exploitation for therapy have received less attention from the research community than those on angiogenesis. Inflammation is a key mediator of both processes, hijacked by many cancers by the aberrant expression of the inflammation-associated enzyme cyclo-oxygenase (COX)-2. In this review, we focus on breast cancer and showed that COX-2 is a major promoter of both events, primarily resulting from the activation of prostaglandin (PG) E receptor EP4 on tumor cells, tumor-infiltrating immune cells, and endothelial cells; and the induction of oncogenic microRNAs. The COX-2/EP4 pathway also promotes additional events in breast cancer progression, such as cancer cell migration, invasion, and the stimulation of stem-like cells. Based on a combination of studies using multiple breast cancer models, we show that EP4 antagonists hold a major promise in breast cancer therapy in combination with other modalities including immune check-point inhibitors.

BTBR mice develop severe diabetes in response to genetically induced obesity due to a failure of the β-cells to compensate for peripheral insulin resistance. In analyzing BTBR islet gene expression patterns, we observed that Pgter3, the gene for the prostaglandin E receptor 3 (EP3), was upregulated with diabetes. The EP3 receptor is stimulated by prostaglandin E2 (PGE2) and couples to G-proteins of the Gi subfamily to decrease intracellular cAMP, blunting glucose-stimulated insulin secretion (GSIS). Also upregulated were several genes involved in the synthesis of PGE2. We hypothesized that increased signaling through EP3 might be coincident with the development of diabetes and contribute to β-cell dysfunction. We confirmed that the PGE2-to-EP3 signaling pathway was active in islets from confirmed diabetic BTBR mice and human cadaveric donors, with increased EP3 expression, PGE2 production, and function of EP3 agonists and antagonists to modulate cAMP production and GSIS. We also analyzed the impact of EP3 receptor activation on signaling through the glucagon-like peptide (GLP)-1 receptor. We demonstrated that EP3 agonists antagonize GLP-1 signaling, decreasing the maximal effect that GLP-1 can elicit on cAMP production and GSIS. Taken together, our results identify EP3 as a new therapeutic target for β-cell dysfunction in T2D.