EP4 Agonist Research Articles

The vasopressin type 2 receptor and prostaglandin receptors EP2 and EP4 can increase aquaporin-2 plasma membrane targeting through a cAMP-independent pathway.

Apical membrane targeting of the collecting duct water channel aquaporin-2 (AQP2) is essential for body water balance. As this event is regulated by Gs coupled 7-transmembrane receptors such as the vasopressin type 2 receptor (V2R) and the prostanoid receptors EP2 and EP4, it is believed to be cAMP dependent. However, on the basis of recent reports, it was hypothesized in the current study that increased cAMP levels are not necessary for AQP2 membrane targeting. The role and dynamics of cAMP signaling in AQP2 membrane targeting in Madin-Darby canine kidney and mouse cortical collecting duct (mpkCCD14) cells was examined using selective agonists against the V2R (dDAVP), EP2 (butaprost), and EP4 (CAY10580). During EP2 stimulation, AQP2 membrane targeting continually increased during 80 min of stimulation; whereas cAMP levels reached a plateau after 10 min. EP4 stimulation caused a rapid and transient increase in AQP2 membrane targeting, but did not significantly increase cAMP levels. After washout of the EP2 agonist or dDAVP, AQP2 membrane abundance remained elevated for at least 80 min, whereas cAMP levels rapidly decreased. Similar effects of the EP2 agonist were also observed for AQP2 constitutively nonphosphorylated at ser-269. The adenylyl cyclase inhibitor SQ22536 did not prevent AQP2 targeting during stimulation of each receptor, nor after dDAVP washout. In conclusion, this study demonstrates that although direct stimulation with cAMP causes AQP2 membrane targeting, cAMP is not necessary for receptor-mediated AQP2 membrane targeting and Gs-coupled receptors can also signal through an alternative pathway that increases AQP2 membrane targeting.

Abstract 3315: Breast cancer stem cell induction by COX-2 via EP4/PI3K-AKT/NOTCH-WNT axis: EP4 as therapeutic target

Abstract Cancer stem-like cells (SLC) resist conventional therapies, necessitating searches for SLC-specific targets. We established that cyclo-oxygenase(COX)-2 expression promotes human breast cancer progression by activation of the prostaglandin(PG)E-2 receptor EP4. Present study revealed that COX-2 induces SLCs by EP4-mediated NOTCH and WNT up-regulation. EP4 antagonist (EP4A) treatment ablated SLCs both in vitro and in vivo. Ectopic COX-2 over-expression in MCF-7 and SKBR-3 human breast cancer cell lines (named MCF-7-COX-2 and SKBR-3-COX-2) resulted in aggressive phenotypes: increased migration/invasion/proliferation, EMT, elevated SLCs, evidenced by spheroid formation for successive generations, increased ALDH activity and co-expression of COX-2/SLC markers. These changes were reversed with COX-2 inhibitor or EP4A, indicating dependence on COX-2/EP4 activities. COX-2 overexpression or EP4 agonist treatment of COX-2 low cells caused up-regulation of NOTCH/WNT pathway genes, blocked with PI3K/AKT inhibitors. Supporting above findings, micro-array analysis showed up-regulation of numerous SLC-regulatory and EMT-associated genes in MCF-7-COX-2 cells. MCF-7-COX-2 cells showed increased orthotopic tumorigenicity and spontaneous multi-organ metastases in NOD/SCID/IL-2Rγ-deficient mice for successive generations with limiting cell inocula. Orthotopic tumors showed significant up-regulation of VEGF-A/C/D, Vimentin and phospho-AKT, down-regulation of E-Cadherin and enrichment of SLC marker positive and spheroid forming cells. MCF-7-COX-2 cells also showed increased lung colonization in NOD/SCID/GUSB-null mice, an effect reversed with EP4 knockdown or EP4A treatment. COX-2, EP4 and ALDH1A expression in situ in human breast cancer tissues were highly correlated with one other, more marked in progressive stage of disease. High COX-2/EP4 expression was linked with poor survival. Thus EP4 represents a novel SLC-ablative target in human breast cancer. (Supported by a grant of the OICR to PKL and a TBCRU fellowship to MM) Citation Format: Mousumi Majumder, Xiping Xin, Ling Liu, Elena Tutunea-Fatan, Mauricio Rodriguez-Torres, Krista Vincent, Andrew Deweyert, Lynne-Marie Postovit, David Hess, Peeyush K. Lala. Breast cancer stem cell induction by COX-2 via EP4/PI3K-AKT/NOTCH-WNT axis: EP4 as therapeutic target. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3315.

This Month in Investigative Urology

No AccessJournal of UrologyThis Month in Investigative Urology1 Aug 2016This Month in Investigative Urology Karl-Erik Andersson Karl-Erik AnderssonKarl-Erik Andersson More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.05.077AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "This Month in Investigative Urology." The Journal of Urology, 196(2), pp. 299–300 References 1 : Promising effects of a novel EP2 and EP3 receptor dual agonist, ONO-8055, on neurogenic underactive bladder in a rat lumbar canal stenosis model. J Urol2016; 196: 609. Abstract, Google Scholar 2 : Role of PAR2 in the development of lower urinary tract dysfunction. J Urol2016; 196: 588. Link, Google Scholar 3 : Urodynamic and immunohistochemical evaluation of intravesical botulinum toxin A delivery using low energy shock waves. J Urol2016; 196: 599. Abstract, Google Scholar © 2016 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 196Issue 2August 2016Page: 299-300 Advertisement Copyright & Permissions© 2016 by American Urological Association Education and Research, Inc.MetricsAuthor Information Karl-Erik Andersson More articles by this author Expand All Advertisement PDF downloadLoading ...

516 Non-steroidal anti-inflammatory drugs exacerbate cutaneous Th2 immune response via a prostaglandin E2-EP2 signaling in keratinocytes

515 A cosmetic formulation with nitric oxide synthase inhibitors shows improvement in facial erythema G Kalahasti, S Burkes, D Gan, T Florence and M Hines SCR, MaryKay, Dallas, TX Facial Erythema (redness) can be a chronic inflammatory skin condition that is characterized by aberrations in immunological responses and cutaneous vasculature. Pathophysiological changes include elevated expression of cytokines, chemokines, impaired skin barrier function, vasodilation of blood vessels and increased blood flow. Elevated levels of proinflammatory cytokines and Nitric Oxide (NO), a key regulator in vasodilation of blood vessels, have been shown in individuals with persistent redness. Currently, there are very few topical formulations available that effectively improve facial redness. Here, we identified four active materials that significantly reduced the expression of pro-inflammatory cytokines and Nitric Oxide Synthase in cultured normal human epidermal keratinocytes (HEKa) and human umbilical vein endothelial cells (HUVEC). A cosmetic formulation containing these ingredients was clinically tested to evaluate visual improvement of facial redness in vivo. Twenty two subjects with persistent facial erythema applied the cream twice daily over 4 weeks. Evaluations from clinical grading revealed a significant reduction in overall facial redness after 4 weeks. In addition, infrared thermograph image analysis showed a significant reduction in skin temperature compared to baseline that correlated with facial redness. This cosmetic formulation effectively reduced facial erythema with improvements in overall reduction of redness and skin temperature. 516 Non-steroidal anti-inflammatory drugs exacerbate cutaneous Th2 immune response via a prostaglandin E2-EP2 signaling in keratinocytes Y Sawada, T Honda, M Nakamura and K Kabashima 1 Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan and 2 Department of Dermatology, University of Occupational and Environmental Health, Kitakyushu, Japan Non-steroidal anti-inflammatory drugs (NSAIDs) have been widely used as anti-inflammatory drugs through an inhibition of the activity of cyclooxygenase (COX) and a consequent suppression of prostanoid synthesis. However, topical application of NSAIDs is known to occasionally aggravate Th2-type cutaneous immune responses, such as atopic dermatitis (AD). It raises a concern about the validity of NSAIDs on the treatment of AD. To address this issue, we used a hapten-induced AD mouse model, in which prostaglandin (PG) E2 is abundantly produced in the skin. We found that the mice treated with topical application of NSAIDs exhibited significantly exacerbated AD-like inflammation, an increased number of IL-4 producing Th2 cells in the skin, and an increased serum IgE level. In addition, treatment with NSAIDs enhanced gene expression of thymic stromal lymphopoietin (TSLP) in keratinocytes (KCs) both in vivo and in vitro. Exacerbation of Th2-type skin immune response by NSAIDs was completely abolished in TSLP receptor-deficient mice, indicating that TSLP mediated this exacerbation. We also found that KCs highly expressed EP2, one of PGE2 receptor subtypes, and that the effects of NSAIDs on skin inflammation and TSLP expression in KCs were completely reversed by administration of an EP2 agonist. Moreover, treatment of KCs with an antagonist for EP2 mimicked the effect of NSAID on TSLP expression on KCs. Intriguingly, surface expression of protease-activated receptor 2 (PAR2), which is known to mediate TSLP production by KCs, was down-regulated by EP2 stimulation in a clathrin-dependent manner. These results suggest that PGE2-EP2 signaling plays inhibitory roles for cutaneous Th2-type immune response by down-regulating PAR2 expression on KCs and subsequent TSLP expression.

Preferred recycling pathway by internalized PGE2 EP4 receptor following agonist stimulation in cultured dorsal root ganglion neurons contributes to enhanced EP4 receptor sensitivity

Prostaglandin E2 (PGE2), a well-known pain mediator abundantly produced in injured tissues, sensitizes nociceptive dorsal root ganglion (DRG) neurons (nociceptors) through its four EP receptors (EP1–4). Our prior study showed that PGE2 or EP4 agonist stimulates EP4 externalization and this event was not only suppressed by the inhibitor of anterograde export, but also by the recycling inhibitor (St-Jacques and Ma, 2013). These data suggest that EP4 recycling also contributes to agonist-enhanced EP4 surface abundance. In the current study, we tested this hypothesis using antibody-feeding-based internalization assay, recycling assay and FITC-PGE2 binding assay. We observed that selective EP4 agonist 1-hydroxy-PGE1 (1-OH-PGE1) or CAY10850 time- and concentration-dependently increased EP4 internalization in cultured DRG neuron. Internalized EP4 was predominantly localized in the early endosomes and recycling endosomes, but rarely in the late endosomes and lysosomes. These observations were confirmed by FITC-PGE2 binding assay. We further revealed that 1-OH-PGE1 or CAY10850 time- and concentration-dependently increased EP4 recycling. Double exposures to 1-OH-PGE1 induced a greater increase in calcitonin gene-related peptide (CGRP) release than a single exposure or vehicle exposure, an event blocked by pre-treatment with the recycling inhibitor monensin. Our data suggest that EP4 recycling contributes to agonist-induced cell surface abundance and consequently enhanced receptor sensitivity. Facilitating EP4 externalization and recycling is a novel mechanism underlying PGE2-induced nociceptor sensitization.

PGE2 – EP3 Receptor Signaling Involves Rho‐kinase Pathway in Hyperoxia‐Induced Airway Hyperresponsiveness

Hyperoxic exposure is associated with airway hyperresponsiveness and it was shown that hyperoxia upregulates synthesis of prostaglandin E2 (PGE2), which induces contraction of airway smooth muscle via EP1 and EP3 receptors. It is not clear through which signaling pathway PGE2 exerts contractile effect on airway smooth muscle under hyperoxic conditions. Therefore, we hypothesized that under hyperoxic condition PGE2 induces contraction of airway smooth muscle via EP3 receptors by activating Rho‐kinase signaling pathway. Tracheal cylinders were obtained from 12 days Wistar rat pups exposed to hyperoxia (≥ 95% O2) or room air for seven days. These cylinders were used to study contractile responses evoked by EP3 agonist (sulprostone, 10−9 – 10−5 M); methacholine (MCh, 10−8 – 10−4 M) in absence or presence of an EP3 antagonist (L‐798106, 10 μM) or Rho‐kinase inhibitors (Y‐27632, 10 μM or fasudil, 10 μM).Both, sulprostone and MCh induced dose‐dependent contractile responses of airway smooth muscle, respectively. The contractile responses were significantly higher in preparations obtained from animals exposed to hyperoxia as compared to those obtained from room air controls. These enhanced contractile responses in hyperoxic tissues were reversed by pre‐incubating tissues with Y‐27632 or fasudil. EP3 antagonist also significantly attenuated contractile responses evoked by sulprostone. The results of this study revealed that PGE2 – EP3 receptor signaling involves Rho‐kinase pathway and we speculate that the use of Rho‐kinase inhibitors or EP3 antagonist could attenuate the contractile effects of PGE2 and this might be an effective therapeutic approach to reverse hyperoxia‐induced airway hyperresponsiveness.Support or Funding InformationSupported by MEST

Structural optimization and structure–functional selectivity relationship studies of G protein-biased EP2 receptor agonists

The modification of the novel G protein-biased EP2 agonist 1 has been investigated to improve its G protein activity and develop a better understanding of its structure–functional selectivity relationship (SFSR). The optimization of the substituents on the phenyl ring of 1, followed by the inversion of the hydroxyl group on the cyclopentane moiety led to compound 9, which showed a 100-fold increase in its G protein activity compared with 1 without any increase in β-arrestin recruitment. Furthermore, SFSR studies revealed that the combination of meta and para substituents on the phenyl moiety was crucial to the functional selectivity.

Prostaglandin E₂ constrains systemic inflammation through an innate lymphoid cell-IL-22 axis.

Systemic inflammation, which results from the massive release of proinflammatory molecules into the circulatory system, is a major risk factor for severe illness, but the precise mechanisms underlying its control are not fully understood. We observed that prostaglandin E2 (PGE2), through its receptor EP4, is down-regulated in human systemic inflammatory disease. Mice with reduced PGE2 synthesis develop systemic inflammation, associated with translocation of gut bacteria, which can be prevented by treatment with EP4 agonists. Mechanistically, we demonstrate that PGE2-EP4 signaling acts directly on type 3 innate lymphoid cells (ILCs), promoting their homeostasis and driving them to produce interleukin-22 (IL-22). Disruption of the ILC-IL-22 axis impairs PGE2-mediated inhibition of systemic inflammation. Hence, the ILC-IL-22 axis is essential in protecting against gut barrier dysfunction, enabling PGE2-EP4 signaling to impede systemic inflammation.

Prostaglandin E2 Stimulates EP2, Adenylate Cyclase, Phospholipase C, and Intracellular Calcium Release to Mediate Cyclic Adenosine Monophosphate Production in Dental Pulp Cells

IntroductionProstaglandin E2 (PGE2) plays a crucial role in pulpal inflammation and repair. However, its induction of signal transduction pathways is not clear but is crucial for future control of pulpal inflammation. MethodsPrimary dental pulp cells were exposed to PGE2 and 19R-OH PGE2 (EP2 agonist) or sulprostone (EP1/EP3 agonist) for 5 to 40 minutes. Cellular cyclic adenosine monophosphate (cAMP) levels were measured using the enzyme-linked immunosorbent assay. In some experiments, cells were pretreated with SQ22536 (adenylate cyclase inhibitor), H89 (protein kinase A inhibitor), dorsomorphin (adenosine monophosphate–activated protein kinase inhibitor), U73122 (phospholipase C inhibitor), thapsigargin (inhibitor of intracellular calcium release), W7 (calmodulin antagonist), verapamil (L-type calcium channel blocker), and EGTA (extracellular calcium chelator) for 20 minutes before the addition of PGE2. ResultsPGE2 and 19R-OH PGE2 (EP2 agonist) stimulated cAMP production, whereas sulprostone (EP1/EP3 agonist) shows little effect. PGE2-induced cAMP production was attenuated by SQ22536 and U73122 but not H89 and dorsomorphin. Intriguingly, thapsigargin and W7 prevented PGE2-induced cAMP production, but verapamil and EGTA showed little effect. ConclusionsThese results indicate that PGE2-induced cAMP production is associated with EP2 receptor and adenylate cyclase activation. These events are mediated by phospholipase C, intracellular calcium release, and calcium-calmodulin signaling. These results are helpful for understanding the role of PGE2 in pulpal inflammation and repair and possible future drug intervention.

Promising Effects of a Novel EP2 and EP3 Receptor Dual Agonist, ONO-8055, on Neurogenic Underactive Bladder in a Rat Lumbar Canal Stenosis Model

We investigated whether the novel EP (prostaglandin E2) receptor agonist ONO-8055 would improve the lower urinary tract dysfunction of neurogenic underactive bladder in a rat lumbar spinal canal stenosis model. First, we studied the agonistic effect of ONO-8055 on EP receptors in EP receptor expressing CHO (Chinese hamster ovary) cells using the increase in the intracellular calcium level and intracellular cAMP (cyclic adenosine monophosphate) production as indicators of receptor activation. The effects of ONO-8055 on bladder and urethral strips from normal rats were then investigated. Finally, the effects of ONO-8055 on bladder and urethral function in rats with lumbar spinal canal stenosis were evaluated by awake cystometry and intraurethral perfusion pressure, respectively. The effects of tamsulosin and distigmine on urethral pressure were also evaluated. ONO-8055 is a highly potent and selective agonist for EP2 and EP3 receptors on CHO cells. While this compound contracted bladder strips, it relaxed urethral strips. Awake cystometry showed that ONO-8055 significantly decreased bladder capacity, post-void residual urine and voiding pressure. Compared with vehicle, tamsulosin and ONO-8055 significantly decreased urethral pressure. ONO-8055 decreased post-void residual urine, probably by decreasing bladder capacity. The decrease in voiding pressure probably resulted from the lowered urethral pressure due to relaxation of the urethra. Thus, the novel EP2 and EP3 receptor dual agonist ONO-8055 has the potential to improve neurogenic underactive bladder.

Prostaglandin E2 possesses different potencies in inducing Vascular Endothelial Growth Factor and Interleukin-8 production in COPD human lung fibroblasts

We studied the role of PGE2, its biosynthetic enzymes and its receptors, in regulating the functions of lung fibroblasts through the production of Vascular Endothelial Growth Factor (VEGF) and Interleukin-8 (IL-8) in COPD subjects.Lung fibroblasts from Control (C) (n=6), Smoker (HS) (n=6) and COPD patients (n=8) were cultured, and basal PGE2, VEGF, and IL-8 measured in supernatants by ELISA. COX-1/COX-2 and EP receptors expression were assessed by western blot and by RT-PCR. Release of VEGF and IL-8 by human fetal lung fibroblasts (HFL-1; lung, diploid, human) was evaluated under different conditions.PGE2, VEGF, and IL-8 levels, COX-2, EP2, and EP4 protein expression and mRNA were increased in COPD when compared to Controls. Low concentrations of synthetic PGE2 increased the release of VEGF in HFL-1, but higher concentrations were needed to induce the release of IL-8. This effect was mimicked by an EP2 agonist and modulated by an EP4 antagonist.In the airways of COPD subjects, fibroblast-derived PGE2 may regulate angiogenesis and inflammation through the production of VEGF and IL-8 respectively, suggesting that the increase in expression of COX-2, EP2 and EP4 observed in COPD fibroblasts may contribute to steering the role of PGE2 from homeostatic to pro-inflammatory.

Abstract TMP51: Activation of PGE2 EP1 Receptor Promotes Microglial Phagocytosis Though CD36 Receptor Recycling

Background and Purpose: We have shown that the deletion of prostaglandin E2 (PGE2) EP1 receptor undermined microglial phagocytosis in vivo. However, the related mechanism still remains unknown. CD36 is a type II scavenger receptor that mediates microglial phagocytosis. It was reported that microglial CD36 contributes to hematoma clearance after intracerebral hemorrhage (ICH). In this study, we sought to identify the effects of two EP1 agonists on microglial phagocytosis and CD36 receptor recycling in primary microglial cultures. Method: The EP1 agonists: 17-phenyl trinor-PGE2 (17-pt-PGE2) and ONO-DI-004 were used to treat primary microglial cultures, respectively. Fluorescent latex beads or CFSE-labeled red blood cells (CFSE-RBCs) were added to microglial cells at a concentration of ten beads/RBCs per cell for live-cell imaging. Immunocytochemistry was also performed with antibody against ionized calcium-binding adapter molecule 1 (Iba1, a marker to microglia). Furthermore, we analyzed CD36 receptor recycling by using an established receptor recycling assay. Result: The primary microglial cells were incubated with fluorescent latex beads and treated with the EP1 agonist 17-pt-PGE2 for 120 min. We found that 17-pt-PGE2 significantly increased the number of attached beads to microglial cells at the concentration of 10 μM from 40 min to 120 min of incubation. Furthermore, we analyzed CD36 receptor recycling using an established receptor recycling assay and the quantitative results showed that 10 μM of 17-pt-PGE2 treatment markedly increased the area of recycled CD36 receptor in microglial cells. We then treated primary microglial cells with another selective EP1 agonist ONO-DI-004 for 120 min at the concentration of 5 μM and 10 μM, respectively. The area of CFSE-RBCs after ice-cold PBS washing was markedly increased with 10 μM of ONO-DI-004 treatment. In the live-cell imaging experiment, 10 μM of ONO-DI-004 treatment also significantly increased the number of attached CFSE-RBCs to microglial cells from 25 min to 60 min of incubation. Conclusions: This study showed that the EP1 agonists: 17-pt-PGE2 and ONO-DI-004 can promote microglial phagocytosis in vitro and enhanced CD36 receptor recycling could mediate some of these effects.

Curcumin Ameliorates the Reduction Effect of PGE2 on Fibrillar β-Amyloid Peptide (1-42)-Induced Microglial Phagocytosis through the Inhibition of EP2-PKA Signaling in N9 Microglial Cells.

Inflammatory activation of microglia and β amyloid (Aβ) deposition are considered to work both independently and synergistically to contribute to the increased risk of Alzheimer’s disease (AD). Recent studies indicate that long-term use of phenolic compounds provides protection against AD, primarily due to their anti-inflammatory actions. We previously suggested that phenolic compound curcumin ameliorated phagocytosis possibly through its anti-inflammatory effects rather than direct regulation of phagocytic function in electromagnetic field-exposed N9 microglial cells (N9 cells). Here, we explored the prostaglandin-E2 (PGE2)-related signaling pathway that involved in curcumin-mediated phagocytosis in fibrillar β-amyloid peptide (1–42) (fAβ42)-stimulated N9 cells. Treatment with fAβ42 increased phagocytosis of fluorescent-labeled latex beads in N9 cells. This increase was attenuated in a dose-dependent manner by endogenous and exogenous PGE2, as well as a selective EP2 or protein kinase A (PKA) agonist, but not by an EP4 agonist. We also found that an antagonist of EP2, but not EP4, abolished the reduction effect of PGE2 on fAβ42-induced microglial phagocytosis. Additionally, the increased expression of endogenous PGE2, EP2, and cyclic adenosine monophosphate (AMP), and activation of vasodilator-stimulated phosphoprotein, cyclic AMP responsive element-binding protein, and PKA were depressed by curcumin administration. This reduction led to the amelioration of the phagocytic abilities of PGE2-stimulated N9 cells. Taken together, these data suggested that curcumin restored the attenuating effect of PGE2 on fAβ42-induced microglial phagocytosis via a signaling mechanism involving EP2 and PKA. Moreover, due to its immune modulatory effects, curcumin may be a promising pharmacological candidate for neurodegenerative diseases.

The influence of prostaglandin E2 on the production of IFN-γ by bovine CD4+, CD8+ and WC1+ T cells

The aim of these studies was to assess the influence of prostaglandin E2 (PGE2) on the production of interferon-gamma (IFN-γ) by bovine CD4+, CD8+, and WC1+ T cells and furthermore, should this effect exist, to identify the E-prostanoid (EP) receptor subtype(s) responsible for this influence. We here report that exposure of bovine peripheral blood mononuclear cells (PBMCs) to PGE2 significantly and dose-dependently decreased the percentage of IFN-γ-producing CD4+ and CD8+ T cells. It was also shown that PGE2 reduced IFN-γ production by WC1+ T cells, but this effect was not dose dependent. The impairment of IFN-γ production should be recognized as an anti-inflammatory and immunosuppressive action, thus the obtained results confirm the paradoxical status of PGE2 as a proinflammatory factor with immunosuppressive activity. The blockade of EP1, EP2, EP3, and EP4 receptors did not prevent PGE2-induced reduction of IFN-γ production by CD4+ and CD8+ T cells, indicating that this effect of PGE2 is not mediated through EP receptors. On the contrary, the blockade of either EP2 or EP4 receptors, but not EP1 or EP3 receptors, prevented the PGE2-induced reduction of percentage of IFN-γ-producing WC1+ T cells. These findings indicate that the ability of PGE2 to impair IFN-γ production by WC1+ T cells is mediated via EP2 and EP4 receptors. These results suggest the possibility of pharmacological manipulation of IFN-γ production by WC1+ T cells via selective antagonists and agonists of EP2 and EP4 receptors.

Discovery of G Protein-Biased EP2 Receptor Agonists.

To identify G protein-biased and highly subtype-selective EP2 receptor agonists, a series of bicyclic prostaglandin analogues were designed and synthesized. Structural hybridization of EP2/4 dual agonist 5 and prostacyclin analogue 6, followed by simplification of the ω chain enabled us to discover novel EP2 agonists with a unique prostacyclin-like scaffold. Further optimization of the ω chain was performed to improve EP2 agonist activity and subtype selectivity. Phenoxy derivative 18a showed potent agonist activity and excellent subtype selectivity. Furthermore, a series of compounds were identified as G protein-biased EP2 receptor agonists. These are the first examples of biased ligands of prostanoid receptors.

Impaired E Prostanoid2 Expression and Resistance to Prostaglandin E2 in Nasal Polyp Fibroblasts from Subjects with Aspirin-Exacerbated Respiratory Disease.

Recurrent, rapidly growing nasal polyps are hallmarks of aspirin-exacerbated respiratory disease (AERD), although the mechanisms of polyp growth have not been identified. Fibroblasts are intimately involved in tissue remodeling, and the growth of fibroblasts is suppressed by prostaglandin E2 (PGE2), which elicits antiproliferative effects mediated through the E prostanoid (EP)2 receptor. We now report that cultured fibroblasts from the nasal polyps of subjects with AERD resist this antiproliferative effect. Fibroblasts from polyps of subjects with AERD resisted the antiproliferative actions of PGE2 and a selective EP2 agonist (P < 0.0001 at 1 μM) compared with nasal fibroblasts from aspirin-tolerant control subjects undergoing polypectomy or from healthy control subjects undergoing concha bullosa resections. Cell surface expression of the EP2 receptor protein was lower in fibroblasts from subjects with AERD than in fibroblasts from healthy control subjects and aspirin-tolerant subjects (P < 0.01 for both). Treatment of the fibroblasts with trichostatin A, a histone deacetylase inhibitor, significantly increased EP2 receptor mRNA in fibroblasts from AERD and aspirin-tolerant subjects but had no effect on cyclooxygenase-2, EP4, and microsomal PGE synthase 1 (mPGES-1) mRNA levels. Histone acetylation (H3K27ac) at the EP2 promoter correlated strongly with baseline EP2 mRNA (r = 0.80; P < 0.01). These studies suggest that the EP2 promotor is under epigenetic control, and one explanation for PGE2 resistance in AERD is an epigenetically mediated reduction of EP2 receptor expression, which could contribute to the refractory nasal polyposis typically observed in this syndrome.

An EP2 Agonist Facilitates NMDA-Induced Outward Currents and Inhibits Dendritic Beading through Activation of BK Channels in Mouse Cortical Neurons.

Prostaglandin E2 (PGE2), a major metabolite of arachidonic acid produced by cyclooxygenase pathways, exerts its bioactive responses by activating four E-prostanoid receptor subtypes, EP1, EP2, EP3, and EP4. PGE2 enables modulating N-methyl-D-aspartate (NMDA) receptor-mediated responses. However, the effect of E-prostanoid receptor agonists on large-conductance Ca2+-activated K+ (BK) channels, which are functionally coupled with NMDA receptors, remains unclear. Here, we showed that EP2 receptor-mediated signaling pathways increased NMDA-induced outward currents (I NMDA-OUT), which are associated with the BK channel activation. Patch-clamp recordings from the acutely dissociated mouse cortical neurons revealed that an EP2 receptor agonist activated I NMDA-OUT, whereas an EP3 receptor agonist reduced it. Agonists of EP1 or EP4 receptors showed no significant effects on I NMDA-OUT. A direct perfusion of 3,5′-cyclic adenosine monophosphate (cAMP) through the patch pipette facilitated I NMDA-OUT, which was abolished by the presence of protein kinase A (PKA) inhibitor. Furthermore, facilitation of I NMDA-OUT caused by an EP2 receptor agonist was significantly suppressed by PKA inhibitor. Finally, the activation of BK channels through EP2 receptors facilitated the recovery phase of NMDA-induced dendritic beading in the primary cultured cortical neurons. These results suggest that a direct activation of BK channels by EP2 receptor-mediated signaling pathways plays neuroprotective roles in cortical neurons.

Discovery of highly potent dual EP(2) and EP(3) agonists with subtype selectivity.

The cyclic carbamate derivatives, 2-{[2-((4S)-4-{(1E,3R)-8-fluoro-3-hydroxy-4,4-dimethyl-1-octenyl}-2-oxo-1,3-oxazolidin-3-yl)ethyl]sulfanyl}-1,3-thiazole-4-carboxylic acid (5) and 2-{[2-((4S)-4-{(1E,3R)-3-[1-(4-fluorobutyl)cyclobutyl]-3-hydroxy-1-propenyl}-2-oxo-1,3-oxazolidin-3-yl)ethyl]sulfanyl}-1,3-thiazole-4-carboxylic acid (7) were identified as the first potent dual EP2 and EP3 agonists with selectivity against the EP1 and EP4 subtypes. Compounds 5 and 7 demonstrated highly potent dual EP2 and EP3 agonist activity with EC50 values of 10nM or less. In addition, these compounds possess structural features distinct from natural prostaglandins, such as a cyclic carbamate moiety, a dimethyl or cyclobutyl group and a terminal fluorine atom.

Prostaglandin E2 Signaling through EP4 Receptor Promotes Hematopoietic Stem Cell Niche Regeneration and Enhances Hematopoietic Recovery

High-dose radiation treatment for hematological malignancies such as leukemia, lymphoma or multiple myeloma induces damage to the bone marrow microenvironment that limits hematopoietic regeneration. We previously demonstrated that single administration of dimethyl prostaglandin E2 (dmPGE2) at 24 hrs post lethal irradiation (796 cGys; LD70/30) in mice increases survival and accelerates hematopoietic recovery. Since bone marrow niches regulate hematopoiesis, we investigated whether the effects of PGE2 administration on hematopoietic recovery and survival were dependent on PGE2 signaling in bone marrow stromal cells. Total body irradiation (TBI) severely disrupts bone marrow niche components including MSC, osteoblasts and endothelial cells. Multivariate flow cytometry revealed that exposure of mice to 650 cGy TBI reduced the total number of bone marrow MSC defined as CD45- Ter119- CD31- PDGFR+ CD51+ MSC by 8 fold and reduced functional fibroblast colony formation (CFU-F) by 8.5 fold. Osteoblast (OB) and endothelial cell (EC) counts were reduced by 6.5 fold and 6 fold respectively. Treatment of mice with dmPGE2 at 24 hours post-irradiation substantially rescued MSC and EC, which were 3.5 and 2.2-fold higher compared to un-treated irradiated mice. Histological and flow cytometric analysis indicated that total OB, OB precursors and mesenchymal progenitor cells (MPC) in bone marrow were also enhanced by PGE2 administration. Interestingly, dmPGE2 failed to rescue MSC and OB from aged (24-27 mo/old) mice compared to young (4-6 mo/old) mice. Since PGE2 signals through four receptors (EP1-4), each with unique signaling pathways, we hypothesized that the hematopoietic niche regeneration was due to activation of PGE2 signaling via one or more EP receptors. Treatment of mice with the EP4 receptor agonist L-902,688 following irradiation enhanced the recovery of bone marrow MSC by 3.1 fold and EC by 2.0 fold compared to untreated irradiated control mice. EP1, EP2 and EP3 receptor agonists failed to enhance hematopoietic niche regeneration in irradiated mice. To further investigate the role of EP4 signaling in bone marrow niche reconstruction and hematopoietic recovery, we transplanted BM cells from wild-type mice into syngeneic wild-type or conditional inducible EP4-/- recipients after lethal irradiation, and analyzed stromal cells recovery and hematopoietic reconstitution. At 15 days post-transplantation, chimeric mice with EP4-/- stroma displayed attenuated niche recovery and hematopoietic reconstitution compared to mice with wild-type stroma. PGE2 or EP4 agonist administration increased the expression of the endogenous anti-apoptotic protein Survivin, and enhanced survival of MSC and EC. In conclusion, our study suggests that PGE2 signaling through the EP4 receptor increases hemotopoietic recovery after TBI by enhancing the survival and expansion of MSC and endothelial cells. Furthermore, our data suggest the modulation/regulation PGE2 signaling in hematopoietic niche components could be beneficial to enhance HSC recovery following clinical hematopoietic transplantation. Disclosures No relevant conflicts of interest to declare.

Low E-prostanoid 2 receptor levels and deficient induction of the IL-1β/IL-1 type I receptor/COX-2 pathway: Vicious circle in patients with aspirin-exacerbated respiratory disease

We hypothesized that the 2 reported alterations in aspirin-exacerbated respiratory disease (AERD), reduced expression/production of COX-2/prostaglandin (PG) E2 and diminished expression of E-prostanoid (EP) 2 receptor, are closely linked. We sought to determine the mechanisms involved in the altered regulation of the COX pathway in patients with AERD. Fibroblasts were obtained from nasal mucosa; samples of control subjects (NM-C, n=8) and from nasal polyps from patients with aspirin-exacerbated respiratory disease (NP-AERD, n=8). Expression of the autocrine loop components regulating PGE2 production and signaling, namely IL-1 type I receptor (IL-1RI), COX-2, microsomal prostaglandin E synthase 1 (mPGES-1), and EP receptors, was assessed at baseline and after stimulation with IL-1β, PGE2, and specific EP receptor agonists. Compared with NM-C fibroblasts, basal expression levels of IL-1RI and EP2 receptor were lower in NP-AERD fibroblasts. IL-1β-induced IL-1RI, COX-2, and mPGES-1 expression levels were also lower in these cells. Levels of IL-1RI positively correlated with COX-2 and mPGES-1 expression in both NM-C and NP-AERD fibroblasts. Incubation with either exogenous PGE2 or selective EP2 agonist significantly increased expression of IL-1RI in NM-C fibroblasts and had hardly any effect on NP-AERD fibroblasts. Alterations in IL-1RI, COX-2, and mPGES-1 expression that were found in NP-AERD fibroblasts were corrected when EP2 receptor expression was normalized by transfection of NP-AERD fibroblasts. Altered expression of EP2 in patients with AERD contributes to deficient induction of IL-1RI, reducing the capacity of IL-1β to increase COX-2 and mPGES-1 expression, which results in low PGE2 production. This impairment in the generation of PGE2 subsequently reduces its ability to induce IL-1RI.