The EP2 agonist, omidenepag, alters the physical stiffness of 3D spheroids prepared from human corneal stroma fibroblasts differently depending on the osmotic pressure.

The objective of the current study was to examine the drug-induced effects of the EP2 agonist, omidenapag (OMD), on human corneal stroma, two- and three-dimensional (2D and 3D) cultures of human corneal stroma fibroblasts (HCSFs). The drug-induced effects on 2D monolayers and 3D spheroids were characterized by examining the ultrastructures by scanning electron microscope (SEM), transendothelial electrical resistance (TEER) measurements, and fluorescein isothiocyanate (FITC)-dextran permeability. The physical properties of 3D spheroids with respect to size and stiffness were also examined. In addition, the gene expressions of extracellular matrix (ECM) molecules, including collagen (COL) 1, 4, and 6, and fibronectin (FN), a tissue inhibitor of metalloproteinase (TIMP) 1-4, matrix metalloproteinase (MMP) 2, 9, and 14, aquaporin1 (AQP1), and several endoplasmic reticulum (ER) stress-related factors were evaluated. In the 2D HCSFs, OMD induced (1) a significant increase in ECM deposits, as evidenced by SEM, the mRNA expression of COL4 and FN, and (2) a decrease in TEER values and a concentration-dependent increase in FITC-dextran permeability. In the case of 3D spheroids, OMD had no effect on size but a substantial increase in stiffness was observed. Furthermore, such OMD-induced effects on stiffness were dramatically modulated by the osmotic pressure of the system. In contrast to the above 2D cultures, among the ECM molecules and the modulators of 3D spheroids, namely, TIMPS and MMPs, the down-regulation of COL1, TIMP1 and 2 and the up-regulation of MMP9 were observed. Interestingly, such diversity in terms of OMD-induced gene expressions between 2D and 3D cultures was also recognized in AQP1 (2D; no significant change, 3D; significant up-regulation) and ER stress-related genes. The findings presented herein suggest that the EP2 agonist, OMD, alters the physical stiffness of 3D spheroids obtained from human corneal stroma fibroblasts and this alteration is dependent on the osmotic pressures. 2D and 3D cell cultures may be useful for evaluating the drug induced effects of OMD toward human corneal stroma.

Prostaglandin F2α and EP2 agonists, and a ROCK inhibitor modulate the formation of 3D organoids of Grave's orbitopathy related human orbital fibroblasts.

3D organoid cultures were used to elucidate the periocular effects of several anti-glaucoma drugs including a prostaglandin F2α analogue (bimatoprost acid; BIM-A), EP2 agonist (omidenepag; OMD) or a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor (ripasudil; Rip) on Grave's orbitopathy (GO) related orbital fatty tissue. 3D organoids were prepared from GO related human orbital fibroblasts (GHOFs) obtained from patients with GO. The effects of either 100 nM BIM-A, 100 nM OMD or 10 µM Rip on the 3D GHOFs organoids were examined with respect to organoid size, physical properties by a micro-squeezer, and the mRNA expression of extracellular matrix (ECM) proteins including collagen (COL) 1, COL 4, COL 6, and fibronectin (FN), ECM regulatory genes including lysyl oxidase (LOX), Connective Tissue Growth Factor (CTGF) and inflammatory cytokines including interleukin-1ß (IL1ß) and interleukin-6 (IL6). The size of the 3D GHOFs organoids decreased substantially in the presence of BIM-A, but also increased substantially in the presence of the others (OMD or Rip). The physical stiffness of the 3D GHOFs organoids was significantly decreased by Rip. BIM-A caused significantly the down-regulation of three ECM genes, Col 1, Col 6 and Fn, and two ECM regulatory genes and the up-regulation of IL6. In the presence of OMD, two ECM genes, Col 1 and Fn, and LOX were significantly down-regulated but IL1ß and IL6 were significantly up-regulated. In the case of Rip, Col 1, FN and CTGF were significant down-regulated. Our present findings indicate that anti-glaucoma drugs modulate the structures and physical properties 3D GHOFs organoids in different manners by modifying the gene expressions of ECM, ECM regulatory factors and inflammatory cytokines. The results indicate that the benefits and demerits of anti-glaucoma medications need to be scrutinized carefully, in cases of patients with GO.

Simultaneous Use of ROCK Inhibitors and EP2 Agonists Induces Unexpected Effects on Adipogenesis and the Physical Properties of 3T3-L1 Preadipocytes.

To elucidate the additive effects of an EP2 agonist, omidenepag (OMD) or butaprost (Buta) on the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) on adipose tissue, two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells were analyzed by lipid staining, the mRNA expression of adipogenesis-related genes, extracellular matrix (ECM) molecules including collagen (Col) -1, -4 and -6, and fibronectin (Fn), and the sizes and physical properties of 3D organoids, as measured by a micro-squeezer. The results indicate that adipogenesis induced (1) an enlargement of the 3D organoids; (2) a substantial enhancement in lipid staining as well as the expression of the Pparγ, Ap2 and Leptin genes; (3) a significant softening of the 3D organoids, the effects of which were all enhanced by Rip except for Pparγ expression; and (4) a significant downregulation in Col1 and Fn, and a significant upregulation in Col4, Col6, the effects of which were unchanged by Rip. When adding the EP2 agonist to Rip, (1) the sizes of the 3D organoids were reduced substantially; (2) lipid staining was increased (OMD), or decreased (Buta); (3) the stiffness of the 3D organoids was substantially increased in Buta; (4-1) the expression of Pparγ was suppressed (2D, OMD) or increased (2D, Buta), and the expressions of Ap2 were downregulated (2D, 3D) and Leptin was increased (2D) or decreased (3D), (4-2) all the expressions of four ECM molecules were upregulated in 2D (2D), and in 3D, the expression of Col1, Col4 was upregulated. The collective findings reported herein indicate that the addition of an EP2 agonist, OMD or Buta significantly but differently modulate the Rip-induced effects on adipogenesis and the physical properties of 2D and 3D cultured 3T3-L1 cells.

Altered PGE2-EP2 is associated with an excessive immune response in HBV-related acute-on-chronic liver failure.

BACKGROUND AND AIMS: Prostaglandin E receptor 2 (EP2) is an immune modulatory molecule that regulates the balance of immunity. Here we investigated the role of EP2 in immune dysregulation in patients with acute-on-chronic liver failure (ACLF). METHODS: Plasma Progstaglandin E2 (PGE2) levels and EP2 expression on immune cells were determined in blood samples collected from patients with chronic hepatitis B related ACLF(HB-ACLF), patients with chronic hepatitis B (CHB), acute decompensated cirrhosis without ACLF (AD) and healthy controls (HC). Cytokine production, bacterial phagocytosis and reactive oxygen species (ROS) production were detected to explore the role of EP2 in regulating immune cell functions. RESULTS: The plasma PGE2 levels were increased and EP2 expression on CD8+ T cells was decreased in HB-ACLF compared with those in controls. The levels of PGE2 and EP2 were associated with systemic inflammation and disease severity. Small molecular chemicals against EP2 increased both cytokine secretion in PBMCs and ROS production in neutrophils and monocytes, but decreased monocytic phagocytosis. By contrast, an EP2-selective agonist reduced the production of a series of cytokines in PBMCs, but increased G-CSF. CONCLUSION: Altered PGE2-EP2 augmented the excessive inflammation of innate and adaptive immune cells in response to LPS or E. coli in HB-ACLF. EP2 might be a new potential target for HB-ACLF treatment.

Prostaglandin E2 suppresses hCAP18/LL-37 expression in human macrophages via EP2/EP4: implications for treatment of Mycobacterium tuberculosis infection.

Prostaglandin (PG)E2 is an arachidonic acid-derived lipid mediator that plays an important role in inflammation and immunity. In this study, we demonstrate that PGE2 suppresses basal and 1,25-dihydroxy vitamin D3 (VD3)-induced expression of hCAP18/LL-37 via E prostanoid (EP)2 and EP4 receptors. In humans, VD3 up-regulates vitamin D receptor (VDR) expression and promotes transcription of the cathelicidin hCAP18/LL-37 gene, whereas PGE2 counteracts this effect. We find that PGE2 induces the cAMP/PKA-signaling pathway and enhances the expression of the inhibitory transcription factor cAMP-responsive modulator/inducible cAMP early repressor, which prevents VDR expression and induction of hCAP18/LL-37 in human macrophages. The negative regulation by PGE2 was evident in M1- and M2-polarized human macrophages, although PGE2 displayed more profound inhibitory effects in M2 cells. PGE2 impaired VD3-induced expression of cathelicidin and concomitant activation of autophagy during Mycobacterium tuberculosis (Mtb) infection and facilitated intracellular Mtb growth in human macrophages. An EP4 agonist also significantly promoted Mtb survival in human macrophages. Our results indicate that PGE2 inhibits hCAP18/LL-37 expression, especially VD3-induced cathelicidin and autophagy, which may reduce host defense against Mtb. Accordingly, antagonists of EP4 may constitute a novel adjunctive therapy in Mtb infection.-Wan, M., Tang, X., Rekha, R. S., Muvva, S. S. V. J. R., Brighenti, S., Agerberth, B., Haeggström, J. Z. Prostaglandin E2 suppresses hCAP18/LL-37 expression in human macrophages via EP2/EP4: implications for treatment of Mycobacterium tuberculosis infection.

Involvement of EP2 and EP4 Receptors in Eosinophilic Esophagitis: A Pilot Study.

BACKGROUND: The prostaglandin D2 receptor DP2 has been implicated in eosinophil infiltration and the development of eosinophilic esophagitis (EoE). AIMS AND METHODS: In this study, we investigated an involvement of PGE2 (EP1-EP4) and PGD2 (DP1) receptors in EoE by measuring their expression in peripheral blood eosinophils and esophageal mucosal biopsies of EoE patients and by performing migration and adhesion assays with eosinophils from healthy donors. RESULTS: Expression of EP2 and EP4, but not EP1 and EP3, was decreased in blood eosinophils of patients with EoE vs. control subjects. Adhesion of eosinophils to esophageal epithelial cells was decreased by EP2 receptor agonist butaprost and EP4 agonist ONO-AE1-329, whereas DP1 agonist BW245C increased adhesion. In chemotaxis assays with supernatant from human esophageal epithelial cells, only ONO-AE1-329 but not butaprost or BW245C inhibited the migration of eosinophils. Expression of EP and DP receptors in epithelial cells and eosinophils was detected in sections of esophageal biopsies from EoE patients by immunohistochemistry. qPCR of biopsies from EoE patients revealed that gene expression of EP4 and DP1 was the highest among PGE2 and PGD2 receptors. Esophageal epithelial cells in culture showed high gene expression for EP2 and EP4. Activation of EP2 and EP4 receptors decreased barrier integrity of esophageal epithelial cells in impedance assays. CONCLUSIONS: Activation of EP2 and EP4 receptors may inhibit eosinophil recruitment to the esophageal mucosa. However, their activation could negatively affect esophageal barrier integrity suggesting that eosinophilic rather than epithelial EP2 and EP4 have a protective role in EoE.

Activation of EP2 receptor suppresses poly(I: C) and LPS-mediated inflammation in primary microglia and organotypic hippocampal slice cultures: Contributing role for MAPKs.

Brain inflammation is a critical factor involved in neurodegeneration. Recently, the prostaglandin E2 (PGE2 ) downstream members were suggested to modulate neuroinflammatory responses accompanying neurodegenerative diseases. In this study, we investigated the protective effects of prostaglandin E2 receptor 2 (EP2 ) during TLR3 and TLR4-driven inflammatory response using in vitro primary microglia and ex vivo organotypic hippocampal slice cultures (OHSCs). Depletion of microglia from OHSCs differentially affected TLR3 and TLR4 receptor expression. Poly(I:C) induced the production of prostaglandin E2 in OHSCs by increasing cyclooxygenase (COX-2) and microsomal prostaglandin E synthase (mPGES)-1. Besides, stimulation of OHSCs and microglia with Poly(I:C) upregulated EP2 receptor expression. Co-stimulation of OHSCs and microglia with the EP2 agonist butaprost reduced inflammatory mediators induced by LPS and Poly(I:C). In Poly(I:C) challenged OHSCs, butaprost almost restored microglia ramified morphology and reduced Iba1 immunoreactivity. Importantly, microglia depletion prevented the induction of inflammatory mediators following Poly(I:C) or LPS challenge in OHSCs. Activation of EP2 receptor reversed the Poly(I:C)/LPS-induced phosphorylation of the mitogen activated protein kinases (MAPKs) ERK, p38 MAPK and c-Jun N-terminal kinase (JNK) in microglia. Collectively, these data identify an anti-inflammatory function for EP2 signaling in diverse innate immune responses, through a mechanism that involves the mitogen-activated protein kinases pathway.

Synthesis and evaluation of a potent, well-balanced EP2/EP3 dual agonist.

A highly potent and well-balanced dual agonist for the EP2 and EP3 receptors is described. Optimization of the lead compound was accomplished in consideration of the relative agonist activity against each EP subtype receptor and the pharmacokinetic profile. As the result, 2-[(2-{(1R,2R)-2-[(1E,4S)-5-cyclopentyl-4-hydroxy-4-methyl-1-penten-1-yl]-5-oxocyclopentyl}eth-yl)thio]-1,3-thiazole-4-carboxylic acid (10) showed excellent potency (human EC50 EP2 = 1.1 nM, EP3 = 1.0 nM) with acceptable selectivity over the EP1 and EP4 subtypes (>2000-fold). Further fine-tuning of compound 10 led to identification of ONO-8055 as a clinical candidate. ONO-8055 was effective at an extremely low dose (0.01 mg/kg, po, bid) in rats, and dose-dependently improved voiding dysfunction in a monkey model of underactive bladder (UAB). ONO-8055 is expected to be a novel and highly promising drug for UAB.

EP2 receptor plays pivotal roles in generating mechanical hyperalgesia after lengthening contractions.

We previously demonstrated that nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) were upregulated after lengthening contractions (LC) in exercised muscle through B2 bradykinin receptor activation and cyclooxygenase (COX)-2 upregulation, respectively, and that these trophic factors sensitized nociceptors resulting in mechanical hyperalgesia (delayed-onset muscle soreness, DOMS). Here, we examined the prostaglandin receptor subtype involved in DOMS. The mechanical withdrawal threshold of the exercised muscle was measured before and after LC in rats administered prostaglandin E2 receptor (EP) antagonists before LC, or in wild-type (WT), EP2 knockout (EP2-/- ), and IP knockout (IP-/- ) mice. The change in expression of NGF, GDNF, or COX-2 mRNA was examined using real-time RT-PCR in the muscle in EP2-/- and WT mice. None of the antagonists to EP1, EP3, and EP4 receptors (ONO-8713, ONO-AE5-599, and ONO-AE3-208, respectively) induced a significant difference in DOMS compared with controls in rats. WT and IP-/- mice developed mechanical hyperalgesia after LC, but EP2-/- mice did not. Upregulation of NGF, GDNF, and COX-2 mRNA was observed after LC in WT mice but not in EP2-/- mice. Injecting an EP2 agonist (ONO-AE1-259-01) into the mouse muscle increased expression of COX-2 mRNA. These results suggest that EP2 contributes to generating mechanical hyperalgesia through positive feedback upregulation of COX-2 expression in muscle after LC.

The prostaglandin E2 receptor PTGER2 and prostaglandin F2α receptor PTGFR mediate oviductal glycoprotein 1 expression in bovine oviductal epithelial cells.

Oviductal glycoprotein 1 (OVGP1), an oviductin, is involved in the maintenance of sperm viability and motility and contributes to sperm capacitation in the oviduct. In this study, the regulatory effects exerted by prostaglandin E2 (PGE2) and F2α (PGF2α) on OVGP1 expression via their corresponding receptors in bovine oviductal epithelial cells (BOECs) were investigated. BOECs were cultured in vitro, and their expression of receptors of PGE2 (PTGER1, PTGER2, PTGER3, and PTGER4) and PGF2α (PTGFR) was measured using RT-qPCR. Ca2+ concentration was determined with a fluorescence-based method and cAMP was quantified by enzyme-linked immunosorbent assays to verify activation of PTGER2 and PTGFR by their corresponding agonists in these cells. OVGP1 mRNA and protein expression was measured using RT-qPCR and western blotting, respectively, following PTGER2 and PTGFR agonist-induced activation. PTGER1, PTGER2, PTGER4, and PTGFR were found to be present in BOECs; however, PTGER3 expression was not detected. OVGP1 expression was significantly promoted by 10-6 M butaprost (a PTGER2 agonist) and decreased by 10-6 M fluprostenol (a PTGFR agonist). In addition, 3 µM H-89 (a PKA inhibitor) and 3 µM U0126 (an ERK inhibitor) effectively inhibited PGE2-induced upregulation of OVGP1, and 5 µM chelerythrine chloride (a PKC inhibitor) and 3 µM U0126 negated OVGP1 downregulation by PGF2α. In conclusion, this study demonstrates that OVGP1 expression in BOECs is enhanced by PGE2 via PTGER2-cAMP-PKA signaling, and reduced by PGF2α through the PTGFR-Ca2+-PKC pathway.

Differential expression of E-type prostanoid receptors 2 and 4 in microglia stimulated with lipopolysaccharide.

BACKGROUND: Cyclooxygenase-2 (COX-2) is induced under inflammatory conditions, and prostaglandin E2 (PGE2) is one of the products of COX activity. PGE2 has pleiotropic actions depending on the activation of specific E-type prostanoid EP1-4 receptors. We investigated the involvement of PGE2 and EP receptors in glial activation in response to an inflammatory challenge induced by LPS. METHODS: Cultures of mouse microglia or astroglia cells were treated with LPS in the presence or absence of COX-2 inhibitors, and the production of PGE2 was measured by ELISA. Cells were treated with PGE2, and the effect on LPS-induced expression of TNF-α messenger RNA (mRNA) and protein was studied in the presence or absence of drug antagonists of the four EP receptors. EP receptor expression and the effects of EP2 and EP4 agonists and antagonists were studied at different time points after LPS. RESULTS: PGE2 production after LPS was COX-2-dependent. PGE2 reduced the glial production of TNF-α after LPS. Microglia expressed higher levels of EP4 and EP2 mRNA than astroglia. Activation of EP4 or EP2 receptors with selective drug agonists attenuated LPS-induced TNF-α in microglia. However, only antagonizing EP4 prevented the PGE2 effect demonstrating that EP4 was the main target of PGE2 in naïve microglia. Moreover, the relative expression of EP receptors changed during the course of classical microglial activation since EP4 expression was strongly depressed while EP2 increased 24 h after LPS and was detected in nuclear/peri-nuclear locations. EP2 regulated the expression of iNOS, NADPH oxidase-2, and vascular endothelial growth factor. NADPH oxidase-2 and iNOS activities require the oxidation of NADPH, and the pentose phosphate pathway is a main source of NADPH. LPS increased the mRNA expression of the rate-limiting enzyme of the pentose pathway glucose-6-phosphate dehydrogenase, and EP2 activity was involved in this effect. CONCLUSIONS: These results show that while selective activation of EP4 or EP2 exerts anti-inflammatory actions, EP4 is the main target of PGE2 in naïve microglia. The level of EP receptor expression changes from naïve to primed microglia where the COX-2/PGE2/EP2 axis modulates important adaptive metabolic changes.

Improvement of metabolic disorders by an EP2 receptor agonist via restoration of the subcutaneous adipose tissue in pulmonary emphysema.

Chronic obstructive pulmonary disease (COPD) is often associated with co-morbidities. Metabolic disorders like hyperlipidemia and diabetes occur also in underweight COPD patients, although the mechanism is uncertain. Subcutaneous adipose tissue (SAT) plays an important role in energy homeostasis, since restricted capacity to increase fat cell number with increase in fat cell size occurring instead, is associated with lipotoxicity and metabolic disorders. The aim of this study is to show the protective role of SAT for the metabolic disorders in pulmonary emphysema of a murine model. We found ectopic fat accumulation and impaired glucose homeostasis with wasting of SAT in a murine model of elastase-induced pulmonary emphysema (EIE mice) reared on a high-fat diet. ONO-AE1-259, a selective E-prostanoid (EP) 2 receptor agonist, improved angiogenesis and subsequently adipogenesis, and finally improved ectopic fat accumulation and glucose homeostasis with restoration of the capacity for storage of surplus energy in SAT. These results suggest that metabolic disorders like hyperlipidemia and diabetes occured in underweight COPD is partially due to the less capacity for storage of surplus energy in SAT, though the precise mechanism is uncertained. Our data pave the way for the development of therapeutic interventions for metabolic disorders in emphysema patients, e.g., use of pro-angiogenic agents targeting the capacity for storage of surplus energy in the subcutaneous adipose tissue.

Prostaglandin E2 facilitates neurite outgrowth in a motor neuron-like cell line, NSC-34.

Prostaglandin E2 (PGE2) exerts various biological effects by binding to E-prostanoid receptors (EP1-4). Although recent studies have shown that PGE2 induces cell differentiation in some neuronal cells such as mouse DRG neurons and sensory neuron-like ND7/23 cells, it is unclear whether PGE2 plays a role in differentiation of motor neurons. In the present study, we investigated the mechanism of PGE2-induced differentiation of motor neurons using NSC-34, a mouse motor neuron-like cell line. Exposure of undifferentiated NSC-34 cells to PGE2 and butaprost, an EP2-selective agonist, resulted in a reduction of MTT reduction activity without increase the number of propidium iodide-positive cells and in an increase in the number of neurite-bearing cells. Sulprostone, an EP1/3 agonist, also significantly lowered MTT reduction activity by 20%; however, no increase in the number of neurite-bearing cells was observed within the concentration range tested. PGE2-induced neurite outgrowth was attenuated significantly in the presence of PF-0441848, an EP2-selective antagonist. Treatment of these cells with dibutyryl-cAMP increased the number of neurite-bearing cells with no effect on cell proliferation. These results suggest that PGE2 promotes neurite outgrowth and suppresses cell proliferation by activating the EP2 subtype, and that the cAMP-signaling pathway is involved in PGE2-induced differentiation of NSC-34 cells.

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

BACKGROUND: 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. OBJECTIVE: We sought to determine the mechanisms involved in the altered regulation of the COX pathway in patients with AERD. METHODS: 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-1ß, PGE2, and specific EP receptor agonists. RESULTS: Compared with NM-C fibroblasts, basal expression levels of IL-1RI and EP2 receptor were lower in NP-AERD fibroblasts. IL-1ß-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. CONCLUSION: Altered expression of EP2 in patients with AERD contributes to deficient induction of IL-1RI, reducing the capacity of IL-1ß 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.

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.

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.

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

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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 ß-arrestin recruitment. Furthermore, SFSR studies revealed that the combination of meta and para substituents on the phenyl moiety was crucial to the functional selectivity.

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.

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 Ca(2+)-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.