Microsomal PGE Synthase-1 Research Articles

Disruption of the PGE2 synthesis / response pathway restrains atherogenesis in programmed cell death-1 (Pd-1) deficient hyperlipidemic mice.

Immune checkpoint inhibitors (ICIs) that target programmed cell death 1 (PD-1) have revolutionized cancer treatment by enabling the restoration of suppressed T-cell cytotoxic responses. However, resistance to single-agent ICIs still limits their clinical utility. Combinatorial strategies enhance the antitumor effects, but may also enhance the risk of immune related adverse effects of ICIs. Prostaglandin (PG) E2, formed by the sequential action of the cyclooxygenase (COX) and microsomal PGE synthase (mPGES-1) enzymes, acting via its E prostanoid (EP) receptors, EPr2 and EPr4, promotes lymphocyte exhaustion, revealing an additional target for ICIs. Thus, COX inhibitors and EPr4 antagonists are currently being combined with ICIs potentially to enhance antitumor efficacy in clinical trials. However, given the cardiovascular (CV) toxicity of COX inhibitors, such combinations may increase the risk particularly of CV AEs. Here, we compared the impact of distinct approaches to disruption of the PGE2 synthesis /response pathway- global or myeloid cell specific depletion of mPges-1 or global depletion of Epr4- on the accelerated atherogenesis in Pd-1 deficient hyperlipidemic (Ldlr KO) mice. All strategies restrained the atherogenesis. While depletion of mPGES-1 suppresses PGE2 biosynthesis, reflected by its major urinary metabolite, PGE2 biosynthesis was increased in mice lacking EPr4, consistent with enhanced expression of aortic Cox-1 and mPges-1. Deletions of mPges-1 and Epr4 differed in their effects on immune cell populations in atherosclerotic plaques; the former reduced neutrophil infiltration, while the latter restrained macrophages and increased the infiltration of T-cells. Consistent with these findings, chemotaxis by bone-marrow derived macrophages from Epr4 KO mice as impaired. in Epr4 depletion also resulted in extramedullary lymphoid hematopoiesis and inhibition of lipoprotein lipase activity (LPL) with coincident spelenomegaly, leukocytosis and dyslipidemia. Targeting either mPGES-1 or EPr4 may restrain lymphocyte exhaustion while mitigating CV irAEs consequent to PD-1 blockade.

Activation of the PGE2-EP2 pathway as a potential drug target for treating eosinophilic rhinosinusitis.

Current treatments of eosinophilic chronic rhinosinusitis (ECRS) involve corticosteroids with various adverse effects and costly therapies such as dupilumab, highlighting the need for improved treatments. However, because of the lack of a proper mouse ECRS model that recapitulates human ECRS, molecular mechanisms underlying this disease are incompletely understood. ECRS is often associated with aspirin-induced asthma, suggesting that dysregulation of lipid mediators in the nasal mucosa may underlie ECRS pathology. We herein found that the expression of microsomal PGE synthase-1 (encoded by PTGES) was significantly lower in the nasal mucosa of ECRS patients than that of non-ECRS subjects. Histological, transcriptional, and lipidomics analyses of Ptges-deficient mice revealed that defective PGE2 biosynthesis facilitated eosinophil recruitment into the nasal mucosa, elevated expression of type-2 cytokines and chemokines, and increased pro-allergic and decreased anti-allergic lipid mediators following challenges with Aspergillus protease and ovalbumin. A nasal spray containing agonists for the PGE2 receptor EP2 or EP4, including omidenepag isopropyl that has been clinically used for treatment of glaucoma, markedly reduced intranasal eosinophil infiltration in Ptges-deficient mice. These results suggest that the present model using Ptges-deficient mice is more relevant to human ECRS than are previously reported models and that eosinophilic inflammation in the nasal mucosa can be efficiently blocked by activation of the PGE2-EP2 pathway. Furthermore, our findings suggest that drug repositioning of omidenepag isopropyl may be useful for treatment of patients with ECRS.

Study on Antipyretic Properties of Phenolics in Lonicerae Japonicae Flos Based on Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry Combined with Network Pharmacology

Objective. To identify and quantify the active phenolic components in Lonicerae japonicae flos (LJF) for fever treatment and their mechanism of action using network pharmacology and molecular docking. Methods. Based on qualitative analysis of LJF, 194 phenolics were obtained, including 81 phenolic acids and 113 flavonoids. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to identify potential targets for these components to interact with fever. Molecular docking with microsomal PGE2 synthase-1, EP1, EP2, EP3, and EP4 targets was used to determine antipyretic components. The antipyretic efficacy of the main components was verified by in vivo experiments. Finally, high-performance liquid chromatography-tandem mass spectrometry was used to quantify the main antipyretic components of LJF. Results. Phenolics in LJF may prevent and treat fever by participating in calcium signaling, regulating TRP channels, and cAMP signaling. Luteolin-7-O-glucoside, apigenin-7-O-glucoside, 3,5-O-dicaffeoylquinic acid, luteolin, and other components have a good docking effect with PGE2 synthase-1 and its four subtypes. 3,5-O-dicaffeoylquinic acid, luteolin-7-O-glucoside, and apigenin-7-O-glucoside have good antipyretic effects in a yeast-induced pyrexia model. The content of these antipyretic components varies with the developmental period of LJF. Phenolic acids are the main components that distinguish the different developmental periods of LJF. Conclusion. The potential antipyretic components and molecular mechanisms of phenolics provide a basis for the traditional medicinal effects and future development and utilization of LJF.

Prostaglandin E2-Transporting Pathway and Its Roles via EP2/EP4 in Cultured Human Dental Pulp

IntroductionProstaglandin E2 (PGE2) exerts biological actions through its transport pathway involving intracellular synthesis, extracellular transport, and receptor binding. This study aimed to determine the localization of the components of the PGE2-transporting pathway in human dental pulp and explore the relevance of PGE2 receptors (EP2/EP4) to angiogenesis and dentinogenesis. MethodsProtein localization of microsomal PGE2 (mPGES)synthase, PGE2 transporters (multidrug resistance-associated protein-4 [MRP4] and prostaglandin transporter [PGT]), and EP2/EP4 was analyzed using double immunofluorescence staining. Tooth slices from human third molars were cultured with or without butaprost (EP2 agonist) or rivenprost (EP4 agonist) for 1 week. Morphometric analysis of endothelial cell filopodia was performed to evaluate angiogenesis, and real-time polymerase chain reaction was performed to evaluate angiogenesis and odontoblast differentiation markers. ResultsMRP4 and PGT were colocalized with mPGES and EP2/EP4 in odontoblasts and endothelial cells. Furthermore, MRP4 was colocalized with mPGES and EP4 in human leukocyte antigen-DR-expressing dendritic cells. In the tooth slice culture, EP2/EP4 agonists induced significant increases in the number and length of filopodia and mRNA expression of angiogenesis markers (vascular endothelial growth factor and fibroblast growth factor-2) and odontoblast differentiation markers (dentin sialophosphoprotein and collagen type 1). ConclusionsPGE2-producing enzyme (mPGES), transporters (MRP4 and PGT), and PGE2-specific receptors (EP2/EP4) were immunolocalized in various cellular components of the human dental pulp. EP2/EP4 agonists promoted endothelial cell filopodia generation and upregulated angiogenesis- and odontoblast differentiation-related genes, suggesting that PGE2 binding to EP2/EP4 is associated with angiogenic and dentinogenic responses.

Modulation of Inflammation-Related Lipid Mediator Pathways by Celastrol During Human Macrophage Polarization.

Background and PurposeCelastrol (CS) is a major active ingredient of the Chinese/Asian herb Tripterygium wilfordii that is frequently used as phytomedicine to treat inflammation and autoimmune diseases. We showed before that short-term exposure to CS (1 µM) favorably impacts the biosynthesis of inflammation-related lipid mediators (LM) in human polarized macrophages by modulating the activities of different lipoxygenases (LOXs). However, whether CS regulates the expression of LOXs and other related LM-biosynthetic enzymes during macrophage polarization is unknown. Here, we investigated how CS affects LM-biosynthetic enzyme expression on the protein level and studied concomitant LM signature profiles during polarization of human monocyte-derived macrophages (MDM) towards M1- and M2-like phenotypes.Methods and ResultsWe used LM metabololipidomics to study the long-term effects of CS on LM profile signatures after manipulation of human monocyte-derived macrophages (MDM) during polarization. Exposure of MDM to low concentrations of CS (ie, 0.2 µM) during polarization to an inflammatory M1 phenotype potently suppressed the formation of pro-inflammatory cyclooxygenase (COX)- and 5-LOX-derived LM, especially prostaglandin (PG)E2. Notably, gene and enzyme expression of COX-2 and microsomal PGE2 synthase (mPGES)-1 as well as M1 markers were strongly decreased by CS during M1-MDM polarization, along with impaired activation of nuclear factor-κB and p38 mitogen-activated protein kinase. During IL-4-induced M2 polarization, CS decreased the capacity of the resulting M2-MDM to generate pro-inflammatory COX and 5-LOX products as well but it also reduced the formation of 12/15-LOX products and specialized pro-resolving mediators, without affecting the levels of liberated fatty acid substrates.ConclusionDepending on the timing and concentration, CS not only favorably affects LOX activities in macrophages but also the expression of LM-biosynthetic enzymes during macrophage polarization connected to changes of inflammation-related LM which might be of relevance for potential application of CS to treat inflammatory disorders.

Imbalanced prostanoid release mediates cigarette smoke-induced human pulmonary artery cell proliferation

BackgroundPulmonary hypertension is a common and serious complication of chronic obstructive pulmonary disease (COPD). Studies suggest that cigarette smoke can initiate pulmonary vascular remodelling by stimulating cell proliferation; however, the underlying cause, particularly the role of vasoactive prostanoids, is unclear. We hypothesize that cigarette smoke extract (CSE) can induce imbalanced vasoactive prostanoid release by differentially modulating the expression of respective synthase genes in human pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs), thereby contributing to cell proliferation.MethodsAqueous CSE was prepared from 3R4F research-grade cigarettes. Human PASMCs and PAECs were treated with or without CSE. Quantitative real-time RT-PCR and Western blotting were used to analyse the mRNA and protein expression of vasoactive prostanoid syhthases. Prostanoid concentration in the medium was measured using ELISA kits. Cell proliferation was assessed using the cell proliferation reagent WST-1.ResultsWe demonstrated that CSE induced the expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostanoid synthesis, in both cell types. In PASMCs, CSE reduced the downstream prostaglandin (PG) I synthase (PGIS) mRNA and protein expression and PGI2 production, whereas in PAECs, CSE downregulated PGIS mRNA expression, but PGIS protein was undetectable and CSE had no effect on PGI2 production. CSE increased thromboxane (TX) A synthase (TXAS) mRNA expression and TXA2 production, despite undetectable TXAS protein in both cell types. CSE also reduced microsomal PGE synthase-1 (mPGES-1) protein expression and PGE2 production in PASMCs, but increased PGE2 production despite unchanged mPGES-1 protein expression in PAECs. Furthermore, CSE stimulated proliferation of both cell types, which was significantly inhibited by the selective COX-2 inhibitor celecoxib, the PGI2 analogue beraprost and the TXA2 receptor antagonist daltroban.ConclusionsThese findings provide the first evidence that cigarette smoke can induce imbalanced prostanoid mediator release characterized by the reduced PGI2/TXA2 ratio and contribute to pulmonary vascular remodelling and suggest that TXA2 may represent a novel therapeutic target for pulmonary hypertension in COPD.

Green, Black and Rooibos Tea Inhibit Prostaglandin E2 Formation in Human Monocytes by Inhibiting Expression of Enzymes in the Prostaglandin E2 Pathway.

The formation of prostaglandin E2 (PGE2) is associated with adverse inflammatory effects. However, long-term treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) comes with risk of severe side effects. Therefore, alternative ways to inhibit PGE2 are warranted. We have investigated the effects of tea extracts and the polyphenols epigallocatechin gallate (EGCG) and quercetin on PGE2 formation, determined by immunoassay, and protein expression, determined by immunoblotting, of cytosolic phospholipase A2 (cPLA2), cyclooxygenase 2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) in human monocytes. Green and black tea extracts, and with a lower potency, Rooibos tea extract, inhibited lipopolysaccharide (LPS) and calcium ionophore-induced PGE2 formation. In addition, all tea extracts inhibited the LPS-induced expression of mPGES-1, and the green and black tea extracts also inhibited, to a lesser extent, COX-2 expression. The tea extracts only marginally reduced cPLA2 expression and had no effect on COX-1 expression. EGCG, present in green and black tea, and quercetin, present in all three teas, also inhibited PGE2 formation and expression of mPGES-1, COX-2 and cPLA2. Cell-based and cell-free assays were also performed to evaluate direct effects on the enzymatic activity of COX and PGE synthases. Mainly, the cell-free assay demonstrated partial inhibition by the tea extracts and polyphenols. However, the inhibition required higher doses compared to the effects demonstrated on protein expression. In conclusion, green and black tea, and to a lesser extent Rooibos tea, are potent inhibitors of PGE2 formation in human monocytes, and mediate their effects by inhibiting the expression of the enzymes responsible for PGE2 formation, especially mPGES-1.

Inducible Prostaglandin E Synthase as a Pharmacological Target for Ischemic Stroke

As the inducible terminal enzyme for prostaglandin E2 (PGE2) synthesis, microsomal PGE synthase-1 (mPGES-1) contributes to neuroinflammation and secondary brain injury after cerebral ischemia via producing excessive PGE2. However, a proof of concept that mPGES-1 is a therapeutic target for ischemic stroke has not been established by a pharmacological strategy mainly due to the lack of drug-like mPGES-1 inhibitors that can be used in relevant rodent models. To this end, we recently developed a series of novel small-molecule compounds that can inhibit both human and rodent mPGES-1. In this study, blockade of mPGES-1 by our several novel compounds abolished the lipopolysaccharide (LPS)-induced PGE2 and pro-inflammatory cytokines interleukin 1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α) in mouse primary brain microglia. Inhibition of mPGES-1 also decreased PGE2 produced by neuronal cells under oxygen–glucose deprivation (OGD) stress. Among the five enzymes for PGE2 biosynthesis, mPGES-1 was the most induced one in cerebral ischemic lesions. Systemic treatment with our lead compound MPO-0063 (5 or 10 mg/kg, i.p.) in mice after transient middle cerebral artery occlusion (MCAO) improved post-stroke well-being, decreased infarction and edema, suppressed induction of brain cytokines (IL-1β, IL-6, and TNF-α), alleviated locomotor dysfunction and anxiety-like behavior, and reduced the long-term cognitive impairments. The therapeutic effects of MPO-0063 in this proof-of-concept study provide the first pharmacological evidence that mPGES-1 represents a feasible target for delayed, adjunct treatment — along with reperfusion therapies — for acute brain ischemia.

Prostaglandin E2 increases the expression of cyclooxygenase-2 in cultured rat microglia.

Prostaglandin E2 (PGE2) plays pivotal roles in controlling microglial activation with the EP2 receptor, a PGE2 receptor subtype. Activated microglia are often reported to increase cyclooxygenase (COX)-2 expression, followed by PGE2 production, but it is unclear whether extracellular PGE2 is involved in microglial PGE2 synthesis. In the present study, we report that PGE2 increases COX-2 protein in microglia. In a culture system, PGE2 at 10−6 M for 3 h increased COX-2 and microsomal PGE synthase (mPGES)-1 mRNA levels, and reduced mPGES-2, but did not affect COX-1 or cytosolic PGE synthase (cPGES) in microglia. PGE2 at 10−6 M for 3 h also increased the COX-2 protein level, but did not affect COX-1, mPGES-1, mPGES-2, or cPGES. An EP2 agonist, ONO-AE1-259-01, also increased COX-2 and mPGES-1 mRNA levels, and reduced mPGES-2, but did not affect COX-1 or cPGES, whereas an EP1 agonist, ONO-DI-004, an EP3 agonist, ONO-AE-248, and an EP4 agonist, ONO-AE1-329, had no effect. Similar to PGE2, ONO-AE1-259-01 increased the COX-2 protein level, but did not affect COX-1, mPGES-1, mPGES-2, or cPGES. In addition, the effects of PGE2 were inhibited by an EP2 antagonist, PF-04418948, but not by an EP1 antagonist, ONO-8713, an EP3 antagonist, ONO-AE3‐240, or an EP4 antagonist, ONO-AE3‐208, at 10−6 M. On the other hand, lipopolysaccharide (LPS) increased PGE2 production, but the LPS-induced PGE2 production was not affected by ONO-8713, PF-04418948, ONO-AE3‐240, or ONO-AE3‐208. These results indicate that PGE2 increases COX-2 protein in microglia through the EP2 receptor supporting the idea that extracellular PGE2 has a triggering aspect for microglial activation.

Monocyte dysfunction in decompensated cirrhosis is mediated by the prostaglandin E2-EP4 pathway

Background & AimsInfection is a major problem in advanced liver disease secondary to monocyte dysfunction. Elevated prostaglandin (PG)E2 is a mediator of monocyte dysfunction in cirrhosis; thus, we examined PGE2 signalling in outpatients with ascites and in patients hospitalised with acute decompensation to identify potential therapeutic targets aimed at improving monocyte dysfunction.MethodsUsing samples from 11 outpatients with ascites and 28 patients hospitalised with decompensated cirrhosis, we assayed plasma levels of PGE2 and lipopolysaccharide (LPS); performed quantitative real-time PCR on monocytes; and examined peripheral blood monocyte function. We performed western blotting and immunohistochemistry for PG biosynthetic machinery expression in liver tissue. Finally, we investigated the effect of PGE2 antagonists in whole blood using polychromatic flow cytometry and cytokine production.ResultsWe show that hepatic production of PGE2 via the cyclo-oxygenase 1–microsomal PGE synthase 1 pathway, and circulating monocytes contributes to increased plasma PGE2 in decompensated cirrhosis. Transjugular intrahepatic sampling did not reveal whether hepatic or monocytic production was larger. Blood monocyte numbers increased, whereas individual monocyte function decreased as patients progressed from outpatients with ascites to patients hospitalised with acute decompensation, as assessed by Human Leukocyte Antigen (HLA)–DR isotype expression and tumour necrosis factor alpha and IL6 production. PGE2 mediated this dysfunction via its EP4 receptor.ConclusionsPGE2 mediates monocyte dysfunction in decompensated cirrhosis via its EP4 receptor and dysfunction was worse in hospitalised patients compared with outpatients with ascites. Our study identifies a potential drug target and therapeutic opportunity in these outpatients with ascites to reverse this process to prevent infection and hospital admission.Lay summaryPatients with decompensated cirrhosis (jaundice, fluid build-up, confusion, and vomiting blood) have high infection rates that lead to high mortality rates. A white blood cell subset, monocytes, function poorly in these patients, which is a key factor underlying their sensitivity to infection. We show that monocyte dysfunction in decompensated cirrhosis is mediated by a lipid hormone in the blood, prostaglandin E2, which is present at elevated levels, via its EP4 pathway. This dysfunction worsens when patients are hospitalised with complications of cirrhosis compared with those in the outpatients setting, which supports the EP4 pathway as a potential therapeutic target for patients to prevent infection and hospitalisation.

The Role of Prostaglandin E2 Synthesized in Rat Lateral Parabrachial Nucleus in LPS-Induced Fever

Introduction: The lateral parabrachial nucleus (LPBN) is considered to be a brain site of the pyrogenic action of prostaglandin (PG) E2 outside of the preoptic area. Yet, the role of the LPBN in fever following a systemic immune challenge remains poorly understood. Methods: We examined the expression of cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) in the LPBN after the intraperitoneal injection of lipopolysaccharide (LPS). We investigated the effects of LPBN NS-398 (COX-2 inhibitor) on LPS-induced fever, the effects of direct LPBN PGE2 administration on the energy expenditure (EE), brown adipose tissue (BAT) thermogenesis, neck muscle electromyographic activity and tail temperature, and the effects of PGE2 on the spontaneous firing activity and thermosensitivity of in vitro LPBN neurons in a brain slice. Results: The COX-2 and mPGES-1 enzymes were upregulated at both mRNA and protein levels. The microinjection of NS-398 in the LPBN attenuated the LPS-induced fever. Direct PGE2 administration in the LPBN resulted in a febrile response by a coordinated response of increased EE, BAT thermogenesis, shivering, and possibly decreased heat loss through the tail. The LPBN neurons showed a clear anatomical distinction in the firing rate response to PGE2, with the majority of PGE2-excited or -inhibited neurons being located in the external lateral or dorsal subnucleus of the LPBN, respectively. However, neither the firing rate nor the thermal coefficient response to PGE2 showed any difference between warm-sensitive, cold-sensitive, and temperature-insensitive neurons in the LPBN. Conclusions: PGE2 synthesized in the LPBN was at least partially involved in LPS-induced fever via its different modulations of the firing rate of neurons in different LPBN subnuclei.

Abstract LB225: Colon cancer preventive efficacy of licofelone and its analogue LFA-9 in PIRC rat model of FAP

Abstract Inflammation is a key hallmark of many cancers and potent target for chemoprevention. Experimental and clinical intervention studies indicate strong cancer preventive efficacy of cyclooxygenase (COX)-2 inhibitors. Their use for chemoprevention is limited due to increased cardiovascular (CV) toxicities. Selective COX-2 inhibition diverts arachidonic acid to the 5-lipoxygenase (5-LOX) pathway resulting in accumulation of prothrombotic leukotrienes while depleting antithrombotic prostaglandin (PG)I2, leading to increased risk of CV events. To overcome side effects, balanced dual inhibitors targeting COX-2/5-LOX or microsomal PGE synthase (mPGES)-1/5-LOX enzymes are being developed for chemoprevention. In this study, the clinically advanced dual COX-2/5-LOX inhibitor, licofelone, and its glycine analogue (LFA-9), the mPGES-1/5-LOX dual inhibitor, were evaluated for colon cancer preventive efficacy in the FAP relevant PIRC rat model. In preclinical dose range finding and preliminary toxicity studies in F344 rats, dietary administration of licofelone <500ppm and LFA9 <1,600ppm for 8 weeks showed no signs of toxicity based on by body weight gain, organs histopathology, blood cell counts, and serum chemistry. For efficacy evaluation, male and female PIRC rats were randomized by age (n≥15/group/gender). All rats had pre-intervention colonoscopies (8 weeks age) to determine baseline colonic polyps and were fed AIN-76A diets containing licofelone (250 ppm), LFA-9 (800ppm) or control until 32 (male) or 40 (female) weeks of age. Colonoscopies were repeated every 8 weeks for longitudinal monitoring of tumor development. At the end of the treatment period, rats were euthanized and intestines were evaluated for tumor multiplicity as an efficacy endpoint. Colonoscopy data suggested no significant difference in pretreatment colonic polyp multiplicity. However, there was a marked delay in colon tumor development in rats in the intervention group compared to control. Colonic tumor multiplicity at termination indicated that licofelone treated male rats developed 32% less tumors (21.87±1.23 (Mean±SEM); p<0.0001), while LFA-9 treated male rats had 31% less tumors (22.13±1.19; p<0.0001) compared to controls (32.25±1.37). There was no significant difference in tumor multiplicity between licofelone and LFA-9 treatment groups. In females, licofelone treatment led to 51% inhibition of colonic tumors (9.18±1.25; p<0.0002) while LFA-9 treatment led to 37% inhibition (11.89±0.86; p<0.002) compared to control (18.91±1.88). Although licofelone trended toward better inhibitory effect in females, this was not significantly different from the LFA-9 group. Colon tumor volume and small intestinal tumor multiplicity was notably inhibited in both licofelone and LFA-9 treated rats when compared to control. Our data suggest both licofelone and LFA-9 exhibit moderate and uniform chemopreventive efficacy against FAP-associated intestinal tumorigenesis in the PIRC rat model. (Supported by NCI HHSN HHSN261201500024I) Citation Format: Venkateshwar Madka, Nagendra S. Yarla, Gopal Pathuri, Yuting Zhang, Anh Bao, Nicole C. Stratton, Anil Singh, David L. McCormick, Altaf Mohammed, Shizuko Sei, Jennifer Fox, Chinthalapally V. Rao. Colon cancer preventive efficacy of licofelone and its analogue LFA-9 in PIRC rat model of FAP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB225.

IL-17-induced inflammation modulates the mPGES-1/PPAR-γ pathway in monocytes/macrophages.

Recent biochemical and pharmacological studies have reported that in several tissues and cell types, microsomal PGE2 synthase (mPGES) and PPAR-γ expression are modulated by a variety of inflammatory factors and stimuli. Considering that very little is known about the biological effects promoted by IL-17 in the context of mPGES-1/PPAR-γ modulation, we sought to investigate the contribution of this unique cytokine on this integrated pathway during the onset of inflammation. We evaluated effects of PF 9184 (mPGES-1 inhibitor) and troglitazone (PPAR-γ agonist) in vitro, using the mouse macrophage cell line J774A.1. In vivo, the dorsal air pouch model in CD1 mice was used, and inflammatory infiltrates were analysed by flow cytometry. Locally produced cyto-chemokines and PGs were assessed using elisa assays. Western blots were also employed to determine the activity of various enzymes involved in downstream signalling pathways. PF 9184 and troglitazone, in a time- and dose-dependent manner, modulated leukocyte infiltration, myeloperoxidase activity, and the expression of COX-2/mPGES-1, NF-кB/IкB-α, and mPTGDS-1/PPAR-γ, induced by IL-17. Moreover, both PF 9184 and troglitazone modulated PG (PGE2 , PGD2 , and PGJ2 ) production, the expression of different pro-inflammatory cyto-chemokines, and the recruitment of inflammatory monocytes, in response to IL-17. Our data suggest that IL-17 may constitute a specific modulator of inflammatory monocytes during later phases of the inflammatory response. The results of this study show, for the first time, that the IL-17/mPGES-1/PPAR-γ pathway could represent a potential therapeutic target for inflammatory-based and immune-mediated diseases. This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.

High mobility group box 1 mediates inflammatory response of astrocytes via cyclooxygenase 2/prostaglandin E2 signaling following spinal cord injury.

High mobility group box 1 (HMGB1) interacts with pattern-recognition receptors of immune cells to activate the inflammatory response. Astrocytes play a positive role in the inflammatory response of the central nervous system by expressing a broad range of pattern-recognition receptors. However, the underlying relationship between HMGB1 and the inflammatory reaction of astrocytes remains unclear. In this study, we established rat models of spinal cord injury via laminectomy at the T8-10 level, and the injured spinal cord was subjected to transcriptome sequencing. Our results showed that the HMGB1/Toll-like receptor 4 (TLR4) axis was involved in the activation of astrocyte inflammatory response through regulation of cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) signaling. Both TLR4 and COX2 were distributed in astrocytes and showed elevated protein levels following spinal cord injury. Stimulation of primary astrocytes with recombinant HMGB1 showed that COX2 and microsomal PGE synthase (mPGES)-1, rather than COX1, mPGES-2, or cytosolic PGE synthase, were significantly upregulated. Accordingly, PGE2 production in astrocytes was remarkably increased in response to recombinant HMGB1 challenges. Pharmacologic blockade of TLR2/4 attenuated HMGB1-mediated activation of the COX2/PGE2 pathway. Interestingly, HMGB1 did not impact the production of tumor necrosis factor-α or interleukin-1β in astrocytes. Our results suggest that HMGB1 mediates the astrocyte inflammatory response through regulating the COX2/PGE2 signaling pathway. The study was approved by the Laboratory Animal Ethics Committee of Nantong University, China (approval No. 20181204-001) on December 4, 2018.

Localization of prostaglandin E2 synthases and E-prostanoid receptors in the spinal cord in a rat model of neuropathic pain

Prostaglandin E2 (PGE2) is a lipid mediator which plays a role in the generation of inflammatory and neuropathic pain. In the peripheral nervous system, PGE2 sensitizes nociceptive afferent neurons through E-prostanoid (EP) receptors. In the central nervous system, PGE2 modulates pain sensitivity and contributes to the development of neuropathic pain. However, the distribution of PGE2 and EP receptors in the spinal cord remains unclear. In the present study, we examined the expression of PGE2 synthases (microsomal PGE synthase [mPGES]-1, mPGES-2, and cytosolic PGE synthase [cPGES]) and EP receptors (EP1-4) in a rat model of neuropathic pain.We identified that mPGES-1 mRNA was upregulated in spinal endothelial cells after nerve injury and exhibited co-localization with cyclooxygenase-2 (COX-2). We detected that mPGES-2 mRNA and cPGES mRNA were expressed in spinal neurons and noted that their expression level was not affected by nerve injury. With respect to EP receptors, EP2 mRNA and EP4 mRNA were expressed in spinal neurons in the dorsal horn. EP3 mRNA was expressed in motor neurons, whereas EP1 mRNA was not detected in the spinal cord. Intrathecal injection of tumor necrosis factor alpha (TNFα) upregulated mPGES-1 mRNA in blood vessels in the spinal cord. Intrathecal injection of a TNFα-neutralizing antibody partially inhibited the upregulation of mPGES-1 mRNA after nerve injury.These results indicate that PGE2 is synthesized by COX-2/mPGES-1 in spinal endothelial cells after nerve injury. These results suggest that in neuropathic pain condition, endothelial cell-derived PGE2 may act on EP2 and EP4 receptors on spinal neurons and modulate pain sensitivity.

Abstract B13: Intrinsic microsomal PGE2 synthase-1 associates with poor patient survival and T-cell infiltration, and regulates immunosuppression in human and mouse melanoma models

Abstract Our research has identified microsomal prostaglandin E2 synthase 1 (mPGES1) as an important downstream effector of the cyclooxygenase 2 (COX2) pathway often expressed by melanoma cells, and largely responsible for tumor-mediated immunosuppression. We observed clear correlations in expression and co-localization of COX2 and mPGES1, for which both are associated with increased expression of immunosuppressive markers in human melanoma. In a syngeneic melanoma mouse model, mPGES1 knockout increased melanoma expression of PD-L1 showing about 25% positive cells, increased infiltration of CD8a+ T cells (around 8 fold) and CD8a+ dendritic cells (around 10 fold) into tumors and suppressed tumor growth (around 70%). Durable tumor regression was observed in mice bearing mPGES1 knockout tumors that were given anti-PD-1 therapy. Analysis of a stage III melanoma tissue microarray revealed significant associations between high mPGES1 expression and low CD8+ infiltration, which correlated with poor survival. Collectively, our results are the first to illustrate a potential role for mPGES1-inhibition and selective targeting in melanoma immune evasion and in supporting the durability of response to PD-1 checkpoint immunotherapy. Research in the development of mPGES1 inhibitors is needed to validate their use as safe treatment options, but they appear attractive and feasible based on data to date. Citation Format: Sun-Hee Kim, Jason Roszik, Sungnam Cho, Dai Ogata, Suhendan Ekmekcioglu, Elizabeth A Grimm. Intrinsic microsomal PGE2 synthase-1 associates with poor patient survival and T-cell infiltration, and regulates immunosuppression in human and mouse melanoma models [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B13.

Regulation of gene expression by MF63, a selective inhibitor of microsomal PGE synthase 1 (mPGES1) in human osteoarthritic chondrocytes.

Background and PurposemPGES1 catalyses the production of PGE2, the most abundant prostanoid related to inflammation and pain in arthritis. mPGES1 is suggested to be a safer and more selective drug target in inflammatory conditions compared to the COX enzymes inhibited by NSAIDs. In the present study, we investigated the effects of the selective mPGES1 inhibitor MF63 on gene expression in primary human chondrocytes from patients with osteoarthritis (OA).Experimental ApproachChondrocytes were isolated from articular cartilage obtained from osteoarthritis patients undergoing knee replacement surgery. The effects of MF63 were studied in the primary chondrocytes with RNA‐sequencing based genome‐wide expression analysis. The main results were confirmed with qRT‐PCR and compared with the effects of the NSAID ibuprofen. Functional analysis was performed with the GO database and interactions between the genes were studied with STRING.Key ResultsMF63 enhanced the expression of multiple metallothionein 1 (MT1) isoforms as well as endogenous antagonists of IL‐1 and IL‐36. The expression of IL‐6, by contrast, was down‐regulated. These genes were also essential in functional and interaction network analyses. The effects of MF63 were consistent in qRT‐PCR analysis, whereas the effects of ibuprofen overlapped only partly with MF63. There were no evident findings of catabolic effects by MF63.Conclusion and ImplicationsMetallothionein 1 has been suggested to have anti‐inflammatory and protective effects in cartilage. Up‐regulation of the antagonists of IL‐1 superfamily and down‐regulation of the pro‐inflammatory cytokine IL‐6 also support novel anti‐inflammatory and possibly disease‐modifying effects of mPGES1 inhibitors in arthritis.

Ellagitannins from Punica granatum leaves suppress microsomal prostaglandin E synthase-1 expression and induce lung cancer cells to undergo apoptosis.

Prostaglandin E2 (PGE2), which is a potent pro-inflammatory lipid mediator, is biosynthesized from arachidonic acid by cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1). Non-steroidal anti-inflammatory drugs (NSAIDs) are used clinically as COX inhibitors, but they have gastrointestinal and cardiovascular side-effects. Thus, the terminal enzyme mPGES-1 holds promise as the next therapeutic target. In this study, we found that the ellagitannins granatin A and granatin B isolated from pomegranate leaves, and geraniin, which is their structural analog, selectively suppressed mPGES-1 expression without affecting COX-2 in non-small cell lung carcinoma A549 cells. The ellagitannins also down-regulated tumor necrosis factor α, inducible nitric oxide synthase, and anti-apoptotic factor B-cell chronic lymphocytic leukemia/lymphoma 2, and induced A549 cells to undergo apoptosis. These findings indicate that the ellagitannins have anti-inflammatory and anti-carcinogenic effects, due to their specific suppression of mPGES-1.Abbreviations: Bcl-2: B-cell chronic lymphocytic leukemia/lymphoma 2; COX: cyclooxygenase; CRE: cAMP response element; DHHDP: dehydrohexahydroxydiphenoyl; Et2O: diethyl ether; EtOAc: ethyl acetate; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; iNOS: inducible nitric oxide synthase; mPGES-1: microsomal prostaglandin E synthase-1; n-BuOH: water-saturated n-butanol; NSAIDs: non-steroidal anti-inflammatory drugs; NF-κB: nuclear factor-κB; PG: prostaglandin; TNF: tumor necrosis factor; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Biological characterization of new inhibitors of microsomal PGE synthase-1 in preclinical models of inflammation and vascular tone.

Microsomal PGE synthase-1 (mPGES-1), the inducible synthase that catalyses the terminal step in PGE2 biosynthesis, is of high interest as therapeutic target to treat inflammation. Inhibition of mPGES-1 is suggested to be safer than traditional NSAIDs, and recent data demonstrate anti-constrictive effects on vascular tone, indicating new therapeutic opportunities. However, there is a lack of potent mPGES-1 inhibitors lacking interspecies differences for conducting in vivo studies in relevant preclinical disease models. Potency was determined based on the reduction of PGE2 formation in recombinant enzyme assays, cellular assay, human whole blood assay, and air pouch mouse model. Anti-inflammatory properties were assessed by acute paw swelling in a paw oedema rat model. Effect on vascular tone was determined with human ex vivo wire myography. We report five new mPGES-1 inhibitors (named 934, 117, 118, 322, and 323) that selectively inhibit recombinant human and rat mPGES-1 with IC50 values of 10-29 and 67-250 nM respectively. The compounds inhibited PGE2 production in a cellular assay (IC50 values 0.15-0.82 μM) and in a human whole blood assay (IC50 values 3.3-8.7 μM). Moreover, the compounds blocked PGE2 formation in an air pouch mouse model and reduced acute paw swelling in a paw oedema rat model. Human ex vivo wire myography analysis showed reduced adrenergic vasoconstriction after incubation with the compounds. These mPGES-1 inhibitors can be used as refined tools in further investigations of the role of mPGES-1 in inflammation and microvascular disease.

Cyclooxygenase-2 Induced the β-Amyloid Protein Deposition and Neuronal Apoptosis Via Upregulating the Synthesis of Prostaglandin E2 and 15-Deoxy-Δ12,14-prostaglandin J2.

Elevated levels of cyclooxygenase-2 (COX-2) and prostaglandins (PGs) have been shown to be involved in the pathogenesis of Alzheimer's disease. Analysis of the underlying mechanisms elucidated a function of sequential PGE2 and PGD2 synthesis in regulating β-amyloid protein (Aβ) deposition by modulating tumor necrosis factor α (TNF-α)-dependent presenilin (PS)1/2 activity in COX-2 and APP/PS1 crossed mice. Specifically, COX-2 overexpression accelerates the expression of microsomal PGE synthase-1 (mPGES-1) and lipocalin-type prostaglandin D synthase (L-PGDS), leading to the synthesis of PGE2 and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) in 6-month-old APP/PS1 mice. Consequently, PGE2 has the ability to increase Aβ production by enhancing the expression of PS1/2 in a TNF-α-dependent manner, which accelerates the cognitive decline of COX-2/APP/PS1 mice. More interestingly, low concentrations of 15d-PGJ2 treatment facilitate the effects of PGE2 on the deposition of Aβ via TNF-α-dependent PS1/2 mechanisms. In contrast, high concentrations of 15d-PGJ2 treatment inhibit the deposition of Aβ via suppressing the expression of TNF-α-dependent PS1/2. In this regard, a high concentration of 15d-PGJ2 appears to be a therapeutic agent against Alzheimer's disease. However, the high 15d-PGJ2 concentration treatment induces neuronal apoptosis via increasing the protein levels of Bax, cleaved caspase-3, and DFF45, which further impairs the learning ability of APP/PS1 mice.