PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function.

Expansion of antigen-experienced CD8+ T cells is critical for the success of tumour-infiltrating lymphocyte (TIL)-adoptive cell therapy (ACT) in patients with cancer1. Interleukin-2 (IL-2) acts as a key regulator of CD8+ cytotoxic T lymphocyte functions by promoting expansion and cytotoxic capability2,3. Therefore, it is essential to comprehend mechanistic barriers to IL-2 sensing in the tumour microenvironment to implement strategies to reinvigorate IL-2 responsiveness and T cell antitumour responses. Here we report that prostaglandin E2 (PGE2), a known negative regulator of immune response in the tumour microenvironment4,5, is present at high concentrations in tumour tissue from patients and leads to impaired IL-2 sensing in human CD8+ TILs via the PGE2 receptors EP2 and EP4. Mechanistically, PGE2 inhibits IL-2 sensing in TILs by downregulating the IL-2Rγc chain, resulting in defective assembly of IL-2Rß-IL2Rγc membrane dimers. This results in impaired IL-2-mTOR adaptation and PGC1α transcriptional repression, causing oxidative stress and ferroptotic cell death in tumour-reactive TILs. Inhibition of PGE2 signalling to EP2 and EP4 during TIL expansion for ACT resulted in increased IL-2 sensing, leading to enhanced proliferation of tumour-reactive TILs and enhanced tumour control once the cells were transferred in vivo. Our study reveals fundamental features that underlie impairment of human TILs mediated by PGE2 in the tumour microenvironment. These findings have therapeutic implications for cancer immunotherapy and cell therapy, and enable the development of targeted strategies to enhance IL-2 sensing and amplify the IL-2 response in TILs, thereby promoting the expansion of effector T cells with enhanced therapeutic potential.

Proliferation of bovine endometrial epithelial cells is promoted by prostaglandin E2-PTGER2 signaling through cell cycle regulation.

It is known that prostaglandin E2 (PGE2) induces proliferation of epithelia in bovine endometrial explants, however, the detailed mechanism of regulation of PGE2 in inducing bovine endometrial epithelial cell (bEEC) proliferation is unclear. In this study, we determined whether proliferation of bEECs is promoted by PGE2-prostaglandin E receptor 2 (PTGER2) signaling activation through cell cycle regulation. The results demonstrated that bEECs proliferation was induced by treatment of PGE2 and PTGER2 agonist butaprost. These processes were down-regulated by PTGER2 antagonist AH6809 and CDK inhibitors (LEE011, CDK2 Inhibitor II and Ro 3306). PGE2 and butaprost induced cyclins (A, B1, D1, D3 and E2), cyclin-dependent kinases (CDKs, 1, 2, 4 and 6), and epidermal growth factor (EGF) expression were inhibited by AH6809 treatment in bEECs. Moreover, proliferating cell nuclear antigen (PCNA), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and PTGER2 expression in bEECs were up-regulated by PGE2 and butaprost treatment. Our data demonstrate that PGE2-PTGER2 signaling activation has a direct molecular association with cell cycle regulation and cell proliferation in bEECs. Collectively, these findings will improve our understanding of the roles for PGE2-PTGER2 signaling activation in the physiological and pharmacological processes of bovine endometrium.

Use of selective PGE2 receptor antagonists on human endometriotic stromal cells and peritoneal macrophages.

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

Restoring metabolism of myeloid cells reverses cognitive decline in ageing.

Ageing is characterized by the development of persistent pro-inflammatory responses that contribute to atherosclerosis, metabolic syndrome, cancer and frailty1-3. The ageing brain is also vulnerable to inflammation, as demonstrated by the high prevalence of age-associated cognitive decline and Alzheimer's disease4-6. Systemically, circulating pro-inflammatory factors can promote cognitive decline7,8, and in the brain, microglia lose the ability to clear misfolded proteins that are associated with neurodegeneration9,10. However, the underlying mechanisms that initiate and sustain maladaptive inflammation with ageing are not well defined. Here we show that in ageing mice myeloid cell bioenergetics are suppressed in response to increased signalling by the lipid messenger prostaglandin E2 (PGE2), a major modulator of inflammation11. In ageing macrophages and microglia, PGE2 signalling through its EP2 receptor promotes the sequestration of glucose into glycogen, reducing glucose flux and mitochondrial respiration. This energy-deficient state, which drives maladaptive pro-inflammatory responses, is further augmented by a dependence of aged myeloid cells on glucose as a principal fuel source. In aged mice, inhibition of myeloid EP2 signalling rejuvenates cellular bioenergetics, systemic and brain inflammatory states, hippocampal synaptic plasticity and spatial memory. Moreover, blockade of peripheral myeloid EP2 signalling is sufficient to restore cognition in aged mice. Our study suggests that cognitive ageing is not a static or irrevocable condition but can be reversed by reprogramming myeloid glucose metabolism to restore youthful immune functions.

Expression of trophoblast derived prostaglandin E2 receptor 2 (EP2) is reduced in patients with recurrent miscarriage and EP2 regulates cell proliferation and expression of inflammatory cytokines.

BACKGROUD: Prostaglandin E2 (PGE2), an inflammatory mediator, modulates cytokines, regulates immune responses in reproductive processes and stimulates inflammatory reactions via the prostaglandin E2 receptor 2 (EP2). However, the regulatory effects of EP2 signaling on trophoblasts and its role in unexplained recurrent miscarriage (uRM) remains unclear. PATIENTS AND METHODS: A total of 19 placentas from patients with a history of more than two consecutive pregnancy losses of unknown cause (uRM group) and placentas of 19 healthy patients following a legal termination of their pregnancy were used for PGE2 receptor (EP1, EP2 and EP4) expression analyses via immunohistochemistry. Double immunofluorescence was also used to identify EP2 expressing cells in the decidua. Finally, HTR-8/SVneo cells were used to clarify the role of EP2 in in vitro experiments. RESULTS: The expression of EP2 and EP4 was found to be reduced in the syncytiotrophoblast and decidua of uRM patients. A selective EP2 receptor antagonist (PF-04,418,948) reduced the proliferation and secretion of ß-hCG, inhibited interleukin -6 (IL-6) and interleukin-8 (IL-8) and up-regulated the production of the tumor necrosis factor-α (TNF-α) and plasminogen activator inhibitor type 1 (PAI-1) in HTR-8/SVneo cells in vitro. CONCLUSION: PGE2-EP2 signaling pathway may represent a novel therapy option for uRM. The involvement of EP2 in uRM acts perhaps via inflammatory cytokines and indicates that the PGE2-EP2 signaling pathway might represent an unexplored etiology for uRM.

Crosstalk between EP2 and PPARα Modulates Hypoxic Signaling and Myopia Development in Guinea Pigs.

Purpose: Cyclic adenosine monophosphate (cAMP) and peroxisome proliferator-activated receptor alpha (PPARα) levels mediate extracellular matrix (ECM) changes by altering the levels of hypoxia-inducible factor 1-alpha (HIF-1α) in various tissues. We aimed to determine, in the sclera of guinea pigs, whether a prostanoid receptor (EP2)-linked cAMP modulation affects PPARα and HIF-1α signaling during myopia. Methods: Three-week-old guinea pigs (n = 20 in each group), were monocularly injected with either an EP2 agonist (butaprost 1 µmol/L/10 µmol/L), an antagonist (AH6809 10 µmol/L/30 µmol/L) or a vehicle solution for two weeks during normal ocular growth. Separate sets of animals received these injections and underwent form deprivation (FD) simultaneously. Refraction and axial length (AL) were measured at two weeks, followed by scleral tissue isolation for quantitative PCR (qPCR) analysis (n = 10) and cAMP detection (n = 10) using a radioimmunoassay. Results: Butaprost induced myopia development during normal ocular growth, with proportional increases in AL and cAMP levels. FD did not augment the magnitude of myopia or cAMP elevations in these agonist-injected eyes. AH6809 suppressed cAMP increases and myopia progression during FD, but had no effect in a normal visual environment. Of the diverse set of 27 genes related to cAMP, PPARα and HIF-1α signaling and ECM remodeling, butaprost differentially regulated 15 of them during myopia development. AH6809 injections during FD negated such differential gene expressions. Conclusion: EP2 agonism increased cAMP and HIF-1α signaling subsequent to declines in PPARα and RXR mRNA levels, which in turn decreased scleral fibrosis and promoted myopia. EP2 antagonism instead inhibited each of these responses. Our data suggest that EP2 suppression may sustain scleral ECM structure and inhibit myopia development.

Manganese exposure caused reproductive toxicity of male mice involving activation of GnRH secretion in the hypothalamus by prostaglandin E2 receptors EP1 and EP2.

Exposure to manganese (Mn) can cause male reproductive damage and lead to abnormal secretion of sex hormones. Gonadotropin-releasing hormone (GnRH) plays an important role in the neuromodulation of vertebrate reproduction. Astrocytes can indirectly regulate the secretion of GnRH by binding paracrine prostaglandin E2 (PGE2) specifically to the EP1 and EP2 receptors on GnRH neurons. Prior studies assessed the abnormal secretion of GnRH caused by Mn exposure, but the specific mechanism has not been reported in detail. This study investigated the effects of Mn exposure on the reproductive system of male mice to clarify the role of PGE2 in the abnormal secretion of GnRH in the hypothalamus caused by exposure to Mn. Our data demonstrate that antagonizing the EP1 and EP2 receptors of PGE2 can restore abnormal levels of GnRH caused by Mn exposure. Mn exposure causes reduced sperm count and sperm shape deformities. These findings suggest that EP1 and EP2, the receptors of PGE2, may be the key to abnormal GnRH secretion caused by Mn exposure. Antagonizing the PGE2 receptors may reduce reproductive damage caused by Mn exposure.

Concerted EP2 and EP4 Receptor Signaling Stimulates Autocrine Prostaglandin E2 Activation in Human Podocytes.

Glomerular hyperfiltration is an important mechanism in the development of albuminuria. During hyperfiltration, podocytes are exposed to increased fluid flow shear stress (FFSS) in Bowman's space. Elevated Prostaglandin E2 (PGE2) synthesis and upregulated cyclooxygenase 2 (Cox2) are associated with podocyte injury by FFSS. We aimed to elucidate a PGE2 autocrine/paracrine pathway in human podocytes (hPC). We developed a modified liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) protocol to quantify cellular PGE2, 15-keto-PGE2, and 13,14-dihydro-15-keto-PGE2 levels. hPC were treated with PGE2 with or without separate or combined blockade of prostaglandin E receptors (EP), EP2, and EP4. Furthermore, the effect of FFSS on COX2, PTGER2, and PTGER4 expression in hPC was quantified. In hPC, stimulation with PGE2 led to an EP2- and EP4-dependent increase in cyclic adenosine monophosphate (cAMP) and COX2, and induced cellular PGE2. PTGER4 was downregulated after PGE2 stimulation in hPC. In the corresponding LC/ESI-MS/MS in vivo analysis at the tissue level, increased PGE2 and 15-keto-PGE2 levels were observed in isolated glomeruli obtained from a well-established rat model with glomerular hyperfiltration, the Munich Wistar Frömter rat. COX2 and PTGER2 were upregulated by FFSS. Our data thus support an autocrine/paracrine COX2/PGE2 pathway in hPC linked to concerted EP2 and EP4 signaling.

Small-molecule inhibition of prostaglandin E receptor 2 impairs cyclooxygenase-associated malignant glioma growth.

BACKGROUND AND PURPOSE: An up-regulation of COX-2 in malignant gliomas causes excessive synthesis of PGE2 , which is thought to facilitate brain tumour growth and invasion. However, which downstream PGE2 receptor subtype (i.e., EP1 -EP4 ) directly contributes to COX activity-promoted glioma growth remains largely unknown. EXPERIMENTAL APPROACH: Using a publicly available database from The Cancer Genome Atlas research network, we compared the expression of PGE2 signalling-associated genes in human lower grade glioma and glioblastoma multiforme (GBM) samples. The Kaplan-Meier analysis was performed to determine the relationship between their expression and survival probability. A time-resolved FRET method was used to identify the EP subtype that mediates COX-2/PGE2 -initiated cAMP signalling in human GBM cells. Taking advantage of a recently identified novel selective bioavailable brain-permeable small-molecule antagonist, we studied the effect of pharmacological inhibition of the EP2 receptor on glioma cell growth in vitro and in vivo. KEY RESULTS: The EP2 receptor is a key Gαs -coupled receptor that mediates COX-2/PGE2 -initiated cAMP signalling pathways in human malignant glioma cells. Inhibition of EP2 receptors reduced COX-2 activity-driven GBM cell proliferation, invasion, and migration and caused cell cycle arrest at G0-G1 and apoptosis of GBM cells. Glioma cell growth in vivo was also substantially decreased by post-treatment with an EP2 antagonist in both subcutaneous and intracranial tumour models. CONCLUSION AND IMPLICATIONS: Taken together, our results suggest that PGE2 signalling via the EP2 receptor increases the malignant potential of human glioma cells and might represent a novel therapeutic target for GBM.

EP2 and EP4 receptor antagonists: Impact on cytokine production and ß2 -adrenergic receptor desensitization in human airway smooth muscle.

Prostaglandin E2 (PGE2 ) is a key prostanoid known to have both proinflammatory and anti-inflammatory impact in the context of chronic respiratory diseases. We hypothesize that these opposing effects may be the result of different prostanoid E (EP) receptor-mediated signaling pathways. In this study, we focus on two of the four EP receptors, EP2 and EP4 , as they are known to induce cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. Using primary human airway smooth muscle (ASM) cells, we first focussed on the PGE2 -induced production of two cAMP-dependent proinflammatory mediators: interleukin 6 (IL-6) and cyclo-oxygenase 2 production. We show that PGE2 -induced IL-6 protein secretion occurs via an EP2 -mediated pathway, in a manner independent of receptor-mediated effects on messenger RNA (mRNA) expression and temporal activation kinetics of the transcription factor cAMP response element binding. Moreover, stimulation of ASM with PGE2 did not establish a positive, receptor-mediated, feedback loop, as mRNA expression for EP2 and EP4 receptors were not upregulated and receptor antagonists were without effect. Our studies revealed that the EP2 , but not the EP4 , receptor is responsible for ß2 -adrenergic desensitization induced by PGE2 . We demonstrate that PGE2 -induced heterologous receptor desensitization responsible for tachyphylaxis to short- (salbutamol) or long- (formoterol) ß2 -agonists (measured by cAMP release) can be reversed by the EP2 receptor antagonist PF-04418948. Importantly, this study highlights that inhibiting the EP2 receptor restores ß2 -adrenergic receptor function in vitro and offers an attractive novel therapeutic target for treating infectious exacerbations in people suffering from chronic respiratory diseases in the future.

Peripherally Restricted, Highly Potent, Selective, Aqueous-Soluble EP2 Antagonist with Anti-Inflammatory Properties.

The prostaglandin E2 receptor, EP2, plays an important role in physiology and in a variety of pathological conditions. Studies indicate that EP2 is pro-inflammatory in chronic peripheral and central nervous system disease and cancer models. Thus, targeting the EP2 receptor with small molecules could be a therapeutic strategy for treating inflammatory diseases and cancer. We recently reported a novel class of competitive antagonists of the EP2 receptor. However, earlier leads displayed low selectivity against the DP1 prostanoid receptor, moderate plasma half-life, and low aqueous solubility, which renders them suboptimal for testing in animal models of disease. We now report a novel compound TG8-69, which has suitable drug-like properties. We present synthesis, lead-optimization studies, pharmacological characterization, and anti-inflammatory properties of this compound that support its use in chronic peripheral inflammatory diseases, including rheumatoid arthritis, endometriosis, and cancer, in which EP2 appears to play a pathogenic role.

Prostanoid EP2 Receptors Are Up-Regulated in Human Pulmonary Arterial Hypertension: A Key Anti-Proliferative Target for Treprostinil in Smooth Muscle Cells.

Prostacyclins are extensively used to treat pulmonary arterial hypertension (PAH), a life-threatening disease involving the progressive thickening of small pulmonary arteries. Although these agents are considered to act therapeutically via the prostanoid IP receptor, treprostinil is the only prostacyclin mimetic that potently binds to the prostanoid EP2 receptor, the role of which is unknown in PAH. We hypothesised that EP2 receptors contribute to the anti-proliferative effects of treprostinil in human pulmonary arterial smooth muscle cells (PASMCs), contrasting with selexipag, a non-prostanoid selective IP agonist. Human PASMCs from PAH patients were used to assess prostanoid receptor expression, cell proliferation, and cyclic adenosine monophosphate (cAMP) levels following the addition of agonists, antagonists or EP2 receptor small interfering RNAs (siRNAs). Immunohistochemical staining was performed in lung sections from control and PAH patients. We demonstrate using selective IP (RO1138452) and EP2 (PF-04418948) antagonists that the anti-proliferative actions of treprostinil depend largely on EP2 receptors rather than IP receptors, unlike MRE-269 (selexipag-active metabolite). Likewise, EP2 receptor knockdown selectively reduced the functional responses to treprostinil but not MRE-269. Furthermore, EP2 receptor levels were enhanced in human PASMCs and in lung sections from PAH patients compared to controls. Thus, EP2 receptors represent a novel therapeutic target for treprostinil, highlighting key pharmacological differences between prostacyclin mimetics used in PAH.

Intra-articular administration of EP2 enhances the articular cartilage repair in a rabbit model.

We have reported the usefulness of chondrocyte sheets on articular cartilage repair in animal experiments. Here, we investigated the regenerative effects of EP2 signalling with or without chondrocyte sheets. Forty-five rabbits were used, with six rabbits in each of the six groups and nine rabbits for chondrocytes and synovial cells harvesting to fabricate triple-layered chondrocyte sheets: osteochondral defect only (control, Group A), EP2 agonist (Group B), EP2 antagonist (Group C), chondrocyte sheets (Group D), EP2 agonist and chondrocyte sheets (Group E), and EP2 antagonist and chondrocyte sheets (Group F). After surgery, the weight distribution ratio was measured as an indicator of pain alleviation. Injections of the EP2 agonist or EP2 antagonist were given from 4 weeks after surgery. The rabbits were sacrificed at 12 weeks, and the repaired tissues were evaluated for histology. The weight distribution ratio and International Cartilage Repair Society grading were as follows: Group A: 40.5% ± 0.2%, 14.8 ± 0.5; Group B: 43.4% ± 0.7%, 25.4 ± 0.8; Group C: 38.7% ± 0.7%, 13.7 ± 0.3; Group D: 48.6% ± 0.6%, 40.2 ± 0.5; Group E: 49.1% ± 0.3%, 40.5 ± 0.4; and Group F; 46.8% ± 0.4%, 38.7 ± 0.5. Significant differences in histology and pain alleviation were observed between groups except between Groups A and C, between Groups D and E, and between Groups D and F. These findings show that the intra-articular administration of an EP2 agonist achieved pain alleviation and tissue repair. However, no synergistic effect with chondrocyte sheets was observed.

The protective effect of the EP2 receptor on TGF-ß1 induced podocyte injury via the PI3K / Akt signaling pathway.

Transforming growth factor ß1 (TGF-ß1) plays a central role in chronic kidney diseases. TGF-ß1 induction causes podocyte injury, which results in proteinuria and renal failure. However, the effect of the prostaglandin E2 /E-prostanoid receptor (EP2) on TGF-ß1-induced podocyte injury remains unknown. Previous studies have shown that phosphoinositide 3-OH kinase (PI3K)/Akt is widespread in cells, and is vital for the regulation of cell proliferation, differentiation, apoptosis and metabolism. In this study, we cultured immortalized mouse podocytes in vitro in different groups: control group; TGF-ß1 (5ng/ml) group; EP2 agonist Butaprost treatment (10-7, 10-6, or 10-5mol/L) +TGF-ß1 group; EP2 antagonist AH6809 treatment (10-7, 10-6, or 10-5mol / L) + TGF-ß1 group. We found that compared with the control group, proliferation of podocytes in the TGF-ß1 group significantly decreased and apoptosis increased. Expression of cAMP decreased, whereas PGE2 increased. Meanwhile, expressions of nephrin, podocin and CD2AP mRNA and protein were dramatically downregulated, activated caspase-3 was increased, and activated PI3K/Akt activity were depressed. Butaprost intervention promoted podocyte proliferation with reduced apoptosis. Conversely, AH6809 intervention led to opposite results (P<0.05). Our findings suggested that EP2 agonist protects podocytes by increasing expression of cAMP, which creates feedback of inhibiting PGE2 expression. This causes the interaction of nephrin, podocin and CD2AP resulting the inhibition of apoptosis induced by activation of the PI3K / Akt signaling pathway.

Effects of an EP2 and EP3 Receptor Dual Agonist, ONO-8055, on a Radical Hysterectomy-Induced Underactive Bladder Model in Monkeys.

OBJECTIVES: The objective was to develop an underactive bladder (UAB) model in primates and to evaluate the potential of prostanoid EP2 and EP3 receptor dual agonist ONO-8055 to become a therapeutic agent for UAB. METHODS: A surgical procedure resembling radical hysterectomy was performed on female cynomolgus monkeys. Subsequently, in vitro muscle strip studies were performed using bladder muscle strips from normal monkeys and monkeys that underwent surgery. In addition, uroflowmetric data were obtained at specified days after the surgery. To evaluate the effects of ONO-8055 and distigmine (DIS) on voiding function in the UAB monkey model, uroflowmetry was performed approximately 1 week after the surgery, before and after the cumulative intravenous administration of the compounds at 2 h intervals. RESULTS: In the bladder muscle strip studies, the responses to potassium chloride at 2 months, and carbachol and electrical field stimulation from 2 weeks decreased significantly. Voided volume (VV), maximum flow rate (Qmax), and average flow rate (Qave) decreased after surgery, while voiding time (VT) increased. In this model, ONO-8055 and DIS significantly increased VV and Qmax. DIS prolonged VT, while ONO-8055 had no effect. The results also showed that ONO-8055 increased Qave. CONCLUSIONS: We developed a neurogenic UAB model in primates. As ONO-8055 improved voiding function in this model to at least the same degree as DIS, this EP2 and EP3 receptor dual agonist has the potential to be a candidate for neurogenic UAB pharmacotherapy.

Prostaglandin EP2 Receptors Mediate Mesenchymal Stromal Cell-Neuroprotective Effects on Dopaminergic Neurons.

Mesenchymal stromal cells (MSCs) have been shown to have useful properties for cell therapy and have been proposed for treatment of neurodegenerative diseases, including Parkinson's disease. However, the mechanisms involved in recovering dopaminergic neurons are not clear. The present study aims to evaluate the pathways and molecules involved in the neuroprotective effect of MSCs. We analyzed the viability of dopaminergic cells from different sources in response to conditioned medium derived from bone marrow MSC (MSC-CM). MSC-CM increased the viability of dopaminergic cells of rat and human origins, having both neuroprotective and neurorescue activities against effects of dopaminergic neurotoxin 6-hydroxydopamine. We found that lipid removal, inhibition of the prostaglandin E2 receptor 2 (EP2), and its signaling pathway were able to block the effects of MSC-CM on a pure population of dopaminergic neurons. Moreover, in primary mesencephalic cultures and hiPSC-derived neurons, inhibition of EP2 signaling caused a reduction in the number of dopaminergic neurons obtained in culture. Taken together, our results demonstrate for the first time the involvement of prostaglandin signaling from MSC in dopaminergic neuron survival through EP2 receptors, and suggest new approaches for treatment of Parkinson's disease.

PGE2 synthesis and signaling in malignant transformation and progression of human hepatocellular carcinoma.

Prostaglandin E 2 (PGE2), which is the most abundant prostaglandin produced in hepatocellular carcinoma (HCC), may be involved in hepatocarcinogenesis. Here, the amount of PGE2 was significantly increased in HCC tissue and adjacent noncancerous tissues relative to normal liver tissue (P<.001). In addition, the expression of EP2 receptor was considerably upregulated in HCC tissue compared with the expression of EP1 (P<.05), EP3 (P<.01), and EP4 (P<.01) receptor. The expression of EP2 receptor was positively correlated with the level of PGE2 in HCC tissue (P<.001). Furthermore, PGE2 significantly increased proliferation and invasion potential of human HCC cells. However, antagonism of EP2 signaling suppressed PGE2-induced growth and invasion in human HCC cells. Taken together, upregulation of PGE2 level was associated with proliferation and invasion potential of HCC, and EP2 receptor predominately mediated the function of PGE2 in the transformation and progression of HCC.

Prostaglandin E2-EP2-NF-κB signaling in macrophages as a potential therapeutic target for intracranial aneurysms.

Intracranial aneurysms are common but are generally untreated, and their rupture can lead to subarachnoid hemorrhage. Because of the poor prognosis associated with subarachnoid hemorrhage, preventing the progression of intracranial aneurysms is critically important. Intracranial aneurysms are caused by chronic inflammation of the arterial wall due to macrophage infiltration triggered by monocyte chemoattractant protein-1 (MCP-1), macrophage activation mediated by the transcription factor nuclear factor κB (NF-κB), and inflammatory signaling involving prostaglandin E2 (PGE2) and prostaglandin E receptor subtype 2 (EP2). We correlated EP2 and cyclooxygenase-2 (COX-2) with macrophage infiltration in human intracranial aneurysm lesions. Monitoring the spatiotemporal pattern of NF-κB activation during intracranial aneurysm development in mice showed that NF-κB was first activated in macrophages in the adventitia and in endothelial cells and, subsequently, in the entire arterial wall. Mice with a macrophage-specific deletion of Ptger2 (which encodes EP2) or macrophage-specific expression of an IκBα mutant that restricts NF-κB activation had fewer intracranial aneurysms with reduced macrophage infiltration and NF-κB activation. In cultured cells, EP2 signaling cooperated with tumor necrosis factor-α (TNF-α) to activate NF-κB and synergistically induce the expression of proinflammatory genes, including Ptgs2 (encoding COX-2). EP2 signaling also stabilized Ccl2 (encoding MCP-1) by activating the RNA-stabilizing protein HuR. Rats administered an EP2 antagonist had reduced macrophage infiltration and intracranial aneurysm formation and progression. This signaling pathway in macrophages thus facilitates intracranial aneurysm development by amplifying inflammation in intracranial arteries. These results indicate that EP2 antagonists may therefore be a therapeutic alternative to surgery.

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.

Topical 0.1% Bromfenac Sodium for Intraoperative Miosis Prevention and Prostaglandin E2 Inhibition in Femtosecond Laser-Assisted Cataract Surgery.

PURPOSE: The purpose of this study was to evaluate the effect of topical 0.1% bromfenac sodium, a nonsteroidal anti-inflammatory drug (NSAID), on intraoperative pupil dilation maintenance and prostaglandin E2 (PGE2) inhibition during femtosecond laser-assisted cataract surgery. METHODS: Sixty patients (30 each in study and control groups) were included in this study. The patients received 0.1% bromfenac ophthalmic solution or control placebo twice a day for 3 days before surgery. Pupil size was measured at the initiation of femtosecond laser pretreatment and phacoemulsification. Aqueous humor was collected at the beginning of routine cataract surgery. PGE2 levels were measured with an enzyme-linked immunoassay. Laser flare photometry was measured preoperatively and at 1 day postoperatively. RESULTS: Compared with untreated patients, the change in pupil size and postoperative day 1 aqueous flare were significantly reduced throughout the operation in the patients treated with 0.1% bromfenac (P < 0.001). Mean PGE2 concentrations were also significantly decreased by treatment with 0.1% bromfenac (P < 0.001). The reduction of the pupil area and postoperative day 1 aqueous flare were significantly correlated with PGE2 levels (P < 0.001). CONCLUSION: NSAID treatment, when administered before femtosecond laser-assisted cataract surgery, was effective in maintaining intraoperative pupil dilation, preventing miosis, and reducing PGE2 levels.