EP Receptor Expression Research Articles

Prostaglandin E2 inhibits the differentiation of T regulatory cells by Peroxisome Proliferator-Activated Receptor-Gamma during allergic rhinitis.

Allergic rhinitis (AR) represents a significant global health concern that can give rise to numerous diseases and result in labor productivity. T regulatory (Treg) cells are pivotal players in the pathogenesis of AR, and their deficiencies are closely related to Prostaglandin E2 (PGE2). However, the downstream mechanisms of this relationship remain poorly understood. This study aims to investigate the inhibitory mechanisms through which PGE2 impacts the differentiation of Treg cells. We compared the differentiation of Treg cells from naïve CD4+ T cells of AR patients and healthy controls, with or without the presence of PGE2 by flow cytometry. Intracellular cAMP concentration, mRNA and protein levels of cyclic-AMP dependent protein kinase A (PKA), as well as their downstream target, Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) were examined in Treg cells from AR and healthy donors. AR mouse model was established by pollen administration. PGE2 suppressed the differentiation of Treg cells from human naïve CD4+ T cells through the EP4 receptor. Furthermore, in AR patients and AR mouse, the expression of EP4 receptor were observed enhanced. The PGE2-EP4 signal was carried out by activating cAMP-PKA signaling pathway. Subsequently, phospholated PKA would suppress PPAR-γ expression. Treatment of Pioglitazone, a PPAR-γ agonist, was demonstrated to rescue the differentiation of Treg and help alleviate inflammation in the AR mouse model. In AR disease, the PGE2-EP4 signaling exerts an inhibitory effect on Treg differentiation by influencing the cAMP-PKA pathway and its downstream target PPAR-γ.

Decreased Expression of EP3 Receptor mRNA in the Brain of Mouse Model of Autism Spectrum Disorder.

Accumulating evidence has implicated the role of neuroinflammation in the pathology of autism spectrum disorder (ASD), a neurodevelopmental disorder. To investigate the expression of prostaglandin EP3 (EP3) receptor mRNA in the brain of ASD mouse model. Pregnant mice were injected with valproic acid (VPA) 500 mg/kg intraperitoneally at 12.5 d gestation. The offspring were tested at the age of 5-6 weeks old for their social interaction behavior. Each mouse was assessed for prostaglandin EP3 receptor expression in the prefrontal cortical, hippocampal and cerebellar areas one day after the behavioral test. Compared to the naive, mice born to dams treated with VPA demonstrated a significantly shorter duration of sniffing behavior, a model of social interaction. Results further showed that the expression of EP3 receptor mRNA was significantly lower in all three brain regions of the mice born to VPA-treated dams. The present study provides further evidence of the relevance of the arachidonic acid cascade as an essential part of neuroinflammation in the pathology of ASD.

Complementary roles of EP2 and EP4 receptors in malignant glioma.

Glioblastoma (GBM) is the most aggressive brain tumour in the central nervous system, but the current treatment is very limited and unsatisfactory. PGE2 -initiated cAMP signalling via EP2 and EP4 receptors is involved in the tumourigenesis of multiple cancer types. However, whether or how EP2 and EP4 receptors contribute to GBM growth largely remains elusive. We performed comprehensive data analysis of gene expression in human GBM samples and determined their expression correlations through multiple bioinformatics approaches. A time-resolved fluorescence energy transfer (TR-FRET) assay was utilized to characterize PGE2 -mediated cAMP signalling via EP2 and EP4 receptors in human glioblastoma cells. Using recently reported potent and selective small-molecule antagonists, we determined the effects of inhibition of EP2 and EP4 receptors on GBM growth in subcutaneous and intracranial tumour models. The expression of both EP2 and EP4 receptors was upregulated and highly correlated with a variety of tumour-promoting cytokines, chemokines, and growth factors in human gliomas. Further, they were heterogeneously expressed in human GBM cells, where they compensated for each other to mediate PGE2 -initiated cAMP signalling and to promote colony formation, cell invasion and migration. Inhibition of EP2 and EP4 receptors revealed that these receptors might mediate GBM growth, angiogenesis, and immune evasion in a compensatory manner. The compensatory roles of EP2 and EP4 receptors in GBM development and growth suggest that concurrently targeting these two PGE2 receptors might represent a more effective strategy than inhibiting either alone for GBM treatment.

Inhibition of cyclooxygenase and EP3 receptor improved long term potentiation in a rat organotypic hippocampal model of repeated blast traumatic brain injury.

Blast-induced traumatic brain injury (bTBI) is a health concern in military service members who are exposed to multiple blasts throughout their training and deployment. Our group has previously reported decreased long term potentiation (LTP) following repeated bTBI in a rat organotypic hippocampal slice culture (OHSC) model. In this study, we investigated changes in inflammatory markers like cyclooxygenase (COX) and tested the efficacy of COX or prostaglandin EP3 receptor (EP3R) inhibitors in attenuating LTP deficits. Expression of COX-2 was increased 48h following repeated injury, whereas COX-1 expression was unchanged. EP3R expression was upregulated, and cyclic adenosine monophosphate (cAMP) concentration was decreased after repeated blast exposure. Post-traumatic LTP deficits improved after treatment with a COX-1 specific inhibitor, SC-560, a COX-2 specific inhibitor, rofecoxib, a pan-COX inhibitor, ibuprofen, or an EP3R inhibitor, L-798,106. Delayed treatment with ibuprofen and L-798,106 also prevented LTP deficits. These findings suggest that bTBI induced neuroinflammation may be responsible for some functional deficits that we have observed in injured OHSCs. Additionally, COX and EP3R inhibition may be viable therapeutic strategies to reduce neurophysiological deficits after repeated bTBI.

Effect of MP-AzeFlu compared to monotherapy on COX-2, PGE2 , and EP2 gene expression in upper airway mucosa.

MP-AzeFlu (intranasal fluticasone and azelastine) has been widely studied and has demonstrated efficacy in Allergic rhinitiswith a superior effect compared to these drugs administered individually; however, the mechanism by which MP-AzeFlu produces this improved clinical effect has not yet been fully explained. In this study, we investigated the effect of MP-AzeFlu and fluticasone propionate (FP) on arachidonic acid metabolism as measured by changes in regulation of cyclooxygenase (COX) isoforms, prostaglandin (PG) D2 , PGE2 , PGE2 receptor (EP) 2, and EP3. Expression of these key inflammation markers was assessed through an in vitro model of upper airway inflammation using fibroblasts derived from both healthy and inflamed upper airway mucosa. Both MP-AzeFlu and FP inhibitedinterleukin-1β-induced COX-2 messengerRNA(mRNA) and protein expression and PGE2 secretion in vitro. MP-AzeFlu and FP both upregulated EP2 mRNA expression, though neither upregulated EP2 protein expression. This downregulation of COX-2 and PGE2 coupled with upregulation of EP2 receptor expression reinforces the anti-inflammatory effect of MP-AzeFlu in upper airway inflammation.

PGE2 inhibits neutrophil phagocytosis through the EP2R-cAMP-PTEN pathway.

Prostaglandin E2 (PGE2) is a potent lipid mediator of inflammation that modulates immune cell function by binding to unique G protein‐coupled receptors (EP receptors). PGE2 production increases during microbial infection and inflammation. In this study, we assessed the effect of PGE2 on the phagocytosis of bacteria by neutrophils, which are key players during infection and inflammation. We also looked for specific EP receptor signaling pathways that contributed to the neutrophil phagocytic activity. PGE2 (50–1000 ng/ml) inhibited the phagocytosis of Escherichia coli by HL‐60 human neutrophils in a concentration‐dependent manner. Inhibition of neutrophil phagocytosis by PGE2 correlated with increased intracellular cyclic adenosine monophosphate (cAMP) production, and forskolin, an adenosyl cyclase agonist, confirmed the inhibitory effect of cAMP stimulation on neutrophil phagocytosis. The expression of EP2 receptors by HL‐60 cells was confirmed by western blot analysis, and selective agonism of EP2 receptors mimicked the inhibition of phagocytosis by PGE2. The EP2 receptor antagonist AH‐6089 partially blocked the inhibition of neutrophil phagocytosis PGE2. Specific inhibition of phosphatase and tensin homolog (PTEN) enzyme attenuated the inhibition of neutrophil phagocytosis by PGE2, and both PGE2 and increased intracellular cAMP increased neutrophil PTEN activity, which was associated with decreased PTEN phosphorylation. The results support negative regulation of the antimicrobial activity of neutrophils (i.e., phagocytosis), which has important implications for the future management of bacterial infections.

Nanomolar EP4 receptor potency and expression of eicosanoid-related enzymes in normal appearing colonic mucosa from patients with colorectal neoplasia

BackgroundAberrations in cyclooxygenase and lipoxygenase (LOX) pathways in non-neoplastic, normal appearing mucosa from patients with colorectal neoplasia (CRN), could hypothetically qualify as predisposing CRN-markers.MethodsTo test this hypothesis, biopsies were obtained during colonoscopy from macroscopically normal colonic mucosa from patients with and without CRN. Prostaglandin E2 (PGE2) receptors, EP1-4, were examined in Ussing-chambers by exposing biopsies to selective EP receptor agonists, antagonists and PGE2. Furthermore, mRNA expression of EP receptors, prostanoid synthases and LOX enzymes were evaluated with qPCR.ResultsData suggest that PGE2 binds to both high and low affinity EP receptors. In particular, PGE2 demonstrated EP4 receptor potency in the low nanomolar range. Similar results were detected using EP2 and EP4 agonists. In CRN patients, mRNA-levels were higher for EP1 and EP2 receptors and for enzymes prostaglandin-I synthase, 5-LOX, 12-LOX and 15-LOX.ConclusionsIn conclusion, normal appearing colonic mucosa from CRN patients demonstrates deviating expression in eicosanoid pathways, which might indicate a likely predisposition for early CRN development and furthermore that PGE2 potently activates high affinity EP4 receptor subtypes, supporting relevance of testing EP4 antagonists in colorectal neoplasia management.

COXIBs and 2,5-dimethylcelecoxib counteract the hyperactivated Wnt/\u03b2-catenin pathway and COX-2/PGE2/EP4 signaling in glioblastoma cells

BackgroundGlioblastoma (GBM) is the deadliest and the most common primary brain tumor in adults. The invasiveness and proliferation of GBM cells can be decreased through the inhibition of Wnt/β-catenin pathway. In this regard, celecoxib is a promising agent, but other COXIBs and 2,5-dimethylcelecoxib (2,5-DMC) await elucidation. Thus, the aim of this study was to analyze the impact of celecoxib, 2,5-DMC, etori-, rofe-, and valdecoxib on GBM cell viability and the activity of Wnt/β-catenin pathway. In addition, the combination of the compounds with temozolomide (TMZ) was also evaluated. Cell cycle distribution and apoptosis, MGMT methylation level, COX-2 and PGE2 EP4 protein levels were also determined in order to better understand the molecular mechanisms exerted by these compounds and to find out which of them can serve best in GBM therapy.MethodsCelecoxib, 2,5-DMC, etori-, rofe- and valdecoxib were evaluated using three commercially available and two patient-derived GBM cell lines. Cell viability was analyzed using MTT assay, whereas alterations in MGMT methylation level were determined using MS-HRM method. The impact of COXIBs, in the presence and absence of TMZ, on Wnt pathway was measured on the basis of the expression of β-catenin target genes. Cell cycle distribution and apoptosis analysis were performed using flow cytometry. COX-2 and PGE2 EP4 receptor expression were evaluated using Western blot analysis.ResultsWnt/β-catenin pathway was attenuated by COXIBs and 2,5-DMC irrespective of the COX-2 expression profile of the treated cells, their MGMT methylation status, or radio/chemoresistance. Celecoxib and 2,5-DMC were the most cytotoxic. Cell cycle distribution was altered, and apoptosis was induced after the treatment with celecoxib, 2,5-DMC, etori- and valdecoxib in T98G cell line. COXIBs and 2,5-DMC did not influence MGMT methylation status, but inhibited COX-2/PGE2/EP4 pathway.ConclusionsNot only celecoxib, but also 2,5-DMC, etori-, rofe- and valdecoxib should be further investigated as potential good anti-GBM therapeutics.

Uterine receptor activation in response to uterotonic agents in late-pregnancy rat myometrium following pretreatment with oxytocin: an in vitro study

Background: Long term exposure to oxytocin reduces the ability of myometrium to respond to oxytocin, leading to oxytocin receptor (OXTR) desensitization. In this study we analyzed the response to other uterotonics such as prostaglandin, as well as investigating prostaglandin E2 receptors (EP3) and prostaglandin F2α receptors (FP). We hypothesized that compensatory mechanisms would increase the expression and activation of FP and EP3 following OXTR desensitization. Methods: Myometrium from late-pregnancy rats was collected in order to assess mRNA expression levels for OXTR, FP, and EP3 using RT-PCR. This was done after 2 hours of pretreatment with 10-6 M oxytocin to induce OXTR desensitization, or equilibration in physiological salt solution (PSS). Myometrium was exposed to increasing concentrations of uterotonic agents (10-10 to 10-5 M) following 2 hours of pretreatment with 10-6 M oxytocin (experimental group) or with PSS (control group). Myometrium from the experimental group was washed with PSS and OXTR expression was assessed using Western blot and RT-PCR. Results: mRNA expression levels for EP3, FP and OXTR were not statistically different between the experimental (OXTR desensitization) and control groups. Compared to the control group, the (mean ± SD) contractile potency of carboprost (pEC50: 7.74 ± 0.56 vs 6.81 ± 0.25, P = 0.03) and maximal contractility of misoprostol (Emax(ratio): 4.44 ± 3.60 vs 1.32 ± 0.22, P = 0.02) were significantly increased in the OXTR desensitization group, while the contractility of oxytocin was significantly reduced (Emax(ratio): 1.62 ± 0.27 vs 2.82 ± 0.98, P = 0.015). No significant differences in myometrial OXTR expression were observed between the PSS, carboprost and misoprostol groups following OXTR desensitization. Discussion: Following OXTR desensitization of myometrium, FP and EP3 activation increased in a compensatory manner, but not FP and EP3 receptor expression.

Thymoquinone Suppresses Migration of Human Renal Carcinoma Caki-1 Cells through Inhibition of the PGE2-Mediated Activation of the EP2 Receptor Pathway.

Renal cell carcinoma (RCC) is likely to metastasize to other organs, and is often resistant to conventional chemotherapies. Thymoquinone (TQ), a phytochemical derived from the seeds of Nigella sativa, has been shown to inhibit migration and metastasis in various cancers. In this study, we assessed the effect of TQ on the migratory activity of human RCC Caki-1 cells. We found that treatment with TQ reduced the proteolytic activity of matrix metalloproteinase-9 (MMP-9) in Caki-1 cells. TQ significantly repressed prostaglandin E2 (PGE2) production, its EP2 receptor expression as well as the activation of Akt and p38, the well-known upstream signal proteins of MMP-9. In addition, treatment with butaprost, a PGE2 agonist, also induced MMP-9 activity and migration/invasion in Caki-1 cells. Moreover, pharmacological inhibitors of PI3K/Akt and p38 remarkably attenuated butaprost-induced Caki-1 cell migration and invasion, implying that activation of PI3K/Akt and p38 is a bridge between the PGE2-EP2 axis and MMP-9-dependent migration and invasion. Taken together, these data suggest that TQ is a promising anti-metastatic drug to treat advanced and metastatic RCC.

The role of EP-2 receptor expression in cervical intraepithelial neoplasia

Prostaglandin induced signalling is involved in different cancers. As previously described, the EP3 receptor expression decreases with increasing stage of cervical intraepithelial lesions (CIN). In addition, in cervical cancer EP3 is an independent prognosticator for overall survival and correlates with FIGO stages. Currently the role of Prostaglandin 2 receptor 2 (EP2) in CIN is unknown. The aim of this study was to analyse the expression of EP2 for potential prognostic value for patients with cervical dysplasia. EP2 expression was analysed by immunohistochemistry in 33 patient samples (CIN1–3) using the immune-reactivity scoring system (IRS). Expression levels were correlated with clinical outcome to analyse prognostic relevance in patients with CIN2. Data analysis was performed using non parametric Kruskal–Wallis and Spearman rank sum test. Cytoplasmic expression levels of EP2 correlated significantly (p < 0.001) with different grades of cervical dysplasia. Median EP2-IRS in CIN1 was 2 (n = 8), 3 in CIN2 (n = 9) and 6 in CIN3 (n = 16). Comparing regressive (n = 3, median IRS = 2) to progressive (n = 6, median IRS = 4) CIN2 cases the median IRS differed significantly (p = 0.017). Staining intensity (p = 0.009) and IRS (p = 0.005) of EP2 and EP3 correlate inversely. EP2 expression level significantly increases with higher grade of CIN and could qualify as a potential prognostic marker for the regressive or progressive course in CIN2 lesions. These findings emphasize the significant role of PGE2 signalling in CIN and could help to identify targets for future therapies.

Effects of alprostadil in β-aminopropanitrile induced aortic dissection in a murine model

Objective: To investigate the protective role of alprostadil on aortic dissection. Methods: 26 C57BL6 male mice were divided into control group (normal drinking water, n=13) and model group (1 g·kg-1·d-1 BAPN via drinking water, n=13). On day 14, mRNA expression of inflammatory-related genes as well as EP receptor families were detected by RT-PCR (n=6 each) and EP4 protein levels were determined by Western blot (n=7 each). Another 88 mice were divided into 3 groups: control group (n=22), model group (n=33) and treatment group (n=33). The mice in model group and treatment group were applied with BAPN (1 g·kg-1·d-1) via drinking water. The mice in treatment group received additional intraperitoneal injection with alprostadil (80 μg·kg-1·d-1) for 28 days. The mice in the control and model group received equal volume intraperitoneal injection with 0.9% saline respectively. The body weight and systolic blood pressure, the mortality and morbidity were monitored from the beginning until the designed end of the study. On day 28, the mice were sacrificed and aorta were fixed, embedded and sliced, followed by staining with HE and Victoria Blue. The distribution of EP4 was determined by immunohistochemistry in control (n=6) and model group (n=6). Furthermore, the concentration of PGE1 were tested among model (n=3) and treatment group (n=4). EP4 protein expression was determined in model group (n=7) and treatment group (n=6). Results: On day 14, mRNA expression level of MCP-1 ((2.74±1.55) vs. (1.00±0.49),<0.05) and MMP2((1.38±0.42) vs. (1.00±0.27), P<0.05) was significantly upregulated in model group compared with control group. Protein expression of EP4 receptor also increased in aorta in model group compared with control group (1.48±0.51 vs. 1.00±0.19, P<0.05). In the dissection area, the EP4 expression was also enriched compared with non-dissection area, particularly in endothelial cells and inflammatory cells on day 28. BAPN applied in drinking water (model and treatment groups) successfully induced the aortic dissection in mice, some mice died of the rupture. The elastic fibers were fractured, and the infiltrated immune cells were visible in dissected tissue. False lumen was formed. There was no dissection and death in the control group. Compared with control group, the morbidity and mortality rates were significantly increased in the model group (60.6%, 20/33, 30.3%, 10/33) and the treatment group (72.7%, 24/33, 24.2%, 8/33). The mortality and morbidity rates were similar between model and treatment groups. There is no difference in terms of SBP among three groups (P>0.05). Further study showed that after alprostadil injection, the blood concentration of PGE1 was increased in treatment group ((0.540±0.041 vs. 0.436±0.012)μmol/L, P<0.05). Besides, the EP4 receptor expression was downregulated in the treatment group compared to model group (0.60±0.30 vs. 1.00±0.20, P<0.05). Conclusion: EP4 expression is upregulated in BAPN induced aortic dissection mouse model. No protective effects are observed post alprostadil treatment in this model probably due to the reduced expression of EP4.

Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem Cells.

Simple SummaryProstaglandin E2 plays a regulatory role in MSC self-renewal, and PGE2 receptor-mediated signalling is involved in cell proliferation and migration. The aim of this work was to establish a genetically modified equine cellular model in which receptor genes EP2 and EP4 were edited by the CRISPR/Cas9 system. We successfully targeted EP2 and EP4 receptors in horse adipose MSCs using the CRISPR/Cas9 system. This targeting did not affect the surface marker phenotype of the adipose MSCs generated. Gene edition significantly lowered the expression of each of the targeted receptors and affected the early migration ability of the edited MSCs. This opens the possibility of using these mutant cell lines as a model system to elucidate the role of EP2 and EP4 in MSCs and, particularly, as therapeutic tools in equine regenerative medicine.In mesenchymal stem cells (MSCs), it has been reported that prostaglandin E2 (PGE2) stimulation of EP2 and EP4 receptors triggers processes such as migration, self-renewal, survival, and proliferation, and their activation is involved in homing. The aim of this work was to establish a genetically modified adipose (aMSC) model in which receptor genes EP2 and EP4 were edited separately using the CRISPR/Cas9 system. After edition, the genes were evaluated as to if the expression of MSC surface markers was affected, as well as the migration capacity in vitro of the generated cells. Adipose MSCs were obtained from Chilean breed horses and cultured in DMEM High Glucose with 10% fetal bovine serum (FBS). sgRNA were cloned into a linearized LentiCRISPRv2GFP vector and transfected into HEK293FT cells for producing viral particles that were used to transduce aMSCs. GFP-expressing cells were separated by sorting to obtain individual clones. Genomic DNA was amplified, and the site-directed mutation frequency was assessed by T7E1, followed by Sanger sequencing. We selected 11 clones of EP2 and 10 clones of EP4, and by Sanger sequencing we confirmed 1 clone knock-out to aMSC/EP2 and one heterozygous mutant clone of aMSC/EP4. Both edited cells had decreased expression of EP2 and EP4 receptors when compared to the wild type, and the edition of EP2 and EP4 did not affect the expression of MSC surface markers, showing the same pattern in filling the scratch. We can conclude that the edition of these receptors in aMSCs does not affect their surface marker phenotype and migration ability when compared to wild-type cells.

Gene expression of prostaglandin EP4 receptor in three canine carcinomas

BackgroundChronic inflammation mediated by the cyclooxygenase enzymes, specifically their product prostaglandin E2 (PGE2), can result in the development of cancer. PGE2 promotes cell proliferation, apoptosis, and angiogenesis through interaction with its specific receptors (EP1 receptor - EP4 receptor [EP1R-EP4R]). In multiple human cancers, the expression of EP4R is associated with the development of malignancy and a poor prognosis. The expression of EP4R has not yet been evaluated in canine tumors. The aim of this study was to characterize the mRNA gene expression of EP4R (ptger4) in canine squamous cell carcinoma (SCC), apocrine gland anal sac adenocarcinoma (AGASACA), and transitional cell carcinoma (TCC). Archived tumor samples of canine cutaneous SCC (n = 9), AGASACA (n = 9), and TCC (n = 9), and matched archived normal tissue controls were evaluated for mRNA expression of canine EP4R using RNA in situ hybridization (RNAscope®). Quantification of RNAscope® signals in tissue sections was completed with an advanced digital pathology image analysis system (HALO). Data was expressed as copy number, H-index, and percent tumor cell expression of EP4R.ResultsIn all canine SCC, AGASACA, and TCC samples evaluated, strong universal positive expression of EP4R was identified. For SCC and AGASACA, mRNA EP4R expression was statistically higher than that of their respective normal tissues. The TCC tissues displayed significantly less mRNA EP4R expression when compared to normal bladder mucosa.ConclusionsThese results confirm the mRNA expression of canine EP4R in all tumor types evaluated, with SCC and AGASACA displaying the highest expression, and TCC displaying the lowest expression. This study also represents the first reported veterinary evaluation of EP4R expression using the novel in situ hybridization technique, RNAscope®.

Regulation of DNA methyltransferase 3a Expression by Prostaglandin E2 in Human Fibroblasts

IntroductionPeriodontitis is common chronic inflammatory condition that not only results in loss of teeth, but contributes to a variety of systemic conditions, including cancer. Previously we showed that exposure of human gingival fibroblasts (HGF) derived from patients with periodontitis to PGE2 decreases expression of DNMT1, DNMT3a and TET1, but increases global levels of methylated and hydroxymethylated DNA, acting predominantly through the EP4 receptor. The current study compares the effects of PGE2 on expression of these genes in non‐inflamed fibroblasts and aims to further elucidate the signaling pathways involved.MethodsReal time PCR and ELISA are used to measure expression of DNMT1, DNMT3a, TET1 and EP receptors 1–4 in untreated HGF and IMR90 lung fibroblasts and those treated with PGE2. The effects of actinomycin D, as well as EP receptor agonists and antagonists and signaling pathway activators and inhibitors on mRNA expression of these genes will also be assessed by qPCR.ResultsResults so far show that PGE2 decreases expression of DNMT1 in IMR90 cells to an extent similar to that observed in HGF, but levels of DNMT3a are increased rather than decreased, and levels of TET1 are unaffected in IMR90 cells. Although all four EP receptors are expressed in both HGF and IMR90, the relative levels of EP4 expression are lower.ConclusionThe ability of this inflammatory mediator to influence DNA methylation is consistent with the association of chronic inflammation with increased risk of cancer. However, the fact that the effects are cell and context dependent indicate that more information about the specific signaling pathways involved in regulating expression of these important enzymes is needed. Further studies will be aimed at elucidating the roles of the PGE2 receptors and their signaling pathways in mediating the cell‐specific effects.

Distribution and Function of Prostaglandin E2Receptors in Mouse Uterus: Translational Value for Human Reproduction

Prostaglandin (PG) E analogues are used clinically to ripen the cervix and induce labour. However, selective receptor agonists may have potential to improve induction response rates or manage unwanted uterine hypercontractility in conditions such as dysmenorrhoea and preterm labour. To characterise their therapeutic value, PGE2 analogues were used to investigate the functional EP receptor population in isolated human uterus. Responsiveness in mouse tissues was also examined to validate its use as a pre-clinical model. Uterine samples were obtained from mice at dioestrus (n=12), term gestation (n=14) and labour (n=12) and from the lower uterus of women undergoing hysterectomy (n=12) or Caesarean section (n=18). Vehicle and agonist effects were assessed using superfusion and immersion techniques. PGE2 evoked predominant excitatory responses in mouse and relaxation in human tissues. Selective EP4 agonists inhibited tissue activity in both non-pregnant species, whilst the EP2 mimetic CP533536 also attenuated uterine contractions throughout gestation. The uterotonic effects of the EP3/1 agonist sulprostone were more pronounced than the EP1 agonist ONO-D1-004, corresponding to abundant EP3 receptor expression in all samples. The contractile phenotype in mouse compared to human uteri may relate to regional differences as well as high expression of EP3 receptor transcripts. Similarities in non-pregnant and gestational tissues across species suggest that EP3 may represent a valuable translational drug target for preventing uterine hypercontractility by employing a selective antagonist. SIGNIFICANCE STATEMENT: This research validates the use of non-pregnant mice for pre-clinical drug discovery of uterine EP receptor targets. To determine the utility of novel drugs and delivery systems at term pregnancy and labour, pharmacological agents interacting with EP3 receptors have clear translational value.

A representative metalloprotease induces PGE2 synthesis in fibroblast-like synoviocytes via the NF-\u03baB/COX-2 pathway with amplification by IL-1\u03b2 and the EP4 receptor

Inflammatory joint conditions are characterized by synovial inflammation, which involves activation of fibroblast-like synoviocytes (FLSs) and production of inflammatory mediators and matrix metalloproteases (MMPs) in joints. This study showed that the snake venom metalloprotease (SVMP) BaP1 activates FLSs to produce PGE2 by a mechanism dependent on COX-2, mPGES-1 and iPLA2s. BaP1 also induces IL-1β release, which up-regulates the production of PGE2 at a late stage of the stimulation. Expression of COX-2 and mPGES-1 are induced by BaP1 via activation of NF-κB pathway. While NF-κB p50 and p65 subunits are involved in up-regulation of COX-2 expression, only p65 is involved in BaP1-induced mPGES-1 expression. In addition, BaP1 up-regulates EP4 receptor expression. Engagement of this receptor by PGE2 triggers a positive feedback loop for its production by up-regulating expression of key components of the PGE2 biosynthetic cascade (COX-2, mPGES-1 and the EP4 receptor), thus contributing to amplification of BaP1-induced effects in FLSs. These data highlight the importance of FLS as a target for metalloproteases in joint inflammation and provide new insights into the roles of MMPs in inflammatory joint diseases. Moreover, our results may give insights into the importance of the catalytic domain, of MMPs for the inflammatory activity of these enzymes.

Prostaglandin E2, a postulated mediator of neurovascular coupling, at low concentrations dilates whereas at higher concentrations constricts human cerebral parenchymal arterioles

There is considerable controversy regarding the vasoactive action of prostaglandin E2 (PGE2). On the one hand, indirect evidence implicates that astrocytic release of PGE2 contributes to neurovascular coupling responses mediating functional hyperemia in the brain. On the other hand, overproduction of PGE2 was also reported to contribute to cerebral vasospasm associated with subarachnoid hemorrhage. The present study was conducted to resolve this controversy by determining the direct vasoactive effects of PGE2 in resistance-sized human cerebral parenchymal arterioles.To achieve this goal PGE2-induced isotonic vasomotor responses were assessed in parenchymal arterioles isolated from fronto-temporo-parietal cortical tissues surgically removed from patients and expression of PGE2 receptors were examined.In functionally intact parenchymal arterioles lower concentrations of PGE2 (from 10−8 to 10-6 mol/l) caused significant, endothelium-independent vasorelaxation, which was inhibited by the EP4 receptor blocker BGC201531. In contrast, higher concentrations of PGE2 evoked significant EP1-dependent vasoconstriction, which could not be reversed by the EP4 receptor agonist CAY10598. We also confirmed previous observations that PGE2 primarily evokes constriction in intracerebral arterioles isolated from R. norvegicus. Importantly, vascular mRNA and protein expression of vasodilator EP4 receptors was significantly higher than that of vasoconstrictor EP1 receptors in human cerebral arterioles.PGE2 at low concentrations dilates whereas at higher concentrations constricts human cerebral parenchymal arterioles. This bimodal vasomotor response is consistent with both the proposed vasodilator role of PGE2 during functional hyperemia and its putative role in cerebral vasospasm associated with subarachnoid hemorrhage in human patients.

Bronchodilation induced by PGE2 is impaired in Group III pulmonary hypertension

In patients with pulmonary hypertension (PH) associated with lung disease and/or hypoxia (Group III), decreased pulmonary vascular tone and tissue hypoxia is therapeutically beneficial. PGE2 and PGI2 induce potent relaxation of human bronchi from non-PH (control) patients via EP4 and IP receptors, respectively. However, the effects of PGE2 /PGI2 and their mimetics on human bronchi from PH patients are unknown. Here, we have compared relaxant effects of several PGI2 -mimetics approved for treating PH Group I with several PGE2 -mimetics, in bronchial preparations derived from PH Group III and control patients. Relaxation of bronchial muscle was assessed in samples isolated from control and PH Group III patients. Expression of prostanoid receptors was analysed by western blot and real-time PCR, and endogenous PGE2 , PGI2 , and cAMP levels were determined by ELISA. Maximal relaxations induced by different EP4 receptor agonists (PGE2 , L-902688, and ONO-AE1-329) were decreased in human bronchi from PH patients, compared with controls. However, maximal relaxations produced by PGI2 -mimetics (iloprost, treprostinil, and beraprost) were similar for both groups of patients. Both EP4 and IP receptor protein and mRNA expressions were significantly lower in human bronchi from PH patients. cAMP levels significantly correlated with PGI2 but not with PGE2 levels. The PGI2 -mimetics retained maximal bronchodilation in PH Group III patients, whereas bronchodilation induced by EP4 receptor agonists was decreased. Restoration of EP4 receptor expression in airways of PH Group III patients with respiratory diseases could bring additional therapeutic benefit.

Suppression of aspirin-mediated eosinophil activation by prostaglandin E2: Relevance to aspirin and nonsteroidal anti-inflammatory drug hypersensitivity

BackgroundAspirin-exacerbated respiratory disease (AERD) is characterized by severe, sometimes life-threatening reactions to nonsteroidal anti-inflammatory drugs (NSAIDs). Mechanisms driving the disease include overproduction of leukotrienes and loss of anti-inflammatory prostaglandin E2 (PGE2) production. Many cell types contribute to the disease; however, eosinophils are markedly elevated and are important drivers of pathologic findings. ObjectiveTo investigate the capacity of aspirin and NSAIDs to drive eosinophil activation and the ability of PGE2 to inhibit this activation. MethodsEosinophils were purified from blood of healthy individuals without AERD and stimulated with lysine aspirin, ketorolac, or sodium salicylate. The role of PGE2 in altering activation was determined by incubating eosinophils with increasing doses of PGE2 before lysine aspirin stimulation. Specific PGE2 receptor use was determined by incubating eosinophils with receptor agonists and antagonists before aspirin stimulation. Cysteinyl leukotrienes (CysLTs), leukotriene B4 (LTB4), and eosinophil-derived neurotoxin (EDN) were quantified by enzyme-linked immunosorbent assay. ResultsStimulation of eosinophils with lysine aspirin, ketorolac, or sodium salicylate resulted in secretion of CysLTs and LTB4 in the absence of EDN release. Low doses of PGE2 inhibited LTB4 and CysLT release, an effect lost at higher PGE2 concentrations. Use of butaprost, an EP2 receptor agonist, suppressed lysine aspirin stimulation. This mechanism was supported by blocking activity of the EP1 and EP3 receptors. ConclusionEosinophils can be directly activated by NSAIDs via cyclooxygenase-independent pathways to produce CysLTs and LTB4. This effect can be inhibited by PGE2 acting through the EP2 receptor. The recognized loss of EP2 receptor expression combined with low PGE2 levels explains in part the sensitivity to NSAIDs.