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The prostaglandin E2 (PGE2) receptor EP3 has been detected in the thick ascending limb (TAL) and the collecting duct of the kidney, where its actions are proposed to inhibit water reabsorption. However, EP3 is also expressed in other cell types, including vascular endothelial cells. The aim here was to determine the contribution of EP3 in renal water handling in male and female adult mice by phenotyping a novel mouse model with doxycycline-dependent deletion of EP3 throughout the kidney tubule (EP3−/− mice). RNAscope demonstrated that EP3 was highly expressed in the cortical and medullary TAL of adult mice. Compared with controls EP3 mRNA expression was reduced by >80% in whole kidney (RT-qPCR) and nondetectable (RNAscope) in renal tubules of EP3−/− mice. Under basal conditions, there were no significant differences in control and EP3−/− mice of both sexes in food and water intake, body weight, urinary output, or clinical biochemistries. No differences were detectable between genotypes in handling of an acute water load or in their response to the vasopressin analog 1-deamino-8- d -arginine-vasopressin (dDAVP). No differences in water handling were observed when PGE2 production was enhanced using 1% NaCl load. Expression of proteins involved in kidney water handling was not different between genotypes. This study demonstrates that renal tubular EP3 is not essential for body fluid homeostasis in males or females, even when PGE2 levels are high. The mouse model is a novel tool for examining the role of EP3 in kidney function independently of potential developmental abnormalities or systemic effects. NEW & NOTEWORTHY: The prostanoid EP3 receptor is proposed to play a key role in the kidney tubule and antagonize the effects of vasopressin on aquaporin-mediated water reabsorption. Here, we phenotyped a kidney tubule-specific inducible knockout mouse model of the EP3 receptor. Our major finding is that, even under physiological stress, tubular EP3 plays no detectable role in renal water or solute handling. This suggests that other EP receptors must be important for renal salt and water handling. [ABSTRACT FROM AUTHOR]

Abstract Aim The progression of atherosclerosis can lead to the occurrence of multiple cardiovascular diseases (coronary heart disease, etc.). E prostanoid receptor‐3 (EP3) is known to participate in the progression of atherosclerosis. This study aimed to investigate the mechanism by which EP3 modulates the development of atherosclerosis. Methods and results ApoE−/− mice were used to construct in vivo model of atherosclerosis. Human aortic smooth muscle cells (HASMCs) were stimulated with oxidized low‐density lipoprotein (ox‐LDL) to construct in vitro model of atherosclerosis. mRNA expressions were assessed by qRT‐PCR, and western blot was applied to assess the protein levels. CCK‐8 assay was applied to assess the cell viability. The inflammatory cytokines levels were assessed by enzyme‐linked immunosorbent assay, and flow cytometry was applied to assess cell apoptosis. In vivo experiment was constructed to investigate the impact of EP3 in atherosclerosis development. L‐798106 (EP3 inhibitor) significantly inhibited the levels of pro‐inflammatory cytokines in atherosclerosis in vivo. EP3 inhibitor (L‐798106) significantly reversed ox‐LDL‐caused HASMCs injury via inhibiting the apoptosis and inflammatory responses (P

Aim: The progression of atherosclerosis can lead to the occurrence of multiple cardiovascular diseases (coronary heart disease, etc.). E prostanoid receptor‐3 (EP3) is known to participate in the progression of atherosclerosis. This study aimed to investigate the mechanism by which EP3 modulates the development of atherosclerosis. Methods and results: ApoE−/− mice were used to construct in vivo model of atherosclerosis. Human aortic smooth muscle cells (HASMCs) were stimulated with oxidized low‐density lipoprotein (ox‐LDL) to construct in vitro model of atherosclerosis. mRNA expressions were assessed by qRT‐PCR, and western blot was applied to assess the protein levels. CCK‐8 assay was applied to assess the cell viability. The inflammatory cytokines levels were assessed by enzyme‐linked immunosorbent assay, and flow cytometry was applied to assess cell apoptosis. In vivo experiment was constructed to investigate the impact of EP3 in atherosclerosis development. L‐798106 (EP3 inhibitor) significantly inhibited the levels of pro‐inflammatory cytokines in atherosclerosis in vivo. EP3 inhibitor (L‐798106) significantly reversed ox‐LDL‐caused HASMCs injury via inhibiting the apoptosis and inflammatory responses (P < 0.05). The levels of interleukin‐17 (IL‐17) and intercellular adhesion molecule‐1 (ICAM‐1) in HASMCs were elevated by ox‐LDL, whereas L‐798106 or knockdown of cyclic AMP (cAMP) response element‐binding protein (CREB) notably restored this phenomenon (P < 0.05). EP3 overexpression further aggravated ox‐LDL‐induced inflammation in HASMCs, and EP3 up‐regulated the levels of IL‐17 and ICAM‐1 in ox‐LDL‐treated HASMCs (P < 0.05). EP3 up‐regulation promoted the inflammatory responses in ox‐LDL‐treated HASMCs through mediation of cAMP/protein kinase A (PKA)/CREB/IL‐17/ICAM‐1 axis (P < 0.05). Conclusions: EP3 inhibitor alleviates ox‐LDL‐induced HASMC inflammation via mediation of cAMP/PKA/CREB/IL‐17/ICAM‐1 axis. Our study might shed new lights on discovering novel strategies against atherosclerosis. [ABSTRACT FROM AUTHOR]

Simple Summary: Due to the lack of effective early diagnostic markers for lung cancer and the rich blood circulation in the lungs, it is very easy to cause lymph node metastasis and distant metastasis of lung cancer, making lung cancer as one of the top ten cancer types with the highest mortality rate in the world. This study found that MAPK15 is highly expressed in the tissues of patients with lung adenocarcinoma lymph node metastasis, and MAPK15 interacts with p50 to regulate the expression of EP3 at the transcriptional level, thereby promoting cancer cell migration. This suggests that MAPK15 plays a key role in the metastasis of lung cancer cells, and MAPK15 can be used as a molecular marker for the early diagnosis or prognosis assessment of lung cancer. Its molecular mechanism for regulating lung cancer metastasis can provide valuable information and insights on novel therapeutic options at molecular levels. Studying the relatively underexplored atypical MAP Kinase MAPK15 on cancer progression/patient outcomes and its potential transcriptional regulation of downstream genes would be highly valuable for the diagnosis, prognosis, and potential oncotherapy of malignant tumors such as lung adenocarcinoma (LUAD). Here, the expression of MAPK15 in LUAD was detected by immunohistochemistry and its correlation with clinical parameters such as lymph node metastasis and clinical stage was analyzed. The correlation between the prostaglandin E2 receptor EP3 subtype (EP3) and MAPK15 expression in LUAD tissues was examined, and the transcriptional regulation of EP3 and cell migration by MAPK15 in LUAD cell lines were studied using the luciferase reporter assay, immunoblot analysis, qRT-PCR, and transwell assay. We found that MAPK15 is highly expressed in LUAD with lymph node metastasis. In addition, EP3 is positively correlated with the expression of MAPK15 in LUAD tissues, and we confirmed that MAPK15 transcriptionally regulates the expression of EP3. Upon the knockdown of MAPK15, the expression of EP3 was down-regulated and the cell migration ability was decreased in vitro; similarly, the mesenteric metastasis ability of the MAPK15 knockdown cells was inhibited in in vivo animal experiments. Mechanistically, we demonstrate for the first time that MAPK15 interacts with NF-κB p50 and enters the nucleus, and NF-κB p50 binds to the EP3 promoter and transcriptionally regulates the expression of EP3. Taken together, we show that a novel atypical MAPK and NF-κB subunit interaction promotes LUAD cell migration through transcriptional regulation of EP3, and higher MAPK15 level is associated with lymph node metastasis in patients with LUAD. [ABSTRACT FROM AUTHOR]

Studying the relatively underexplored atypical MAP Kinase MAPK15 on cancer progression/patient outcomes and its potential transcriptional regulation of downstream genes would be highly valuable for the diagnosis, prognosis, and potential oncotherapy of malignant tumors such as lung adenocarcinoma (LUAD). Here, the expression of MAPK15 in LUAD was detected by immunohistochemistry and its correlation with clinical parameters such as lymph node metastasis and clinical stage was analyzed. The correlation between the prostaglandin E2 receptor EP3 subtype (EP3) and MAPK15 expression in LUAD tissues was examined, and the transcriptional regulation of EP3 and cell migration by MAPK15 in LUAD cell lines were studied using the luciferase reporter assay, immunoblot analysis, qRT-PCR, and transwell assay. We found that MAPK15 is highly expressed in LUAD with lymph node metastasis. In addition, EP3 is positively correlated with the expression of MAPK15 in LUAD tissues, and we confirmed that MAPK15 transcriptionally regulates the expression of EP3. Upon the knockdown of MAPK15, the expression of EP3 was down-regulated and the cell migration ability was decreased in vitro; similarly, the mesenteric metastasis ability of the MAPK15 knockdown cells was inhibited in in vivo animal experiments. Mechanistically, we demonstrate for the first time that MAPK15 interacts with NF-κB p50 and enters the nucleus, and NF-κB p50 binds to the EP3 promoter and transcriptionally regulates the expression of EP3. Taken together, we show that a novel atypical MAPK and NF-κB subunit interaction promotes LUAD cell migration through transcriptional regulation of EP3, and higher MAPK15 level is associated with lymph node metastasis in patients with LUAD.

Pathological cardiac hypertrophy induced by activation of the renin–angiotensin–aldosterone system (RAAS) is one of the leading causes of heart failure. However, in current clinical practice, the strategy for targeting the RAAS is not sufficient to reverse hypertrophy. Here, we investigated the effect of prostaglandin E1 (PGE1) on angiotensin II (AngII)-induced cardiac hypertrophy and potential molecular mechanisms underlying the effect. Adult male C57 mice were continuously infused with AngII or saline and treated daily with PGE1 or vehicle for two weeks. Neonatal rat cardiomyocytes were cultured to detect AngII-induced hypertrophic responses. We found that PGE1 ameliorated AngII-induced cardiac hypertrophy both in vivo and in vitro. The RNA sequencing (RNA-seq) and expression pattern analysis results suggest that Netrin-1 (Ntn1) is the specific target gene of PGE1. The protective effect of PGE1 was eliminated after knockdown of Ntn1. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the PGE1-mediated signaling pathway changes are associated with the mitogen-activated protein kinase (MAPK) pathway. PGE1 suppressed AngII-induced activation of the MAPK signaling pathway, and such an effect was attenuated by Ntn1 knockdown. Blockade of MAPK signaling rescued the phenotype of cardiomyocytes caused by Ntn1 knockdown, indicating that MAPK signaling may act as the downstream effector of Ntn1. Furthermore, inhibition of the E-prostanoid (EP) 3 receptor, as opposed to the EP1, EP2, or EP4 receptor, in cardiomyocytes reversed the effect of PGE1, and activation of EP3 by sulprostone, a specific agonist, mimicked the effect of PGE1. In conclusion, PGE1 ameliorates AngII-induced cardiac hypertrophy through activation of the EP3 receptor and upregulation of Ntn1, which inhibits the downstream MAPK signaling pathway. Thus, targeting EP3, as well as the Ntn1–MAPK axis, may represent a novel approach for treating pathological cardiac hypertrophy. [Display omitted] • PGE1 treatment attenuates AngII-induced cardiac hypertrophy without altering blood pressure. • PGE1 mainly targets cardiomyocytes but not cardiac fibroblasts or CMECs. • The activation of EP3 receptor by PGE1 leads to up-regulation of Ntn1, which inhibits the MAPK signaling pathway. • PGE1-EP3 as well as targeting the Ntn1–MAPK axis may represent a novel treatment for pathological cardiac hypertrophy. [ABSTRACT FROM AUTHOR]

Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) is an acute inflammatory vesiculobullous reaction of the mucosa of the ocular surface, oral cavity, and genitals, and of the skin. Severe ocular complications (SOC) are observed in about half of SJS/TEN patients diagnosed by dermatologists and in burn units. Ophthalmologists treat SOC, and they tend to encounter the patients not only in the acute stage, but also in the chronic stage. Our investigation of the pathogenesis of SJS/TEN with SOC led us to suspect that abnormal innate mucosal immunity contributes to the ocular surface inflammation seen in SJS/TEN with SOC. We confirmed that cold medicines such as NSAIDs and multi-ingredient cold medications are the main causative drugs for SJS/TEN with SOC. Single nucleotide polymorphism (SNP) association analysis of cold medicine-related SJS/TEN with SOC showed that the Toll-like receptor 3 (TLR3)-, the prostaglandin-E receptor 3 (PTGER3)-, and the IKZF1 gene were significantly associated with SNPs and that these genes could regulate mucocutaneous inflammation including that of the ocular surface. We also examined the tear cytokines of SJS/TEN with SOC in the chronic stage and found that IL-8, IL-6, IFN-γ, RANTES, eotaxin, and MIP-1β were significantly upregulated in SJS/TEN with SOC in the chronic stage. Only IP-10 was significantly downregulated in SJS/TEN with SOC in the chronic stage. This mini-review summarizes the pathological mechanisms that we identified as underlying the development of SJS/TEN with SOC.

Although commonly thought to produce prostacyclin (prostaglandin I2; PGI2) that evokes vasodilatation and protects vessels from the development of diseases, the endothelial cyclooxygenase (COX)‐mediated metabolism has also been found to release substance(s) called endothelium‐derived contracting factor(s) (EDCF) that causes endothelium‐dependent contraction and implicates in endothelial dysfunction of disease conditions. Various mechanisms have been proposed for the process; however, the major endothelial COX metabolite PGI2, which has been classically considered to activate the I prostanoid receptor (IP) that mediates vasodilatation and opposes the effects of thromboxane (Tx) A2 produced by COX in platelets, emerges as a major EDCF in health and disease conditions. Our recent studies from genetically altered mice further suggest that vasomotor reactions to PGI2 are collectively modulated by IP, the vasoconstrictor Tx‐prostanoid receptor (TP; the prototype receptor of TxA2) and E prostanoid receptor‐3 (EP3; a vasoconstrictor receptor of PGE2) although with differences in potency and efficacy; a contraction to PGI2 reflects activities of TP and/or EP3 outweighing that of the concurrently activated IP. Here, we discuss the history of endothelium‐dependent contraction, evidences that support the above hypothesis, proposed mechanisms for the varied reactions to endothelial PGI2 synthesis as well as the relation of its dilator activity to the effect of another NO‐independent vasodilator mechanism, the endothelium‐derived hyperpolarizing factor. Also, we address the possible pathological and therapeutic implications as well as questions remaining to be resolved or limitations of our above findings obtained from genetically altered mouse models. [ABSTRACT FROM AUTHOR]

Anna Hester,* Barbara Salzmann,* Martina Rahmeh, Thomas Kolben, Bastian Czogalla, Nina Ditsch, Sven Mahner, Udo Jeschke, Theresa Maria KolbenDepartment of Obstetrics and Gynecology, University Hospital, LMU Munich, Munich, Germany*These authors contributed equally to this workPurpose: COX-2 overexpression and elevated levels of prostaglandin E2 (PGE2) play an important role in breast cancer carcinogenesis. Recently, expression of the PGE2 receptor EP3 has been shown to be a positive prognostic factor in breast cancer. This study analyzes the functional aspects of targeting EP3 in breast cancer cell lines.Material and methods: EP3 and EP1 expressions were determined in five breast cancer cell lines on the mRNA- and the protein-level. The selected cell lines were subsequently stimulated for 24–72 hrs with 10–1,000 nM of PGE2, the EP1/EP3 agonist sulprostone and the EP3 antagonist L798,106. Cell proliferation was determined via BrdU-assay, migration via scratch assay, EP3, Gi-protein and p-ERK1/2 expressions via Western blot and cAMP concentrations via ELISA. The Mann–Whitney-U-test was used to test for statistical significance.Results: The cell lines T-47D (EP3 expression 77.7%) and SK-BR-3 (EP3 expression 48.7%) were chosen. EP3 antagonism reduced its expression on SK-BR-3 significantly, while no effect was observed on T-47D. The proliferation and migration of SK-BR-3 cells were significantly reduced due to treatment with the EP1/3 agonist, the EP3 antagonist or a combination of both. Neither agonism nor antagonism influenced cell proliferation or migration in T-47D. In SK-BR-3, EP3 antagonism showed a significant decrease in Gi-protein levels, an increase in cAMP levels, and no significant change in p-ERK1/2 expression.Conclusion: Antagonism of the EP3 receptor results in a reduced proliferation and migration of SK-BR-3 breast cancer cells, potentially mediated via a Gi-protein-cAMP pathway. The results suggest that EP3 plays a role in tumorigenesis. This is in accordance with the cell culture data of other gynecological tumors, but it is conflicting in so far, as positive EP3 expression is clinically a positive prognostic marker in breast cancer. Therefore, other factors may be important in explaining this contradiction.Keywords: carcinoma of the breast, prostaglandin E2 receptor 3, cell growth, cell traffic, signal transduction, in vitro experiments

Vasomotor reactions of prostacyclin (prostaglandin I2; PGI2) can be collectively modulated by thromboxane prostanoid receptor (TP), E‐prostanoid receptor‐3 (EP3), and the vasodilator I prostanoid receptor (IP). This study aimed to determine the direct effect of PGI2 on renal arteries and/or the whole renal vasculature and how each of these receptors is involved. Experiments were performed on vessels or perfused kidneys of wild‐type mice and/or mice with deficiency in TP (TP−/−) and/or EP3. Here we show that PGI2 did not evoke relaxation, but instead resulted in contraction of main renal arteries (from ~0.001‐0.01 µM) or reduction of flow in perfused kidneys (from ~1 µM); either of them was reversed into a dilator response in TP−/−/EP3−/− counterparts. Also, we found that in renal arteries although it has a lesser effect than TP−/− on the maximal contraction to PGI2 (10 µM), EP3−/− but not TP−/− resulted in relaxation to the prostanoid at 0.01‐1 µM. Meanwhile, TP−/− only significantly reduced the contractile activity evoked by PGI2 at ≥0.1 µM. These results demonstrate that PGI2 may evoke an overall vasoconstrictor response in the mouse renal vasculature, reflecting activities of TP and EP3 outweighing that of the vasodilator IP. Also, our results suggest that EP3, on which PGI2 can have a potency similar to that on IP, plays a major role in the vasoconstrictor effect of the prostanoid of low concentrations (≤1 µM), while TP, on which PGI2 has a lower potency but higher efficacy, accounts for a larger part of its maximal contractile activity. [ABSTRACT FROM AUTHOR]

Abstract Background In various cancers, overexpression of cyclooxygenase (COX)-2 and elevated prostaglandin (PG) E2 synthesis have been associated with tumor development and progression. The potential of COX-2 inhibitors in cancer prevention and treatment has been shown repeatedly; however, their clinical use is limited due to toxicity. PGE2 signals via EP receptors 1–4, whose functions are analyzed in current research in search for targeted anti-PG therapies. EP2 and EP4 rather promote tumorigenesis, while the role of EP3, especially in breast cancer, is not yet clear and both pro- and anti-tumorigenic effects have been described. Our study evaluates EP3 receptor expression in sporadic breast cancer and its association with clinicopathological parameters, progression-free and overall survival. Methods Two hundred eighty-nine sporadic breast cancer samples without primary distant metastasis were immunohistochemically analyzed for EP3 receptor expression. Tissue was stained with primary anti-EP3-antibodies. Immunoreactivity was quantified by the immunoreactivity-score (IRS); samples with an IRS ≥ 2 scored as EP3 positive. Chi-squared and Mann-Whitney-U test were used for comparison of data; Kaplan-Meier estimates and Cox-regression were used for survival analyses. Results EP3 receptor was expressed in 205 of 289 samples analyzed (70.9%). EP3 receptor expression was not associated with clinicopathological parameters (e. g. tumor size, hormone receptors, lymph node status). Kaplan-Meier estimates showed a significant association of EP3 positivity with improved progression-free survival (p = 0.002) and improved overall survival (p = 0.001) after up to 10 years. Cox regression analysis confirmed EP3 positivity as a significant prognostic factor even when other known prognosticators were accounted for. Conclusions In sporadic breast cancer, EP3 receptor expression is not significantly associated with clinicopathological parameters but is a significant prognostic factor for improved progression-free and overall survival. However, the functional aspects of EP3 receptor in breast cancer and the way how EP3 may oppose the pro-tumorigenic effects of PGE2 elevation and COX-2 overexpression are not fully understood so far. Further studies aiming at identification of the factors regulated by EP3 are necessary to evaluate the possibility of targeting EP3 in future anti-tumor therapy in breast cancer.

Angiotensin II (ANG II) plays a key role in regulating blood pressure and inflammation. Prostaglandin E2 (PGE2) signals through four different G protein-coupled receptors, eliciting a variety of effects. We reported that activation of the EP3 receptor reduces cardiac contractility. More recently, we have shown that overexpression of the EP4 receptor is protective in a mouse myocardial infarction model. We hypothesize in this study that the relative abundance of EP3 and EP4 receptors is a major determinant of end-organ damage in the diseased heart. Thus EP3 is detrimental to cardiac function and promotes inflammation, whereas antagonism of the EP3 receptor is protective in an ANG II hypertension (HTN) model. To test our hypothesis, male 10- to 12-wk-old C57BL/6 mice were anesthetized with isoflurane and osmotic minipumps containing ANG II were implanted subcutaneously for 2 wk. We found that antagonism of the EP3 receptor using L798,106 significantly attenuated the increase in blood pressure with ANG II infusion. Moreover, antagonism of the EP3 receptor prevented a decline in cardiac function after ANG II treatment. We also found that 10- to 12-wk-old EP3-transgenic mice, which overexpress EP3 in the cardiomyocytes, have worsened cardiac function. In conclusion, activation or overexpression of EP3 exacerbates end-organ damage in ANG II HTN. In contrast, antagonism of the EP3 receptor is beneficial and reduces cardiac dysfunction, inflammation, and HTN. [ABSTRACT FROM AUTHOR]

Although recognized to have an in vivo vasodepressor effect blunted by the vasoconstrictor effect of E‐prostanoid receptor‐3 (EP3), prostaglandin E2 (PGE2) evokes contractions of many vascular beds that are sensitive to antagonizing the thromboxane prostanoid receptor (TP). This study aimed to determine the direct effect of PGE2 on renal arteries and/or the whole renal vasculature and how each of these two receptors is involved in the responses. Experiments were performed on isolated vessels and perfused kidneys of wild‐type mice and/or mice with deficiency in TP (TP−/−), EP3 (EP3−/−), or both TP and EP3 (TP−/−/EP3−/−). Here we show that PGE2 (0.001‐30 μM) evoked not only contraction of main renal arteries, but also a decrease of flow in perfused kidneys. EP3‐/‐ diminished the response to 0.001‐0.3 μM PGE2, while TP−/− reduced that to the prostanoid of higher concentrations. In TP−/−/EP3−/− vessels and perfused kidneys, PGE2 did not evoke contraction but instead resulted in vasodilator responses. These results demonstrate that PGE2 functions as an overall direct vasoconstrictor of the mouse renal vasculature with an effect reflecting the vasoconstrictor activities outweighing that of dilation. Also, our results suggest that EP3 dominates the vasoconstrictor effect of PGE2 of low concentrations (≤0.001‐0.3 μM), but its effect is further added by that of TP, which has a higher efficacy, although activated by higher concentrations (from 0.01 μM) of the same prostanoid PGE2. [ABSTRACT FROM AUTHOR]

Purpose: Prostaglandin-mediated inflammatory reactions play a major role in different cancers. Recently, it has been observed that prostaglandin E2-receptor 3 (EP3) might be an independent prognostic factor for overall survival in cervical and endometrial cancer. The role of EP3 expression in ovarian cancer is currently unknown. Methods: EP3 expression was analyzed by immunohistochemistry in 156 patient samples using the IR-scoring system. Expression levels were correlated with clinical and pathological parameters and with overall survival (OS) to assess for prognostic relevance. Data analysis was performed using Spearman's correlations, Kruskal–Wallis test and Kaplan–Meier estimates. Results: EP3 expression was significantly higher in clear-cell carcinoma (p < 0.001) compared to the other histological subtypes. No further correlations with clinical parameters could be found. EP3 expression correlated significantly with FSH-receptor expression (p < 0.001), galectin-1 expression in the tumor (p = 0.012) and with cytoplasmatic TA-MUC1 expression (p = 0.001). None of these parameters showed significant correlation with OS. In the TA-MUC1 negative subgroup, EP3 negative patients showed significantly longer OS (median OS: 102 months vs. 34 months in EP3 positive patients, p = 0.035), while EP3 did not appear to have prognostic relevance in the TA-MUC1-positive subgroup. Conclusion: The potential prognostic relevance of EP3 expression for OS in TA-MUC1 negative patients might reflect an interplay between the COX and the MUC1 pathway, as it has been shown that MUC1 could induce COX2 expression. Our findings support the importance of the prostanoid signaling in TA-MUC1 negative ovarian cancer; however, future studies are necessary to characterize specific pathways and possible interactions. [ABSTRACT FROM AUTHOR]

Purpose: Prostaglandin-mediated inflammatory reactions play a major role in different cancers. Prostaglandin E2-receptor 3 (EP3) expression correlates with FIGO stages in cervical cancer and has been shown to be an independent prognostic factor for overall survival. EP3 expression levels in cervical intraepithelial neoplasia (CIN) as the precursor lesion of cervical cancer are currently unknown.Methods: EP3 expression was analyzed by immunohistochemistry in 124 patient samples (CIN 1-3 and healthy controls) using the IR-scoring system. Expression levels were correlated with clinical outcome to assess for prognostic relevance in patients with CIN 2. Data analysis was performed using Kruskal-Wallis and Mann-Whitney U test.Results: EP3 expression levels significantly correlated with different grades of cervical dysplasia. Median EP3-IRS in healthy cervical tissue was 12 (n = 13) compared to 9 in CIN 1 (n = 38; p = 0.031 vs. healthy control), 6 in CIN 2 (n = 45; p < 0.001 vs. CIN 1) and 4 in CIN 3 (n = 28, p = 0.008 vs. CIN 2). The percentage of EP3 expressing cells in CIN 2 lesions was significantly lower in progressive than in regressive cases (mean percentage of EP3 positive cells in progress: 3.8%, n = 18; in regress: 9.3%, n = 20; p = 0.040).Conclusion: EP3 expression significantly decreases with higher grades of cervical intraepithelial neoplasia—which is in line with published IR scores in cervical cancer patients—and seems to be a prognostic marker for regression or progression of CIN 2 lesions. Our findings support the importance of the prostanoid pathway in cervical cancer and could help to identify targets for future therapies. [ABSTRACT FROM AUTHOR]

Abstract Aims Sphingosine-1-phosphate (S1P) has been implicated lately in inflammatory bowel disease which has diarrhea as one of its symptoms. Diarrhea is due to altered water movements as a result of altered electrolyte transport, and in particular sodium. Sodium movements are geared by the sodium gradient established by the Na+/K+ ATPase. The aim of this work was to investigate if S1P can modulate the activity of the ATPase, using Caco-2 cells as a model and the S1P analogue, FTY720P. Materials and methods The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain. Protein expression of the various S1P receptors was studied by western blot analysis. Key findings Caco-2 cells were found to express mainly S1PR2 and S1PR3. FTY720P (7.5 nM) reduced significantly the activity of the Na+/K+ ATPase when applied for 15 min. This inhibitory effect disappeared in presence of JTE-013, a specific blocker of S1PR2, and indomethacin, an inhibitor of cyclooxygenase enzymes, and was mimicked by CYM5520, a S1PR2 agonist and by exogenous PGE2. The inhibitory effect of PGE2 did not appear when EP3 receptors were blocked or when a nitric oxide scavenger was added. RpcAMP, a PKA inhibitor, reduced the activity of the Na+/K+ ATPase, while dbcAMP, a PKA activator was without any effect and when added, abrogated the effect of PGE2. Significance It was concluded that FTY720P inhibits the Na+/K+ ATPase via activation of S1PR2 and generation of PGE2 nitric oxide. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Background: In various cancers, overexpression of cyclooxygenase (COX)-2 and elevated prostaglandin (PG) E2 synthesis have been associated with tumor development and progression. The potential of COX-2 inhibitors in cancer prevention and treatment has been shown repeatedly; however, their clinical use is limited due to toxicity. PGE2 signals via EP receptors 1-4, whose functions are analyzed in current research in search for targeted anti-PG therapies. EP2 and EP4 rather promote tumorigenesis, while the role of EP3, especially in breast cancer, is not yet clear and both pro- and anti-tumorigenic effects have been described. Our study evaluates EP3 receptor expression in sporadic breast cancer and its association with clinicopathological parameters, progression-free and overall survival.Methods: Two hundred eighty-nine sporadic breast cancer samples without primary distant metastasis were immunohistochemically analyzed for EP3 receptor expression. Tissue was stained with primary anti-EP3-antibodies. Immunoreactivity was quantified by the immunoreactivity-score (IRS); samples with an IRS ≥ 2 scored as EP3 positive. Chi-squared and Mann-Whitney-U test were used for comparison of data; Kaplan-Meier estimates and Cox-regression were used for survival analyses.Results: EP3 receptor was expressed in 205 of 289 samples analyzed (70.9%). EP3 receptor expression was not associated with clinicopathological parameters (e. g. tumor size, hormone receptors, lymph node status). Kaplan-Meier estimates showed a significant association of EP3 positivity with improved progression-free survival (p = 0.002) and improved overall survival (p = 0.001) after up to 10 years. Cox regression analysis confirmed EP3 positivity as a significant prognostic factor even when other known prognosticators were accounted for.Conclusions: In sporadic breast cancer, EP3 receptor expression is not significantly associated with clinicopathological parameters but is a significant prognostic factor for improved progression-free and overall survival. However, the functional aspects of EP3 receptor in breast cancer and the way how EP3 may oppose the pro-tumorigenic effects of PGE2 elevation and COX-2 overexpression are not fully understood so far. Further studies aiming at identification of the factors regulated by EP3 are necessary to evaluate the possibility of targeting EP3 in future anti-tumor therapy in breast cancer. [ABSTRACT FROM AUTHOR]

Endothelial injury or dysfunction is an early event in the pathogenesis of atherosclerosis. Epidemiological and animal studies have shown that 2, 3, 7, 8-tetrachlorodibenzo- p-dioxin (TCDD) exposure increases morbidity and mortality from chronic cardiovascular diseases, including atherosclerosis. However, whether or how TCDD exposure causes endothelial injury or dysfunction remains largely unknown. Cultured human umbilical vein endothelial cells (HUVECs) were exposed to different doses of TCDD, and cell apoptosis was examined. We found that TCDD treatment increased caspase 3 activity and apoptosis in HUVECs in a dose-dependent manner,at doses from 10 to 40 nM. TCDD increased cyclooxygenase enzymes (COX)-2 expression and its downstream prostaglandin (PG) production (mainly PGE2 and 6-keto-PGF1α) in HUVECs. Interestingly, inhibition of COX-2, but not COX-1, markedly attenuated TCDD-triggered apoptosis in HUVECs. Pharmacological inhibition or gene silencing of the PGE2 receptor subtype 3 (EP3) suppressed the augmented apoptosis in TCDD-treated HUVECs. Activation of the EP3 receptor enhanced p38 MAPK phosphorylation and decreased Bcl-2 expression following TCDD treatment. Both p38 MAPK suppression and Bcl-2 overexpression attenuated the apoptosis in TCDD-treated HUVECs. TCDD increased EP3-dependent Rho activity and subsequently promoted p38MAPK/Bcl-2 pathway-mediated apoptosis in HUVECs. In addition, TCDD promoted apoptosis in vascular endothelium and delayed re-endothelialization after femoral artery injury in wild-type (WT) mice, but not in EP3−/− mice. In summary, TCDD promotes endothelial apoptosis through the COX-2/PGE2/EP3/p38MAPK/Bcl-2 pathway. Given the cardiovascular hazard of a COX-2 inhibitor, our findings indicate that the EP3 receptor and its downstream pathways may be potential targets for prevention of TCDD-associated cardiovascular diseases. [ABSTRACT FROM AUTHOR]

The increase in bovine dairy consumption has been correlated with the development of many inflammatory diseases. Emerging evidence points to milk exosomes as potential causative agents. The cargo of exosome consists of cytokines that are elevated in inflammatory bowel disease and modulate the activity of the colonic Na+/K+ ATPase. Whether a cause effect relationship exists between the exosomes and the Na+/K+ ATPase is a question that has not been addressed before and constitutes the aim of this work, using Caco-2 cells as a model. Exosomes were extracted from skim milk by a series of ultra-centrifugations and filtrations. The activity of the pump was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain. The exosomes increased dose and time-dependently the activity of the ATPase with a maximal effect observed at 1 h. This increase was mediated via PGE2 and still appeared in presence of a blocker of EP1 and EP2, was enhanced further when EP4 was blocked, and changed to an inhibition in presence of an EP3 antagonist, suggesting an involvement of both EP3 and EP4. The activation of the Na+/K + ATPase was significantly enhanced or reduced when cells were treated respectively with an inhibitor or an activator of PKA. Since EP4 and EP3 act via Gs and Gi respectively, they exert opposite effects on PKA and consequently on the ATPase. The stimulatory effect of EP3 on the ATPase predominates over that of EP4 resulting in a net activation of the pump. [ABSTRACT FROM AUTHOR]

EP3 is a receptor for prostaglandin E2 (PGE2), and although its effect on bladder excitability has attracted considerable attention, the underlying mechanism remains unclear. To investigate whether the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in the interstitial cells of Cajal (ICCs) of the bladder are involved in the effect of EP3 activation on bladder excitability, wild-type mice, HCN1 knockout (HCN1 −/− ) mice and rats were used in our study. Double immunofluorescence staining and immunoprecipitation assays demonstrated the interaction between EP3 and the HCN channels. Sulprostone is a selective agonist of EP3. The current density of HCN channels was enhanced by sulprostone or PGE2 using whole-cell patch clamping. Western blot analyses showed that the expression levels of HCN1 and HCN4 were higher in bladders that had undergone intravesical instillation with sulprostone than in bladders treated with normal saline (NS). Both PGE2 and sulprostone increased the calcium concentration of the ICCs, and their effects were inhibited by ZD7288 (antagonist of HCN channels) treatment. In bladder detrusor strip testing, both PGE2 and sulprostone enhanced the amplitude of the bladder detrusor in HCN1 −/− mice; however, these effects were less than those in the wild-type mice. Furthermore, the effects of PGE2 and sulprostone were inhibited by ZD7288. Taken together, our results indicate that EP3 is expressed in bladder ICCs and facilitates bladder excitability via HCN channels. This study provides more comprehensive insights into the mechanism between inflammation and bladder excitability and highlights methods that can resolve bladder hyperactivity. [ABSTRACT FROM AUTHOR]

Prostaglandin E2 (PGE2) is a lipid hormone that signals through 4 different G-protein coupled receptor subtypes which act to regulate key physiological processes. Our laboratory has previously reported that PGE2 through its EP3 receptor reduces cardiac contractility at the level of isolated cardiomyocytes and in the isolated working heart preparation. We therefore hypothesized that cardiomyocyte specific overexpression of the PGE2 EP3 receptor further decreases cardiac function in a mouse model of heart failure produced by myocardial infarction. Our study tested this hypothesis using EP3 transgenic mice (EP3 TG), which overexpress the porcine analogue of human EP3 in the cardiomyocytes, and their wildtype (WT) littermates. Mice were analyzed 2 wks after myocardial infarction (MI) or sham operation by echocardiography, RT-PCR, immunohistochemistry, and histology. We found that the EP3 TG sham controls had a reduced ejection fraction, reduced fractional shortening, and an increased left ventricular dimension at systole and diastole compared to the WT sham controls. Moreover, there was a further reduction in the EP3 TG mice after myocardial infarction. Additionally, single-cell analysis of cardiomyocytes isolated from EP3 TG mice showed reduced contractility under basal conditions. Overexpression of EP3 significantly increased cardiac hypertrophy, interstitial collagen fraction, macrophage, and T-cell infiltration in the sham operated group. Interestingly, after MI, there were no changes in hypertrophy but there were changes in collagen fraction, and inflammatory cell infiltration. Overexpression of EP3 reduces cardiac function under basal conditions and this is exacerbated after myocardial infarction. [ABSTRACT FROM AUTHOR]

Neurons interact closely with astrocytes via glutamate; this neuron-glia circuit may play a pivotal role in synaptic transmission. On the other hand, astrocytes contact vascular endothelial cells with their end-feet. It is becoming obvious that non-neuronal cells play a critical role in regulating the neuronal activity in the brain. We find that kainic acid (KA) administration induces the expression of microsomal prostaglandin E synthase-1 (mPGES-1) in venous endothelial cells and the prostaglandin E2 (PGE2) receptor prostaglandin E receptor (EP)-3 on astrocytes. Endothelial mPGES-1 exacerbates KA-induced neuronal damage in in vivo experiments. In in vitro experiments, mPGES-1 produces PGE2, which enhances astrocytic Ca2+ levels via the EP3 receptor and increases Ca2+-dependent glutamate release, thus aggravating neuronal injury. This novel endothelium-astrocyte-neuron signaling pathway may be crucial for driving neuronal damage after repetitive seizures and could be a new therapeutic target for epilepsy and other brain disorders.

Purpose: To compare the prostaglandin E2 receptor subtype 3 (EP3) distribution in the lacrimal glands of normals, non-specific dacryoadenitis, and chronic Stevens-Johnson syndrome (SJS) patients., Methods: Biopsies from lacrimal glands of four chronic SJS patients with severe dry eye disease, four dacryoadenitis patients, and five fresh body donors were assessed for EP3 expression using immunohistochemistry., Results: In normal main and accessory lacrimal glands, EP3 is expressed strongly in nuclei and cytoplasm of majority (>75%) of acini with no ductular expression. In dacryoadenitis, EP3 expression was similar to normal glands. However, lacrimal glands from SJS patients (5-20/HPF mononuclear cells) showed a weak and reduced (<10% acini) EP3 expression within acinar cells. The reduction in intensity was more in glands with higher mononuclear cell infiltration (>10/HPF)., Conclusion: There is downregulation of EP3 expression in the lacrimal glands of SJS patients, whereas EP3 expression is preserved in non-specific lacrimal gland inflammations.

Exposure to aerosolized citric acid (CA, 150 mM) and prostaglandin E 2 (PGE 2 , 0.43 mM) for 10 min in guinea pigs reportedly produces the distinct cough patterns (Type I vs. II) and ventilatory responses (long-lasting hyperventilation vs. brief tachypnea) even though triggering the same cough numbers. Type I and II coughs are primarily mediated by activation of TRPV1 and EP3 receptors (a PGE 2 receptor) of vagal C-fibers respectively. Substance P (SP) and neurokinin A (NKA) released by vagal pulmonary sensory fibers peripherally are capable of affecting CA-induced cough and ventilation via preferentially activating neurokinin 1 and 2 receptors (NK 1 R and NK 2 R) respectively. This study aimed to define the impacts of CA- and PGE 2 -exposure on pulmonary SP and NKA levels and the roles of NK 1 R and NK 2 R in modulating CA- and PGE 2 -evoked cough and ventilatory responses. In unanesthetized guinea pigs, we determined: (1) pulmonary SP and NKA contents induced by the CA- or PGE 2 -exposure; (2) effects of CP-99994 and SR-48968 (a NK 1 R and a NK 2 R antagonist respectively) given by intraperitoneal injection (IP) or aerosol inhalation (IH) on the CA- and PGE 2 -evoked cough and ventilatory responses; and (3) immunocytochemical expressions of NK 1 R/NK 2 R in vagal C-neurons labeled by TRPV1 or EP3 receptors. We found that CA- and PGE 2 -exposure evoked Type I and II cough respectively associated with different degrees of increases in pulmonary SP and NKA. Applications of CP-99994 and SR-48968 via IP and IH efficiently suppressed the cough responses to CA with less impact on the cough response to PGE 2. These antagonists inhibited or blocked the ventilatory response to CA and caused hypoventilation in response to PGE 2. Moreover, NK 1 R and NK 2 R were always co-expressed in vagal C-neurons labeled by TRPV1 or EP3 receptors. These results suggest that SP and NKA endogenously released by CA- and PGE 2 -exposure play important roles in generating the cough and ventilatory responses to CA and PGE 2 , at least in part, via activation of NK 1 R and NK 2 R expressed in vagal C-neurons (pulmonary C-neurons). • Substance P and neurokinin A affect citric acid (CA)-induced cough and ventilation via activating NK 1 R and NK 2 R respectively. • However, the roles of these receptors in prostaglandin E 2 (PGE 2)-produced cough and ventilatory responses are unexplored. • Our data show that CA- and PGE 2 -exposure promote pulmonary release of substance P and neurokinin A in awake guinea pigs. • IV injection or aerosol inhalation of NK 1 R or NK 2 R antagonist inhibits cough and ventilatory responses to CA and PGE 2. • Both NK 1 R and NK 2 R are expressed in vagal C-neurons labeled by TRPV1 or EP3 receptors. [ABSTRACT FROM AUTHOR]

Partial and generalized status epilepticus (pSE and gSE) trigger the same level of progenitor cell proliferation in adult dentate gyrus, but survival of new neurons is poor after gSE. Here, we show markedly elevated levels of prostaglandin E2 (PGE2) and brain-derived neurotrophic factor (BDNF) in rat hippocampal formation at 7 days following pSE but not gSE. Administration of the cyclooxygenase (COX) inhibitor flurbiprofen for 1 week, starting at day 8 post-SE, abated PGE2 and decreased BDNF levels, but did not affect survival of new neurons 4 weeks later. Thus, high PGE2 and BDNF levels induced by pSE are probably not of major importance for survival of new neurons during the first days after formation. We propose that they modulate other aspects of synaptic and cellular plasticity, and thereby may influence epileptogenesis.

EP3 is prostaglandin E2 receptor subtype 3 and mediates the activation of several signaling pathways, changing in cAMP levels, calcium mobilization, and activation of phospholipase C. Previous studies demonstrated a direct role for EP3 in various neurodegenerative disorders, such as stroke and Alzheimer disease. However, the distribution and function of EP3 in ICH diseases remain unknown. Here, we demonstrate that EP3 may be involved in neuronal apoptosis in the processes of intracerebral hemorrhage (ICH). From the results of Western blot and immunohistochemistry, we obtained a significant up-regulation of EP3 in neurons adjacent to the hematoma following ICH. Up-regulation of EP3 was found to be accompanied by the increased expression of active caspase-3 and pro-apoptotic Bcl-2-associated X protein (Bax) and decreased expression of anti-apoptotic protein B cell lymphoma-2 (Bcl-2) in vivo and vitro studies. Furthermore, the expression of these three proteins reduced active caspase-3 and Bax expression, while increased Bcl-2 were changed after knocking down EP3 by RNA interference in PC12 cells, further confirmed that EP3 might exert its pro-apoptotic function on neuronal apoptosis. Thus, EP3 may play a role in promoting the neuronal apoptosis following ICH. [ABSTRACT FROM AUTHOR]

We examined the involvement of adipocyte cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2)-prostaglandin E receptor (EP)3-mediated signaling during hypertrophy and hypoxia in the development of obesity-associated adipose tissue (AT) inflammation and insulin resistance. The experiments were conducted with high-fat diet (HFD)-induced obese rats, db/db mice, human subjects, and 3T3-L1 and the human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes; the groups were treated with selective inhibitors of COX-2 [celecoxib 30 mg/kg, half maximal inhibitory concentration (IC50) 0.04 μM] and EP3 (L-798106 100 μg/kg, IC50 0.5 μM) or a short interfering RNA. There were strong, positive correlations between adipocyte COX-2 and EP3 gene expressions and the AT TNF-a and monocyte chemotactic protein-1 contents and the homeostatic model assessment for insulin resistance in HFD-induced obese rats, as well as body mass index in human subjects. Treatment with COX-2 and EP3 inhibitors significantly reversed AT inflammatory gene and protein expressions (-50%) and impaired glucose and insulin tolerance in db/db mice. COX-2 inhibition diminished the chemotaxis of adipocytes isolated from HFD rats to macrophages and T cells. Targeting inhibition of adipocyte COX-2 and EP3 during hypertrophy and hypoxia reversed the release of the augmented proinflammatory adipokines and the diminished adiponectin and also suppressed NF-κB and hypoxia-inducible factor-1a transcription activation. These findings suggest that adipocyte COX-2 PGE2-EP3-mediated signaling is crucially involved in the development of obesity-associated AT inflammation and insulin resistance. [ABSTRACT FROM AUTHOR]

The prostanoid E2 (PGE2) is known to modulate the aggregative response of platelets to their conventional agonists such as ADP, TXA2, thrombin or collagen. Through the activation of its receptor EP3, PGE2 sensitizes platelets to their agonists but also inhibits them through its two other receptors, EP2 and EP4. In mice, the net result of these opposed actions is the EP3-mediated potentiation of platelet aggregation and the in vivo aggravation of murine atherothrombosis. Since the pathway PGE2/EP3 is not involved in murine hemostasis, we propose a “platelet EP3 paradigm” to describe this apparently paradoxical association between the facilitating impact on atherothrombosis and the unaltered hemostasis. Consistent with this paradigm, a drug blocking EP3 dramatically decreased atherothrombosis without inducing bleeding in mice. In humans, several studies did not agree on the effect of PGE2 on platelets. Reinterpreting these data with the notion of “potentiation window” and taking the platelet initial cAMP level into account reconciled these inconsistent results. Thereby, the in vitro potentiating effect of PGE2 on human platelets becomes clear. In addition, the EP3 blocking drug DG-041 abrogated the potentiating effect of PGE2 in whole human blood but did not prolong bleeding times in volunteers. Thus, the murine “platelet EP3 paradigm” would apply to humans if the aggravating role of PGE2 on atherothrombosis is shown in patients. Therefore, testing an EP3 blocker in a phase III trial would be of high interest to fulfill the unmet medical need which is to control atherothrombosis without impacting hemostasis and thus to improve the prevention of myocardial infarction. [ABSTRACT FROM AUTHOR]

Background: Inflammation following intracerebral hemorrhage (ICH) significantly contributes to secondary brain damage and poor outcomes. Prostaglandin E2 (PGE2) is known to modulate neuroinflammatory responses and is upregulated in response to brain injury as a result of changes in inducible cyclooxygenase 2 (COX-2) and the membrane-bound type of PGE synthase. Inhibition of COX-2 activity has been reported to attenuate ICH-induced brain injury; however, the clinical utility of such drugs is limited due to the potential for severe side effects. Therefore, it is now important to search for downstream targets capable of preferentially modulating PGE2 signaling, and the four E prostanoid receptors, EP1-4, which are the main targets of PGE2, remain a viable therapeutic option. We have previously shown that EP1 receptor deletion aggravates ICH-induced brain injury and impairs functional recovery, thus the current study aimed to elaborate on these results by including a pharmacologie approach targeting the EP1 receptor. Results: Chronic post-treatment with the selective EP1 receptor antagonist, SC-51089, increased lesion volume by 30.1 ± 14.5% (p < 0.05) and treatment with the EP1 agonist, 17-pt-PGE2, improved neuromuscular functional recovery on grip strength (p < 0.01) and hanging wire (p < 0.05) behavioral testing. To begin identifying the mechanisms involved in EP1-mediated neuroprotection after ICH, histology was performed to assess ferric iron content, neuroinflammation, leukocyte transendothelial migratory potential, and peripheral neutrophil and immunoglobulin infiltration. Following ICH, mice treated with the antagonist displayed increased ferric iron (p < 0.05) and cortical microgliosis (p < 0.05), whereas treatment with the agonist decreased cortical (p < 0.01) and striatal (p < 0.001) astrogliosis, leukocyte transendothelial migratory potential (p < 0.01), neutrophil infiltration (p < 0.05), and blood brain barrier breakdown (p < 0.05). Conclusions: In agreement with our previous results, selective antagonism of the EP1 receptor aggravated ICH-induced brain injury. Furthermore, EP1 receptor agonism improved anatomical outcomes and functional recovery. Thus, the present data continues to reinforce a putative role for EPI as a new and more selective therapeutic target for the treatment of ICH that could reduce the side effects associated with COX-2 inhibition while still exploiting the beneficial effects. [ABSTRACT FROM AUTHOR]

Background: Inflammation following intracerebral hemorrhage (ICH) significantly contributes to secondary brain damage and poor outcomes. Prostaglandin E2 (PGE2) is known to modulate neuroinflammatory responses and is upregulated in response to brain injury as a result of changes in inducible cyclooxygenase 2 (COX-2) and the membrane- bound type of PGE synthase. Inhibition of COX-2 activity has been reported to attenuate ICH-induced brain injury; however, the clinical utility of such drugs is limited due to the potential for severe side effects. Therefore, it is now important to search for downstream targets capable of preferentially modulating PGE2 signaling, and the four E prostanoid receptors, EP1-4, which are the main targets of PGE2, remain a viable therapeutic option. We have previously shown that EP1 receptor deletion aggravates ICH-induced brain injury and impairs functional recovery, thus the current study aimed to elaborate on these results by including a pharmacologic approach targeting the EP1 receptor. Results: Chronic post-treatment with the selective EP1 receptor antagonist, SC-51089, increased lesion volume by 30.1 ± 14.5% (p < 0.05) and treatment with the EP1 agonist, 17-pt-PGE2, improved neuromuscular functional recovery on grip strength (p < 0.01) and hanging wire (p < 0.05) behavioral testing. To begin identifying the mechanisms involved in EP1-mediated neuroprotection after ICH, histology was performed to assess ferric iron content, neuroinflammation, leukocyte transendothelial migratory potential, and peripheral neutrophil and immunoglobulin infiltration. Following ICH, mice treated with the antagonist displayed increased ferric iron (p < 0.05) and cortical microgliosis (p < 0.05), whereas treatment with the agonist decreased cortical (p < 0.01) and striatal (p < 0.001) astrogliosis, leukocyte transendothelial migratory potential (p < 0.01), neutrophil infiltration (p < 0.05), and blood brain barrier breakdown (p < 0.05). Conclusions: In agreement with our previous results, selective antagonism of the EP1 receptor aggravated ICHinduced brain injury. Furthermore, EP1 receptor agonism improved anatomical outcomes and functional recovery. Thus, the present data continues to reinforce a putative role for EP1 as a new and more selective therapeutic target for the treatment of ICH that could reduce the side effects associated with COX-2 inhibition while still exploiting the beneficial effects. [ABSTRACT FROM AUTHOR]

Neurons interact closely with astrocytes via glutamate; this neuron-glia circuit may play a pivotal role in synaptic transmission. On the other hand, astrocytes contact vascular endothelial cells with their end-feet. It is becoming obvious that non-neuronal cells play a critical role in regulating the neuronal activity in the brain. We find that kainic acid (KA) administration induces the expression of microsomal prostaglandin E synthase-1 (mPGES-1) in venous endothelial cells and the prostaglandin E2 (PGE2) receptor prostaglandin E receptor (EP)-3 on astrocytes. Endothelial mPGES-1 exacerbates KA-induced neuronal damage in in vivo experiments. In in vitro experiments, mPGES-1 produces PGE2, which enhances astrocytic Ca2+ levels via the EP2 receptor and increases Ca2+dependent glutamate release, thus aggravating neuronal injury. This novel endothelium-astrocyte-neuron signaling pathway may be crucial for driving neuronal damage after repetitive seizures and could be a new therapeutic target for epilepsy and other brain disorders. [ABSTRACT FROM AUTHOR]

Background and Purpose The guinea pig trachea ( GPT) is commonly used in airway pharmacology. The aim of this study was to define the expression and function of EP receptors for PGE2 in GPT as there has been ambiguity concerning their role. Experimental Approach Expression of mRNA for EP receptors and key enzymes in the PGE2 pathway were assessed by real-time PCR using species-specific primers. Functional studies of GPT were performed in tissue organ baths. Key Results Expression of mRNA for the four EP receptors was found in airway smooth muscle. PGE2 displayed a bell-shaped concentration-response curve, where the initial contraction was inhibited by the EP1 receptor antagonist ONO-8130 and the subsequent relaxation by the EP2 receptor antagonist PF-04418948. Neither EP3 ( ONO- AE5-599) nor EP4 ( ONO- AE3-208) selective receptor antagonists affected the response to PGE2. Expression of COX-2 was greater than COX-1 in GPT, and the spontaneous tone was most effectively abolished by selective COX-2 inhibitors. Furthermore, ONO-8130 and a specific PGE2 antibody eliminated the spontaneous tone, whereas the EP2 antagonist PF-04418948 increased it. Antagonists of other prostanoid receptors had no effect on basal tension. The relaxant EP2 response to PGE2 was maintained after long-term culture, whereas the contractile EP1 response showed homologous desensitization to PGE2, which was prevented by COX-inhibitors. Conclusions and Implications Endogenous PGE2, synthesized predominantly by COX-2, maintains the spontaneous tone of GPT by a balance between contractile EP1 receptors and relaxant EP2 receptors. The model may be used to study interactions between EP receptors. [ABSTRACT FROM AUTHOR]

Abstract: Recent preclinical studies demonstrate a role for the prostaglandin E2 (PGE2) subtype 1 (EP1) receptor in mediating, at least in part, the pathophysiology of hypertension and diabetes mellitus. A series of amide and N-acylsulfonamide analogs of a previously described picolinic acid-based human EP1 receptor antagonist (7) were prepared. Each analog had improved selectivity at the mouse EP1 receptor over the mouse thromboxane receptor (TP). A subset of analogs gained affinity for the mouse PGE2 subtype 3 (EP3) receptor, another potential therapeutic target. One analog (17) possessed equal selectivity for EP1 and EP3, displayed a sufficient in vivo residence time in mice, and lacked the potential for acyl glucuronide formation common to compound 7. Treatment of mice with 17 significantly attenuated the vasopressor activity resulting from an acute infusion of EP1 and EP3 receptor agonists. Compound 17 represents a potentially novel therapeutic in the treatment of hypertension and diabetes mellitus. [Copyright &y& Elsevier]

Abstract: Prostaglandin E and F regulate diverse physiological functions including gastrointestinal motility, fever induction and reproduction. This multitude of biological effects is mediated via their four E receptor subtypes (EP1, EP2, EP3 and EP4) and F receptor (FP), respectively. Majority of these studies was performed in mammalian species, while investigations on their roles were impeded by inadequate information on their receptors in avian species. In present study, full-length cDNAs of chicken EP3 (cEP3) and two isoforms of FP – cFPa and cFPb – were cloned from adult hen ovary. The putative cEP3 and cFPa share high amino acid sequence identity with their respective orthologs, while the predicted cFPb is a novel middle-truncated splice variant which lacks 107 amino acids between transmembrane domains 4 and 6. RT-PCR showed that cEP3, cFPa and cFPb are widely expressed in adult tissues examined, including ovary and oviduct. Using a pGL3-CRE luciferase reporter system, cEP3-expressing DF1 cells inhibited forskolin-induced luciferase activity (EC50: <1.9pM) upon PGE2 treatment, suggesting that cEP3 may functionally couple to Gi protein. Upon PGF2α addition, cFPa was shown to potentially couple to intracellular Ca2+-signaling pathway by pGL3-NFAT-RE reporter assay (EC50: 2.9 nM), while cFPb showed no response. Using a pGL4-SRE reporter system, both cEP3 and cFPa exhibited potential MAPK activation by PGE2 and PGF2α at EC50 0.34 and 13nM, respectively. Molecular characterization of these receptors paved the road to the better understanding of PGE2 and PGF2α roles in avian physiology and comparative endocrinology studies. [Copyright &y& Elsevier]

Abstract: Arterial vascularization of the spinal cord may be mechanically or functionally altered during thoraco-abdominal surgery/intravascular procedures. Increased arterial pressure has been shown to restore spinal perfusion and function probably by increasing the blood flow through the intercostal arteries. The regulation of human intercostal artery (HICA) vascular tone is not well documented. Prostaglandin (PG)E2 concentration is increased during inflammatory conditions and has been shown to regulate vascular tone in many preparations. In this context, the pharmacological response of HICA to PGE2 and the characterization of the PGE2 receptor subtypes (EP1, EP2, EP3 or EP4) involved are of importance and that is the aim of this study. Rings of HICA were prepared from 29 patients and suspended in organ baths for isometric recording of tension. Cumulative concentration–response curves were performed in these preparations with various EP receptor agonists in the absence or presence of different receptor antagonists or inhibitors. PGE2 induced the contraction of HICA (Emax =7.28±0.16g; pEC50 value=0.79±0.18; n=17); contractions were also observed with the EP3 receptor agonists, sulprostone, 17-phenyl-PGE2, misoprostol or ONO-AE-248. In conclusion, PGE2 induced vasoconstriction of HICA via EP3 receptor subtypes and this result was confirmed by the use of selective EP receptor antagonists (L-826266, ONO-8713, SC-51322) and by a strong detection of EP3 mRNA. These observations suggest that in the context of perioperative inflammation, increased PGE2 concentrations could trigger vasoconstriction of HICA and possibly alter spinal vascularization. [Copyright &y& Elsevier]

Abstract: Prostaglandin E2 (PGE2) is considered to be a key mediator in migraine pathophysiology. PGE2 acts via four receptors (EP1–EP4) but their distribution in the brain districts implicated in migraine has yet to be delineated. We quantified amount of mRNA and protein expression for the EP receptors in both peripheral and central structures involved in pain transmission and perception in migraine: dura mater, cerebral arteries, trigeminal ganglion, trigeminal nucleus caudalis, periaqueductal grey, thalamus, hypothalamus, cortex, pituitary gland, hippocampus and cerebellum. In the trigeminal-vascular system (TVS) we found highest expression of EP1 and EP2 protein in the trigeminal nucleus caudalis. EP3 and EP4 mRNA expression were highest in the trigeminal ganglion. Within intracranial structures EP1 mRNA and protein expression were significantly higher in pituitary gland and cerebellum than in dorsal root ganglia (peripheral control), whereas the EP2 mRNA and protein were highly abundant in the pituitary gland. EP3 mRNA was mainly found in thalamus and hypothalamus. The most robust mRNA and protein expression for EP4 receptor was seen in the dorsal root ganglion. In conclusion, all four receptors are located in areas implicated in migraine supporting the possible involvement of PGE2 in this disease. [Copyright &y& Elsevier]

Summary: The overexpression of cyclooxygenase (COX)-2 is a frequent event in squamous cell carcinomas of the head and neck (HNSCC), and non-steroidal anti-inflammatory drugs, which are potent inhibitors of COX-1 and COX-2, exert chemopreventive effects on HNSCC cancer development. COX-2 promotes the release of the pro-inflammatory mediator prostaglandin E2 (PGE2), which acts on its cell surface G protein-coupled receptors EP1, EP2, EP3, and EP4. Here, we investigated the role of PGE2 and its receptors in cellular proliferation in HNSCC. The expression of COX-2 and EP1–4 was examined in immortalized oral epithelial cells and in a representative panel of HNSCC cell lines, and based on these data EP1–EP3 and COX-2 expression were evaluated by immunohistochemistry in a large clinical sample collection using HNSCC tissue microarrays. The ability of selective COX-2 inhibition to block PGE2 secretion was measured by ELISA specific assays. The effects of PGE2 on cell proliferation were evaluated using PGE2, its stable analog, and EP2 and EP3-specific synthetic agonists. The results presented here show that HNSCC tumoral lesions and their derived cell lines constitutively express COX-2 and the EP1, EP2 and EP3 receptors for PGE2. HNSCC cells secrete PGE2, which can be suppressed by low concentrations of COX-2 selective inhibitors, without inhibiting cell proliferation. Exogenously added stable PGE2 and EP3-specific agonists induce DNA synthesis in all HNSCC cell lines tested. Overall, our study supports the emerging notion that PGE2 produced in the tumor microenvironment by the overexpression of COX-2 in tumoral and inflammatory cells may promote the growth of HNSCC cells in an autocrine and paracrine fashion by acting on PGE2 receptors that are widely expressed in most HNSCC cancer cells. In particular, our findings suggest that EP3 receptor may play a more prominent role in HNSCC cell growth promotion, thus providing a rationale for the future evaluation of this PGE2 receptor as a target for HNSCC prevention strategies. [Copyright &y& Elsevier]

Abstract: A series of 3-urea-1-(phenylmethyl)-pyridones was discovered as novel EP3 antagonists via high-throughput screening and subsequent optimization. The synthesis, structure–activity relationships, and optimization of the initial hit that resulted in potent and selective EP3 receptor antagonists such as 11g are described. [ABSTRACT FROM AUTHOR]

Abstract: Gene expression profiling can be of benefit in identifying critical factors in the process of disease initiation and development. However, in endometriosis it has proven difficult to identify common genes between DNA microarray studies, presumably because of tissue homogeneity in lesions and diversity in the patients’ conditions. We attempted DNA microarray analysis in a mouse model for endometriosis with stable lesions and a homogeneous genetic background. Data extracted from the mouse model was then evaluated in human tissues. Mice of the ddY strain underwent surgery to remove the left side of the uterine horn, and the uterine tissue was then minced into small segments and auto-transplanted onto the left peritoneum. After 8 weeks, most of the uterine grafts were enlarged and had regenerated lumens. Comparison of the intensity of mRNA expression between grafts and normal uteri showed that genes encoding immune regulators (e.g. CXCL10) and metabolic factors (e.g. calbindin D-28K) were highly up-regulated in the grafts. Strongly inhibited genes in the grafts included prostaglandin-related factors [e.g. prostaglandin E receptor 3 (subtype EP3) and prostaglandin I2 synthase]. Variation in some candidate factors detected in the mouse model was observed by immunohistochemical studies in human adenomyosis tissues. The gene list in the present study is available for re-evaluation of past studies and provides new candidate factors potentially involved in the pathogenesis of endometriosis. [Copyright &y& Elsevier]

Accumulating evidence suggests that overproduction of prostaglandin (PG) E2 attributable to induction of cyclooxygenase-2 plays an important role in the development of lung adenocarcinoma. Recently, we have reported that a PGE2 receptor, EP3 is involved in appearance of malignant phenotype of a lung adenocarcinoma cell (A549 cell). In line with our previous study, here we investigated if Src signaling could be involved in PGE2-stimulated growth of A549 cells via EP3. PGE2-dependent cell growth in A549 cell positively related to the activation of Src. A specific antagonist against EP3 abrogated the cell growth and Src activation in the cells stimulated with PGE2. Also, the inhibition of Src activity suppressed its downstream signaling related to cell growth as well as the cell growth in the cells treated with PGE2. These results indicate that PGE2-dependent activation of Src signaling via EP3 plays an important role in growth of A549 cells.

Abstract: This Letter discloses a series of 2-aminothiadiazole amides as selective EP3 receptor antagonists. SAR optimization resulted in compounds with excellent functional activity in vitro. In addition, efforts to optimize DMPK properties in the rat are discussed. These efforts have resulted in the identification of potent, selective EP3 receptor antagonists with excellent DMPK properties suitable for in vivo studies. [Copyright &y& Elsevier]

Objective. To examine mRNA expression of prostaglandin E2 receptor isoforms EP3-II, III and VI in lower uterine segment myometrium in the non-pregnant and pregnant state using quantitative real-time RT-PCR. Methods. Myometrial samples were obtained at the time of cesarean delivery or hysterectomy. Pregnant subjects were categorised based on the presence or absence of labour. Labour was defined as regular uterine contractions resulting in cervical change. Quantification for EP3 isoforms II, III and VI mRNA was performed using real-time RT-PCR. Results. There were no differences between non-pregnant and non-labouring pregnant subjects in mRNA expression of EP3-II, III and VI. However, when compared to pregnant subjects not in labour, labouring subjects had a 3.8-fold reduction in EP3-II expression (p < 0.001) and 5.3-fold reduction in EP3-VI expression (p = 0.022). Conclusions. In human parturition, there is decreased mRNA expression of lower-uterine segment EP3 receptor isoforms II and VI during labour. This may reflect differential relaxation of the lower segment of the uterus allowing dilatation and descent of the fetus. [ABSTRACT FROM AUTHOR]

Abstract: Partial and generalized status epilepticus (pSE and gSE) trigger the same level of progenitor cell proliferation in adult dentate gyrus, but survival of new neurons is poor after gSE. Here, we show markedly elevated levels of prostaglandin E2 (PGE2) and brain-derived neurotrophic factor (BDNF) in rat hippocampal formation at 7 days following pSE but not gSE. Administration of the cyclooxygenase (COX) inhibitor flurbiprofen for 1 week, starting at day 8 post-SE, abated PGE2 and decreased BDNF levels, but did not affect survival of new neurons 4 weeks later. Thus, high PGE2 and BDNF levels induced by pSE are probably not of major importance for survival of new neurons during the first days after formation. We propose that they modulate other aspects of synaptic and cellular plasticity, and thereby may influence epileptogenesis. [Copyright &y& Elsevier]

Intrauterine growth restriction (IUGR) has a multifactorial pathogenesis and is an important cause of perinatal mortality. The relationship between fetal weight and placental blood flow in an animal model of IUGR has been investigated, showing that fetal growth is regulated by placental blood flow. The aim of the present study was to determine whether ischemia–reperfusion (I/R) injury stimulates the prostaglandin E2 (PGE2) system or the vascular endothelial growth factor (VEGF) system in the placenta of a rat IUGR model. COX-2 is reported to be involved in ischemic damage in many organs. There are 4 types of PGE2 receptor (EP1, EP2, EP3 and EP4). It is well known that EP1 and EP3 is associated with vasoconstriction. In the present study, vessels were occluded in the right uterine horn on day 17 of pregnancy in rats, and the clamps were removed after 30min of ischemia. At 24h, 48h, and 5 days after I/R injury, the live fetuses and placentas were obtained by cesarean section. This study revealed that I/R injury caused IUGR 5 days after the treatment. COX-2 expression and EP3 receptor expression were significantly elevated at 24h after I/R injury, but VEGF mRNA expression was not altered in the placenta from the ischemic horn compared with the non-ischemic horn. These results suggested that induction of the COX-2-EP3 system in the placenta may be one of the causes of IUGR induced by uterine ischemia, because the EP3 receptor and PGE2 are well known to mediate vasoconstriction in many organs. [Copyright &y& Elsevier]

Abstract: Prostaglandin E2 (PGE2), the principal pro-inflammatory prostanoid, is known to play versatile roles in pain transmission via four PGE receptor subtypes, EP1–EP4. We recently demonstrated that continuous production of nitric oxide (NO) by neuronal NO synthase (nNOS) following phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) and NMDA receptor NR2B subunits is essential for neuropathic pain. These phosphorylation and nNOS activity visualized by NADPH-diaphorase histochemistry were blocked by indomethacin, a PG synthesis inhibitor. To clarify the interaction between cyclooxygenase and nNOS pathways in the spinal cord, we examined the effect of EP subtype-selective agonists on NO production. NO formation was stimulated in the spinal superficial layer by EP1, EP3, and EP4 agonists. While the EP1- and the EP4-stimulated NO formation was markedly blocked by MK-801, an NMDA receptor antagonist, the EP3-stimulated one was completely inhibited by H-1152, a Rho-kinase inhibitor. Phosphorylation of MARCKS and NADPH-diaphorase activity stimulated by the EP3 agonist were also blocked by H-1152. These results suggest that PGE2 stimulates NO formation by Rho-kinase via EP3, a mechanism(s) different from EP1 and EP4. [Copyright &y& Elsevier]

Accumulating evidence suggests that overproduction of prostaglandin (PG) E2 attributable to induction of cyclooxygenase-2 plays an important role in the development of lung adenocarcinoma. Recently, we have reported that a PGE2 receptor, EP3 is involved in appearance of malignant phenotype of a lung adenocarcinoma cell (A549 cell). In line with our previous study, here we investigated if Src signaling could be involved in PGE2-stimulated growth of A549 cells via EP3. PGE2-dependent cell growth in A549 cell positively related to the activation of Src. A specific antagonist against EP3 abrogated the cell growth and Src activation in the cells stimulated with PGE2. Also, the inhibition of Src activity suppressed its downstream signaling related to cell growth as well as the cell growth in the cells treated with PGE2. These results indicate that PGE2-dependent activation of Src signaling via EP3 plays an important role in growth of A549 cells. [Copyright &y& Elsevier]