Prostaglandin EP4 Receptor Research Articles

Engraftment and Reconstitution of Hematopoiesis Is Dependent on Endothelial Cell Expressed Prostaglandin EP4 Receptor Signaling Mediated Regeneration of Vascular Niche

Defining the cellular and molecular components of the bone marrow (BM) niche that regulate the homeostasis and regeneration of hematopoietic stem and progenitor cells (HSPCs) can facilitate targeted interventions to improve hematopoietic recovery after injury and enhance stem cell engraftment after transplantation. We have recently shown that BM niche prostaglandin E2 (PGE2) signaling via the EP4 receptor is crucial for hematopoietic reconstitution, as transplantation of wild-type donor cells into EP4 receptor deficient recipient mice shows reduced stem cell engraftment. However, the specific contribution of EP4 signaling on different niche constituents in hematopoietic regeneration is unknown. To investigate which niche constituent/stromal population EP4 signaling functionally contributes to hematopoietic regeneration, we utilized genetic mouse models, in which EP4 could be deleted in specific stromal cell types.We conditionally deleted EP4 expression in nestin expressing mesenchymal stem cell (MSC), leptin receptor expressing mesenchymal stromal cell or tie 2 expressing endothelial cell (EC) invivo using a cre-recombinase approach. At steady-state, selective deletion of EP4 from nestin+ MSC (nestin-cre EP4flox/flox mice), leptin receptor+stromal cell (leptin R-cre EP4flox/flox mice) or tie 2+EC (tie 2-cre EP4flox/flox mice) showed no change in BM MSC (CD45-Ter119-CD31-CD51+PDGFR+), leptin receptor+stromal cells (CD45-Ter119-leptin R+) and EC (CD45-ter119-CD31+VE-cadherin+) number and survival. We next determined whether loss of EP4 expression/signaling in stromal cell specific genetic knockout mouse models affects BM niche constituents after exposure to ionization radiation. Interestingly, loss of EC but not nestin/leptin EP4 expression severely disrupted the BM vasculature and reduced EC and MSC survival at 24 hours post total body lethal (TBI) irradiation, compared to wild-type irradiated mice, and was associated with reduced expression of Survivin and increased expression of Bax, anti-apoptotic and pro-apoptotic factors respectively. In addition, niche EC and MSC regeneration at 2-month post irradiation was substantially reduced in tie 2-cre EP4flox/flox mice compared to wild-type mice. Nestin-cre EP4flox/flox mice and leptin R-cre EP4flox/flox niche restoration was equivalent to wild-type mice. To determine whether loss of EP4 receptor in different niche cells differentially regulates hematopoietic regeneration, we transplanted wild-type donor cells into nestin-cre EP4flox/flox or tie 2-cre EP4flox/flox recipient mice. Chimeric mice made with EC-specific EP4 deficient stroma showed reduced HSPC engraftment compared to chimeric mice made with wild-type stroma. In contrast, stem cell engraftment in chimeric mice made with MSC-specific EP4 deficient stroma was not reduced. In addition, in vivo administration of PGE2 into wild-type recipient mice at six-hour post irradiation enhanced donor cell engraftment. However, PGE2 treatment failed to enhance donor cell engraftment into tie 2-cre EP4flox/flox recipient mice. These data suggest that EC EP4 expression is important for niche restoration and hematopoietic regeneration after transplantation. To identify the mechanism involved in EP4 signaling mediated niche restoration, we measured angiocrine factors in the BM niche of lethally irradiated wild-type and tie 2-cre EP4flox/flox mice. Interestingly, we found that vascular endothelial growth factor (VEGF) expression was substantially reduced in the BM of tie 2-cre EP4flox/flox mice compared to wild-type mice. To investigate whether EP4 signaling regulates niche restoration by stimulating VEGF expression/production, we treated lethally irradiated tie 2-cre EP4flox/flox mice with recombinant VEGF (20 ng/mouse/day) for 3 days. In vivo administration of recombinant VEGF substantially enhanced niche restoration in tie 2-cre EP4flox/floxmice compared to control tie 2-cre EP4flox/flox. In conclusion, our study suggests that EC specific EP4 receptor signaling-mediated increased expression of VEGF promotes vascular niche restoration after myelosuppression and supports hematopoietic regeneration. DisclosuresNo relevant conflicts of interest to declare.

EP4 knockdown alleviates glomerulosclerosis through Smad and MAPK pathways in mesangial cells

Prostaglandin E2 has exhibited pleiotropic effects in the regulation of glomerulosclerosis progression through its four receptors. The current study aimed to evaluate the effect of prostaglandin receptor EP4 on mesangial cell proliferation. Invivo, 5/6 nephrectomy was introduced into EP4+/‑ and wild‑type (WT) mice. Clinical parameters were monitored post‑surgery. At 8weeks post‑surgery, glomerular fibrosis‑associated indicators were measured by immunohistochemical staining and trichrome staining. Invitro, mesangial cells in different groups (transfected with green fluorescent protein, AD‑EF4 or AD‑CRE) were exposed to transforming growth factor (TGF)‑β1 for 24h to detect the level of downstream signaling. Corresponding signaling inhibitors were also used to validate the signaling effects. Following surgery, EP4+/‑ mice presented a higher survival rate and normal urine volume compared with the WT group, and serum creatinine level and 24h urine protein were lower in the EP4+/‑ mice. Furthermore, associated profibrotic indicators were identified to have decreased at 8weeks post‑surgery along with less tubule‑interstitial fibrosis. Invivo, the inhibition of extracellular signal‑regulated kinase and P38 phosphorylation alleviated the accumulation of mesangial matrix, and these signals were enhanced when EP4 was overexpressed. EP4 enhancement aggravated imbalanced mesangial cell proliferation stimulated by TGF‑β1 and GS of mice treated with 5/6 nephrectomy through the Smad and mitogen‑activated protein kinase pathways.

Pharmacological properties of ASP7657, a novel, potent, and selective prostaglandin EP4 receptor antagonist.

We determined the pharmacologic profile of ASP7657, trans-4-[({[1-(quinolin-2-ylmethyl)-5-(trifluoromethyl)-1H-indol-7 yl] carbonyl} amino) methyl] cyclohexanecarboxylic acid methanesulfonate (1:1), a newly synthesized selective E-type prostaglandin (EP)4 receptor antagonist using several in vitro and in vivo experiments. ASP7657 exhibited high affinity for rat and human EP4 receptors, with Ki values of 6.02nM and 2.21nM, respectively. In addition, ASP7657 potently inhibited the PGE2-induced cyclic adenosine monophosphate (cAMP) increase in Chinese hamster ovary (CHO) cells expressing rat EP4 receptors and human lymphoblastoid T (Jurkat) cells, with IC50 values of 0.86nM and 0.29nM, respectively. In contrast, ASP7657 did not inhibit the PGE2-induced intracellular calcium increase in HEK293 cells expressing rat EP1 and EP3 receptors, or cAMP increase in CHO cells expressing rat EP2 receptors. ASP7657 showed good pharmacokinetic properties following oral dosing and dose-dependently antagonized the prostaglandin (PG)E2-mediated inhibition of lipopolysaccharide-induced tumor necrosis factor-α release from rat whole blood culture. In addition, 4weeks repeated oral administration of ASP7657 dose-dependently attenuated albuminuria in type 2 diabetic mice; these effects were significant at doses of 0.01mg/kg or higher. These results demonstrate that ASP7657 is a potent and selective EP4 receptor antagonist that may be useful in future studies to help clarify the physiological and pathophysiological roles of PG.

A luciferase complementation assay system using transferable mouse artificial chromosomes to monitor protein\u2013protein interactions mediated by G protein-coupled receptors

G protein-coupled receptors (GPCRs) are seven-transmembrane domain receptors that interact with the β-arrestin family, particularly β-arrestin 1 (ARRB1). GPCRs interact with 33% of small molecule drugs. Ligand screening is promising for drug discovery concerning GPCR-related diseases. Luciferase complementation assay (LCA) enables detection of protein–protein complementation via bioluminescence following complementation of N- and C-terminal luciferase fragments (NEluc and CEluc) fused to target proteins, but it is necessary to co-express the two genes. Here, we developed LCAs with mouse artificial chromosomes (MACs) that have unique characteristics such as stable maintenance and a substantial insert-carrying capacity. First, an NEluc-ARRB1 was inserted into MAC4 by Cre-loxP recombination in CHO cells, named ARRB1-MAC4. Second, a parathyroid hormone receptor 2 (PTHR2)-CEluc or prostaglandin EP4 receptor (hEP4)-CEluc were inserted into ARRB1-MAC4, named ARRB1-PTHR2-MAC4 and ARRB1-hEP4-MAC4, respectively, via the sequential integration of multiple vectors (SIM) system. Each MAC was transferred into HEK293 cells by microcell-mediated chromosome transfer (MMCT). LCAs using the established HEK293 cell lines resulted in 35,000 photon counts upon somatostatin stimulation for ARRB1-MAC4 with transient transfection of the somatostatin receptor 2 (SSTR2) expression vector, 1800 photon counts upon parathyroid hormone stimulation for ARRB1-PTHR2-MAC4, and 35,000 photon counts upon prostaglandin E2 stimulation for ARRB1-hEP4-MAC4. These MACs were maintained independently from host chromosomes in CHO and HEK293 cells. HEK293 cells containing ARRB1-PTHR2-MAC4 showed a stable reaction for long-term. Thus, the combination of gene loading by the SIM system into a MAC and an LCA targeting GPCRs provides a novel and useful platform to discover drugs for GPCR-related diseases.

The prostaglandin EP4 receptor is a master regulator of the expression of PGE2 receptors following inflammatory activation in human monocytic cells

Prostaglandin E2 (PGE2) is responsible for inflammatory symptoms. However, PGE2 also suppresses pro-inflammatory cytokine production. There are at least 4 subtypes of PGE2 receptors, EP1–EP4, but it is unclear which of these specifically control cytokine production. The aim of this study was to determine which of the different receptors, EP1R–EP4R modulate production of tumor necrosis factor-α (TNF-α) in human monocytic cells.Human blood, or the human monocytic cell line THP-1 were stimulated with LPS. The actions of PGE2, alongside selective agonists of EP1–EP4 receptors, were assessed on LPS-induced TNF-α, IL-1β and IL-10 release. The expression profiles of EP2R and EP4R in monocytes and THP-1 cells were characterised by RT-qPCR. In addition, the production of cytokines was evaluated following knockdown of the receptors using siRNA and over-expression of the receptors by transfection with constructs.PGE2 and also EP2 and EP4 agonists (but not EP1 or EP3 agonists) suppressed TNF-α production in blood and THP-1 cells. LPS also up regulated expression of EP2R and EP4R but not EP1 or EP3. siRNA for either EP2R or EP4R reversed the suppressive actions of PGE2 on cytokine production and overexpression of EP2R and EP4R enhanced the suppressive actions of PGE2.This indicates that PGE2 suppression of TNF-α by human monocytic cells occurs via EP2R and EP4R expression. However EP4Rs also control their own expression and that of EP2 whereas the EP2R does not affect EP4R expression. This implies that EP4 receptors have an important master role in controlling inflammatory responses.

The renal vasodilatory effect of prostaglandins is ameliorated in isolated-perfused kidneys of endotoxemic mice.

Endotoxemia-related acute kidney injury (AKI) is associated with increased formation of prostaglandins, which may serve as a compensatory mechanism to maintain renal function. We hypothesized that an increase of renal EP2 or EP4 receptors and/or a downregulation of renal EP1 and EP3 receptors enhances PGE2-induced renal vasodilatation. Injection of lipopolysaccharide (LPS; 3mg/kg i.p.) increased microsomal prostaglandin E synthase (mPGES)-1 and prostacyclin synthase expression, whereas mPGES-2 expression was unaltered. Further, LPS increased the mRNA abundance for the prostaglandin EP4 receptor, whereas the expressions of the EP1 and EP3 receptors were decreased. In isolated-perfused kidneys from control mice, PGE2 exerted a dual effect on renal vascular tone, inducing vasodilatation at lower concentrations and vasoconstriction at higher concentrations. In kidneys from endotoxemic mice, the vasodilatory component was more pronounced, whereas the vasoconstriction at higher PGE2 concentrations was absent. Similarly, prostacyclin (PGI2)-induced vasodilatation was more pronounced in endotoxemic kidneys. The enhanced vasodilatory effect was paralleled by an increase in renal vascular EP4 and prostacyclin IP receptor mRNA expression. Further, stimulation of renin secretion rate by PGE2 and PGI2 was enhanced in endotoxemic kidneys. Pretreatment with the cyclooxygenase (COX)-2 inhibitor SC-236 (10mg/kg) did not alter the basal GFR, but augmented the LPS-induced decline in GFR, and attenuated the LPS-induced increase in plasma renin concentration in vivo. Our data suggest that an activation of the COX-2/mPGES-1 synthetic pathway is responsible for the increased renal formation of PGE2 in response to LPS and that the vasodilatory effect of PGE2 and PGI2 is enhanced during endotoxemia.

Prostaglandin EP2 receptor: Novel therapeutic target for human cancers (Review).

Prostaglandin E2 (PGE2) receptor 2 subtype (EP2), which is a metabolite of arachidonic acid that binds with and regulates cellular responses to PGE2, is associated with numerous physiological and pathological events in a wide range of tissues. As a stimulatory G protein‑coupled receptor, PGE2‑induced EP2 activation can activate adenylate cyclase, leading to increased cytoplasmic cAMP levels and activation of protein kinase A. The EP2 receptor can also activate the glycogen synthase kinase 3β and β‑catenin pathways. The present study aimed to review the roles of the EP2 receptor in tumor development, including immunity, chronic inflammation, angiogenesis, metastasis and multidrug resistance. Furthermore, the involvement of the EP2 receptor signaling pathway in cancer was discussed. Understanding the role and mechanisms of action of the EP2 receptor, and its importance in targeted therapy, may help identify novel methods to improve management of numerous types of cancer.

FOXC1 Regulates Expression of Prostaglandin Receptors Leading to an Attenuated Response to Latanoprost.

This study examines the effect of FOXC1 on the prostaglandin pathway in order to explore FOXC1's role in the prostaglandin-resistant glaucoma phenotype commonly seen in Axenfeld-Rieger syndrome. Binding and transcriptional activity of FOXC1 to the gene coding for the EP3 prostaglandin receptor (PTGER3) were evaluated through ChIP-qPCR and luciferase-based assays. Immortalized trabecular meshwork cells (TM1) and HeLa cells had FOXC1 mRNA reduced via siRNA interference. qPCR and Western blot experiments were conducted to examine the changes in prostaglandin receptor expression brought about by lowered FOXC1. TM1 cells were then treated with 10 μM latanoprost acid and/or an siRNA for FOXC1. The expression of fibronectin and matrix metalloproteinase 9 were evaluated via qPCR in each treatment condition. ChIP-qPCR and luciferase experiments confirmed that FOXC1 binds to and activates transcription of the EP3 gene prostaglandin receptor. qPCR and Western experiments in HeLa and TM1 cells showed that FOXC1 siRNA knockdown results in significantly lowered EP3 levels (protein and RNA). In addition, RNA levels of the other prostaglandin receptor genes EP1 (PTGER1), EP2 (PTGER2), EP4 (PTGER4), and FP (PTGFR) were altered when FOXC1 was knocked down in TM1 and HeLa cells. Analysis of fibronectin expression in TM1 cells after treatment with 10 μM latanoprost acid showed a statistically significant increase in expression; this increase was abrogated by cotreatment with a siRNA for FOXC1. We show the abrogation of latanoprost signalling when FOXC1 is knocked down via siRNA in a trabecular meshwork cell line. We propose that the lower levels of active FOXC1 in Axenfeld-Rieger syndrome patients with glaucoma account for the lack of response to prostaglandin-based medications.

Abstract 348: The Blood-Pressure-Lowering Effect of Moxonidine May Be Partially Through the Prostaglandin Pathway in the Kidney

Objectives: Moxonidine is believed to decrease blood pressure via inhibiting sympathetic activity in the brain and consequently decreasing vascular tone. However, whether direct peripheral mechanisms are involved in this process is unknown. This study aimed to investigate possible peripheral mechanisms of moxonidine in lowering blood pressure. Methods: Two groups (N = 10 in each group) of apolipoprotein E-deficient mice were used. These animals were treated with or without moxonidine (18 mg/kg body weight per day) via drinking water throughout the experiment. Three days after the initiation of moxonidine treatment, hypertension was induced by subcutaneous infusion of angiotensin II (1 μg/kg body weight per min) for 28 days. Blood pressure was measured by the tail cuff method. Size and area of kidney tubules and glomeruli were analyzed by morphometric analysis. Gene expression was assessed by quantitative PCR. Results: Moxonidine treatment significantly decreased blood pressure (P<0.05). It did not change the size or area of either kidney tubules or glomeruli (P>0.05), nor the renal expression of inflammatory markers. Both types of moxonidine receptors (imidazoline 1 and alpha-2 adrenergic receptors) were found to be expressed in the kidney, and their gene expression was not altered by moxonidine treatment. However, moxonidine treatment significantly increased the expression of prostaglandin EP3 receptor (P<0.05). Increased prostaglandin EP3 receptor has been reported to be associated with promoting diuresis and natriuresis and thus decreasing blood pressure. Summary: The results indicate that moxonidine may lower blood pressure partially through an unrecognized non-central pathway by upregulating prostaglandin EP3 receptor in the kidney in mice.

Discovery of 2-Piperidinyl Phenyl Benzamides and Trisubstituted Pyrimidines as Positive Allosteric Modulators of the Prostaglandin Receptor EP2.

Prostaglandin E2 (PGE2) via its Gαs-coupled EP2 receptor protects cerebral cortical neurons from excitotoxic and anoxic injury, though EP2 receptor activation can also cause secondary neurotoxicity in chronic inflammation. We performed a high-throughput screen of a library of 292 000 small molecules and identified several compounds that have a 2-piperidinyl phenyl benzamide or trisubstituted pyrimidine core as positive modulators for human EP2 receptor. The most active compounds increased the potency of PGE2 on EP2 receptor 4-5-fold at 20 μM without altering efficacy, indicative of an allosteric mechanism. These compounds did not augment the activity of the other Gαs-coupled PGE2 receptor subtype EP4 and showed neuroprotection against N-methyl-d-aspartate (NMDA)-induced excitotoxicity. These newly developed compounds represent second-generation allosteric potentiators for EP2 receptor and shed light on a promising neuroprotective strategy. They should prove valuable as molecular tools to achieve a better understanding of the dichotomous action of brain EP2 receptor activation.

Chapter 54 - Antipyretic therapy: clinical pharmacology

Fever depends on a complex physiologic response to infectious agents and other conditions. To alleviate fever, many medicinal agents have been developed over a century of trying to improve upon aspirin, which was determined to work by inhibiting prostaglandin synthesis. We present the process of fever induction through prostaglandin synthesis and discuss the development of pharmaceuticals that target enzymes and receptors involved in prostaglandin-mediated signal transduction, including prostaglandin H2 synthase (also known as cyclooxygenase), phospholipase A2, microsomal prostaglandin E2 synthase-1, EP receptors, and transient potential cation channel subfamily V member 1. Clinical use of established antipyretics will be discussed as well as medicinal agents under clinical trials and future research.

Selective inhibition of histamine-evoked Ca2+ signals by compartmentalized cAMP in human bronchial airway smooth muscle cells

Intracellular Ca2+ and cAMP typically cause opposing effects on airway smooth muscle contraction. Receptors that stimulate these pathways are therapeutic targets in asthma and chronic obstructive pulmonary disease. However, the interactions between different G protein-coupled receptors (GPCRs) that evoke cAMP and Ca2+ signals in human bronchial airway smooth muscle cells (hBASMCs) are poorly understood. We measured Ca2+ signals in cultures of fluo-4-loaded hBASMCs alongside measurements of intracellular cAMP using mass spectrometry or [3H]-adenine labeling. Interactions between the signaling pathways were examined using selective ligands of GPCRs, and inhibitors of Ca2+ and cAMP signaling pathways. Histamine stimulated Ca2+ release through inositol 1,4,5-trisphosphate (IP3) receptors in hBASMCs. β2-adrenoceptors, through cAMP and protein kinase A (PKA), substantially inhibited histamine-evoked Ca2+ signals. Responses to other Ca2+-mobilizing stimuli were unaffected by cAMP (carbachol and bradykinin) or minimally affected (lysophosphatidic acid). Prostaglandin E2 (PGE2), through EP2 and EP4 receptors, stimulated formation of cAMP and inhibited histamine-evoked Ca2+ signals. There was no consistent relationship between the inhibition of Ca2+ signals and the amounts of intracellular cAMP produced by different stimuli. We conclude that β-adrenoceptors, EP2 and EP4 receptors, through cAMP and PKA, selectively inhibit Ca2+ signals evoked by histamine in hBASMCs, suggesting that PKA inhibits an early step in H1 receptor signaling. Local delivery of cAMP within hyperactive signaling junctions mediates the inhibition.

Prostaglandin receptor EP3 regulates cell proliferation and migration with impact on survival of endometrial cancer patients.

BackgroundProstaglandin E2 (PGE2) receptor 3 (EP3) regulates tumor cell proliferation, migration, and invasion in numerous cancers. The role of EP3 as a prognostic biomarker in endometrial cancer remains unclear. The primary aim of this study was to analyze the prognostic significance of EP3 expression in endometrial cancer.MethodsWe analyzed the EP3 expression of 140 endometrial carcinoma patients by immunohistochemistry. RL95-2 endometrial cancer cell line was chosen from four endometrial cancer cell lines (RL95-2, Ishikawa, HEC-1-A, and HEC-1-B) according to EP3 expression level. Treated with PGE2 and EP3 antagonist, RL95-2 cells were investigated by MTT, BrdU, and wound healing assay for functional assessment of EP3.ResultsEP3 staining differed significantly according to WHO tumor grading in both whole cohort (p = 0.01) and the subgroup of endometrioid carcinoma (p = 0.01). Patients with high EP3 expression in their respective tumors had impaired progression-free survival as well as overall survival in both cohorts above. EP3 expression in the overall cohort was identified as an independent prognostic marker for progression-free survival (HR 1.014, 95%CI 1.003-1.024, p = 0.01) when adjusted for age, stage, grading, and recurrence. Treatment with EP3 antagonists induced upregulation of estrogen receptor β and decreased activity of Ras and led to attenuated proliferation and migration of RL95-2 cells.ConclusionsEP3 seems to play a crucial role in endometrial cancer progression. In the context of limited systemic treatment options for endometrial cancer, this explorative analysis identifies EP3 as a potential target for diagnostic workup and therapy.

PDTM-17. IDENTIFICATION OF HUMAN PEDIATRIC BRAIN TUMORS CELLS IN PATIENT DERIVED ORTHOTOPIC XENOGRAFT MOUSE MODELS BY USING SPECIES-SPECIFIC PTGER2 GENE qPCR

AbstractBACKGROUNDPediatric brain tumors continue to be among the most difficult to cure and are the leading cause of cancer-related deaths in children. Research investigating pediatric brain cancer is still impaired by the lack of cancer models. We established a large panel of PDOX models as well as several paired permanent cell lines using and culture techniques. However, an inherent risk of xenograft models is the contamination of isolates of human tumors with host cells. Therefor strategies to identify the human/mouse ratio are essential. In this study we investigated Prostaglandin Receptor EP2 (PTGER2) as a useful target to identifying mouse cell contamination in PDOX models.METHODSpecies specific primers based on Alcoster et al., 2011 were used to detect PTGER2 in xenograft and cultured cells. RT-qPCR was carried out using a StepOnePlus™ RT-PCR System. Reaction mix contained SYBR Green master mix, respective primers and 5ng of total gDNA in a total reaction volume of 10μL. The qPCR conditions were as follows: 50°C-2min, 95°C-10min, 40 cycles of (95°C-15sec, 61°C-1min). Primers were additionally controlled by analysis of amplified products on a 1.2% agarose gel. FACS was used to isolate human cells according to anti-Human HLA-ABC Ab and mouse-specific Ab cocktail surface staining and subjected to qPCR.RESULTSVarious models of pediatric brain cancer established in our group were investigated for mouse cell contamination. Xenograft as well as cultured cells were routinely analyzed. Our study confirms PTGER2 as a useful target for accurately estimate mouse cell in PDOX models. This enabled us to improve detection and characterization of HLA-ABC/mouse-cocktail double negative population as seen in FACS.CONCLUSIONIn conclusion we have successfully implemented an reliable qRT-PCR approach to determine the human/mouse cell ration in any mixed PDOX cell population.

PTGER2 activation induces PTGS-2 and growth factor gene expression in endometrial epithelial cells of cattle

The prostaglandin E2 receptor 2 (PTGER2) is present in the endometrium and its gene expression is accompanied with endometrial growth, however, it is unknown whether there is endometrial repair through stimulation of growth factor gene expression that is promoted by PTGER2 activation in cattle. The aim of this study was to investigate whether PTGER2 activation can induce prostaglandin-endoperoxide synthase-2 (PTGS-2) and growth factor gene expression by activating PKA and ERK signaling pathways in endometrial epithelial cells of cattle. Results demonstrated that the PTGER2 agonist, butaprost, induced cAMP/PKA and ERK activation and up-regulated PTGS-2, VEGF, CTGF, TGF-β1 and IL-8 gene expression. These activations were less after PTGER2 antagonist, AH6809, treatment. Data suggested that PTGS-2 gene expression was induced by PTGER2 activation through the PKA and ERK pathways. Furthermore, PTGER2 activation promoted several growth factor gene expressions in endometrial epithelial cells. One potential implication of this finding is that PTGER2 activation in the endometrium of cattle could induce endometrial repair by stimulating VEGF, CTGF, TGF-β1 and IL-8 gene expression.

Prostaglandins in thiazide-induced hyponatraemia: do they hold water?

Thiazide diuretics lower blood pressure and cardiovascular risk but can cause severe hyponatraemia. Researchers now demonstrate that thiazide-induced hyponatraemia is associated with a genetic variant in a distal nephron prostaglandin transporter. They propose that reduced prostaglandin reabsorption through this transporter facilitates activation of the prostaglandin EP4 receptor to increase water reabsorption.

Abstract P473: Group IV Cytosolic Phospholipase A2α is Critical for Norepinephrine-Induced Hypertension

Previously we reported that angiotensin (Ang) II-induced hypertension and associated cardiovascular and renal dysfunction are mediated by cytosolic phospholipase A 2 α (cPLA 2 α) activation, the release of arachidonic acid (AA), and production of eicosanoids predominantly with pro-hypertensive effects ( Hypertension. 2015; 65: 784-792; 2016; 29: 258-265 ). We have also shown that norepinephrine (NE) by activating cPLA 2 releases AA, and production of prostanoids in vascular smooth muscle cells ( J Biol Chem. 1996; 217:30149-30157; J. Pharmacol. Exp. Ther. 1993; 266: 1113–1124 ). This study was conducted to determine the contribution of cPLA 2 α in NE-induced hypertension. Eight weeks old male wild-type (cPLA 2 α +/+ ) and cPLA 2 α gene disrupted (cPLA 2 α -/- ) mice were infused with NE (10 mg/kg/day, s.c.) or its vehicle using mini-osmotic pumps for 2 weeks, and the systolic blood pressure (SBP) was measured by tail-cuff. Infusion of NE increased the SBP in cPLA 2 α +/+ mice (148±3 vs. 118±3 mmHg, P<0.05, n=4-5); but not in cPLA 2 α -/- mice (122±5 mmHg, n=5). The NE-induced increase in SBP was minimized by treatment with AA metabolism inhibitor, 5,8,11,14-eicosatetraynoic acid (ETYA) (25 mg/kg, i.p., every 3 rd day) in cPLA 2 α +/+ mice (125±5 vs. 148±3 mmHg, P<0.05, n=4-5). Prostaglandin (PG) E2-EP1 and EP3 receptor activation that increase blood pressure have been implicated in Ang II-induced hypertension. In our study antagonists of the EP3 receptor (L-798106) (10 mg/kg, i.p. every 3 rd day) decreased the NE-induced increase in SBP (130±5 vs. 148±3 mmHg, P<0.05, n=5/group). These data suggest that cPLA 2 α contributes to NE-induced increase in SBP via cPLA 2 α activation, the release of AA and generation of eicosanoids, most likely PGE2 that exerts pro-hypertensive effects by stimulating EP3 receptors. Therefore, the development of agents that selectively inhibit the cPLA 2 α activity or block EP3 receptors could be useful in treating hypertension and its pathogenesis.

Design, Synthesis, and Pharmacokinetics of a Bone-Targeting Dual-Action Prodrug for the Treatment of Osteoporosis.

A dual-action bone-targeting prodrug has been designed, synthesized, and evaluated for in vitro and in vivo metabolic stability, in vivo tissue distribution, and rates of release of the active constituents after binding to bones through the use of differentially double-labeled derivatives. The conjugate (general structure 7) embodies the merger of a very potent and proven anabolic selective agonist of the prostaglandin EP4 receptor, compound 5, and alendronic acid, a potent inhibitor of bone resorption, optimally linked through a differentially hydrolyzable linker unit, N-4-carboxymethylphenyl-methyloxycarbonyl-leucinyl-argininyl-para-aminophenylmethylalcohol (Leu-Arg-PABA). Optimized conjugate 16 was designed so that esterase activity will liberate 5 and cathepsin K cleavage of the Leu-Arg-PABA element will liberate alendronic acid. Studies with doubly radiolabeled 16 provide a proof-of-concept for the use of a cathepsin K cleavable peptide-linked conjugate for targeting of bisphosphonate prodrugs to bone and slow release liberation of the active constituents in vivo. Such conjugates are potential therapies for the treatment of bone disorders such as osteoporosis.

The Prostaglandin EP3 Receptor Is an Independent Negative Prognostic Factor for Cervical Cancer Patients.

We know that one of the main risk factors for cervical cancer is an infection with high-risk human papillomavirus (HR-HPV). Prostaglandins and their receptors are very important for the tumour growth and tumour-associated angiogenesis. Little is known about the expression of the Prostaglandin E receptor type 3 (EP3) or the Prostaglandin (PG)E2-EP3 signalling in cervical cancer, so the aim of the study was to analyse the expression of the EP3 receptor in cervical cancer and find prognostic factors in relation to survival; EP3 immunohistological staining of 250 cervical cancer slides was performed and analysed with a semi-quantitative score. The statistical evaluation was performed with Statistical Package for the Social Sciences (SPSS) to evaluate the staining results and the survival analyses of the cervical cancer cases. A significant difference was observed in EP3 expression in Fédération Internationale de Gynécologie et d’Obstétrique (FIGO) stadium I versus FIGO stadium II–IV cases. High expression of EP3 (IRS ≥ 1.5) in cervical cancer patients was correlated with poor prognosis in overall survival rates. Survival in adenocarcinoma (AC) of the cervix was lower than in squamous cell carcinoma (SCC). Cox regression analysis shows that EP3 is an independent prognosticator. In this study we could show that the membrane-bound prostaglandin receptor EP3 is an independent prognosticator for cervical cancer patient survival. Targeting the EP3 receptor seems to be an interesting candidate for endocrine therapy. Therefore, more research is needed on the influence of the receptor system and its influence on cervical cancer growth.

Compartmentalized cAMP responses to prostaglandin EP2 receptor activation in human airway smooth muscle cells.

Previous studies indicate that prostaglandin EP2 receptors selectively couple to AC2 in non-lipid raft domains of airway smooth muscle (ASM) cells, where they regulate specific cAMP-dependent responses. The goal of the present study was to identify the cellular microdomains where EP2 receptors stimulate cAMP production. FRET-based cAMP biosensors were targeted to different subcellular locations of primary human ASM cells. The Epac2-camps biosensor, which expresses throughout the cell, was used to measure bulk cytoplasmic responses. Epac2-MyrPalm and Epac2-CAAX were used to measure responses associated with lipid raft and non-raft regions of the plasma membrane respectively. Epac2-NLS was used to monitor responses at the nucleus. Activation of AC with forskolin or β2 -adrenoceptors with isoprenaline increased cAMP in all subcellular locations. Activation of EP2 receptors with butaprost produced cAMP responses that were most readily detected by the non-raft and nuclear sensors, but only weakly detected by the cytosolic sensor and not detected at all by the lipid raft sensor. Exposure to rolipram, a PDE4 inhibitor, unmasked the ability of EP2 receptors to increase cAMP levels associated with lipid raft domains. Overexpression of AC2 selectively increased EP2 receptor-stimulated production of cAMP in non-raft membrane domains. EP2 receptor activation of AC2 leads to cAMP production in non-raft and nuclear compartments of human ASMs, while β2 adrenoceptor signalling is broadly detected across microdomains. The activity of PDE4 appears to play a role in maintaining the integrity of compartmentalized EP2 receptor responses in these cells.