PGE2 accounts for bidirectional changes in alveolar macrophage self-renewal with aging and smoking.

Alveolar macrophages (AMs) are resident immune cells of the lung that are critical for host defense. AMs are capable of proliferative renewal, yet their numbers are known to decrease with aging and increase with cigarette smoking. The mechanism by which AM proliferation is physiologically restrained, and whether dysregulation of this brake contributes to altered AM numbers in pathologic circumstances, however, remains unknown. Mice of advanced age exhibited diminished basal AM numbers and contained elevated PGE2 levels in their bronchoalveolar lavage fluid (BALF) as compared with young mice. Exogenous PGE2 inhibited AM proliferation in an E prostanoid receptor 2 (EP2)-cyclic AMP-dependent manner. Furthermore, EP2 knockout (EP2 KO) mice exhibited elevated basal AM numbers, and their AMs resisted the ability of PGE2 and aged BALF to inhibit proliferation. In contrast, increased numbers of AMs in mice exposed to cigarette smoking were associated with reduced PGE2 levels in BALF and were further exaggerated in EP2 KO mice. Collectively, our findings demonstrate that PGE2 functions as a tunable brake on AM numbers under physiologic and pathophysiological conditions.

The prostaglandin receptor EP2 determines prognosis in EP3-negative and galectin-3-high cervical cancer cases.

Recently our study identified EP3 receptor and galectin-3 as prognosticators of cervical cancer. The aim of the present study was the analysis of EP2 as a novel marker and its association to EP3, galectin-3, clinical pathological parameters and the overall survival rate of cervical cancer patients. Cervical cancer tissues (n = 250), as also used in our previous study, were stained with anti-EP2 antibodies employing a standardized immunohistochemistry protocol. Staining results were analyzed by the IRS scores and evaluated for its association with clinical-pathological parameters. H-test of EP2 percent-score showed significantly different expression in FIGO I-IV stages and tumor stages. Kaplan-Meier survival analyses indicated that EP3-negative/EP2-high staining patients (EP2 IRS score ≥2) had a significantly higher survival rate than the EP3-negative/EP2-low staining cases (p = 0.049). In the subgroup of high galectin-3 expressing patients, the group with high EP2 levels (IRS ≥2) had significantly better survival rates compared to EP2-low expressing group (IRS <2, p = 0.044). We demonstrated that the EP2 receptor is a prognostic factor for the overall survival in the subgroup of negative EP3 and high galectin-3 expressed cervical cancer patients. EP2 in combination with EP3 or galectin-3 might act as prognostic indicators of cervical cancer. EP2, EP3, and galectin-3 could be targeted for clinical diagnosis or endocrine treatment in cervical cancer patients, which demands future investigations.

Enhancement of acid-sensing ion channel activity by prostaglandin E2 in rat dorsal root ganglion neurons.

Prostaglandin E2 (PGE2) and proton are typical inflammatory mediators. They play a major role in pain processing and hypersensitivity through activating their cognate receptors expressed in terminals of nociceptive sensory neurons. However, it remains unclear whether there is an interaction between PGE2 receptors and proton-activated acid-sensing ion channels (ASICs). Herein, we show that PGE2 enhanced the functional activity of ASICs in rat dorsal root ganglion (DRG) neurons through EP1 and EP4 receptors. In the present study, PGE2 concentration-dependently increased ASIC currents in DRG neurons. It shifted the proton concentration-response curve upwards, without change in the apparent affinity of proton for ASICs. Moreover, PGE2 enhancement of ASIC currents was partially blocked by EP1 or EP4 receptor antagonist. PGE2 failed to enhance ASIC currents when simultaneous blockade of both EP1 and EP4 receptors. PGE2 enhancement was partially suppressed after inhibition of intracellular PKC or PKA signaling, and completely disappeared after concurrent blockade of both PKC and PKA signaling. PGE2 increased significantly the expression levels of p-PKCε and p-PKA in DRG cells. PGE2 also enhanced proton-evoked action potentials in rat DRG neurons. Finally, peripherally administration of PGE2 dose-dependently exacerbated acid-induced nocifensive behaviors in rats through EP1 and EP4 receptors. Our results indicate that PGE2 enhanced the electrophysiological activity of ASICs in DRG neurons and contributed to acidosis-evoked pain, which revealed a novel peripheral mechanism underlying PGE2 involvement in hyperalgesia by sensitizing ASICs in primary sensory neurons.

The role of prostaglandin E2 receptor EP1 in 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced neonatal hydronephrosis in mice.

Prostaglandin E2 (PGE2) is a critical factor in the pathogenesis of dioxin-induced neonatal hydronephrosis. Since the PGE2 receptor has four subtypes, EP1 - EP4, this study was aimed to challenge the hypothesis that at least one of the four subtypes is responsible for the pathogenesis of dioxin-induced hydronephrosis. To this end, we used mouse pups, with a C57BL/6 J background, genetically lacking EP1, EP2, or EP3, and wild-type pups in whom EP4 was suppressed by administering ONO-AE3-208 (ONO), an EP4 antagonist, from postnatal day 1 (PND 1) to PND 13. To expose the pups to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via lactation, the dams were administered TCDD at an oral dose of 20 µg/kg on PND 1. The pups' urine and kidneys were collected on PND 14 for urinalysis and histological examination, respectively. We found that the incidence of hydronephrosis was 80% in the EP1+/+ group, but was markedly reduced to 28.6% in the EP1-/- group despite the fact that PGE2 concentration in the urine was similarly increased in the both groups. In contrast, the incidence of hydronephrosis was 80% and 100% in the EP2+/+ and EP2-/-groups, respectively, and 88.9% and 100% in the EP3+/+ and EP3-/- groups, respectively. With regard to EP4, the incidence of hydronephrosis in vehicle (saline)-treated groups and ONO-treated was 88.9% and 100%, respectively. Therefore, we concluded that among PGE2 receptor subtypes, EP1 plays a predominant role in the onset of TCDD-induced neonatal hydronephrosis in mouse pups.

Regulation of the porcine corpus luteum during pregnancy.

The new corpora lutea (CLs) in pigs are formed from the preovulatory follicles after the luteinizing hormone (LH) surge. However, total autonomy and independence of CLs from LH up to Day 12 of cycle has recently been questioned. Transformation of estrous cycle CL to CL of pregnancy initiated by embryonic signals requires not only the cessation of prostaglandin F2 (PGF2α) supply to the luteal tissue but also needs the CL to overcome luteolytic acquisition and/or changing its sensitivity to PGF2α during Days 12-14 of pregnancy. The luteolytic cascade is prevented by inhibition of lymphocyte infiltration and leucocyte recruitment, limitation of cell apoptosis, upregulation of pregnancy-associated genes and an enhanced antiluteolytic role of PGE2 Our 'two-signal switch hypothesis' highlights the importance of post PGF2α and PGE2 receptor signaling pathways activation in CLs during luteolysis and rescue. The 'luteolytic switch' involves increased expression of many regression mediators and activation of the post PTGFR signaling pathway. The 'rescue switch' initiated by embryonic signals - estradiol 17ß and PGE2 - induces post PTGER2/4 pathway, turning the 'luteolytic switch' off and triggering activity of genes responsible for CL maintenance. In mid and late pregnancy, CLs are maintained by LH and the synergistic action of metabolic hormones. This paper provides an outline of recent views on CL regression, rescue and maintenance during pregnancy in pigs that conflict with previous paradigms and highlights new findings regarding the actions of prostaglandins, role of microRNAs (miRNA) and immune system and signaling pathways governing the life cycle of porcine CL.

Investigation on the regulatory effect of PGE2 on ESCC cells through the trans-activation of EGFR by EP2 and the relevant mechanism.

OBJECTIVE: To determine whether the combination of prostaglandin E2 (PGE2) and EP2, the subtype receptor of PGE2, could trans-activate the epidermal growth factor receptor (EGFR). PATIENTS AND METHODS: In this experiment, we selected epithelial cells from normal esophageal mucosa as the negative control group, and the ESCC EC109 and TE-1 cell strain as the observation group. Real-time PCR and Western-blotting were used to detect the expression of EP2, EGFR and phosphorylated EGFR (p-EGFR). The pre-treatment of ESCC cell strains was carried out using Butaprost (special agonist of PGE2 and EP2) and RNAi of EP2, and we observed the expression of EP2, EGFR, and p-EGFR. WST-8 (CCK-8) was applied for the detection of the cell proliferation rate. The transwell invasion experiment was conducted for the detection of the invasion capability of cells. The expression of MMP-9 (matrix metalloproteinase-9), VEGF (vascular endothelial growth factor), pro-inflammatory factors (IL-6 and TNF-α) in the cell supernatant were detected using ELISA. RESULTS: The high mRNA and protein expression of EP2, EGFR, and p-EGFR were found in the EC109 and TE-1 cell strains in the observation group, which were higher than those in the control group (p < 0.05). After the intervention of PGE2, EP2 expression was decreased and the p-EGFR expression was increased (p < 0.05). There was no variation found in the expression of EGFR (p > 0.05). After cells were intervened using Butaprost, the expressions of EP2 and p-EGFR were increased (p < 0.05), and there were no changes identified in the expression of EGFR (p > 0.05). After the intervention of RNAi, the expression of EP2 and p-EGFR was decreased (p < .05), and no changes were identified in the expression of EGFR (p > 0.05). After the intervention of PGE2 and Butaprost, great increases were seen in the cell proliferation rate, invasion capability, and the expression of MMP-9, VEGF, IL-6, and TNF-α in EC109 and TE-1 cell strains (p < 0.05), however, the intervention of RNAi could reduce above indexes (p < 0.05). CONCLUSIONS: Through cell experiments, we verified that the combination of prostaglandin E2 (PGE2) and EP2, the subtype receptor of PGE2, could trans-activate the epidermal growth factor receptor (EGFR) to regulate the proliferation and invasion capability of esophageal squamous cell carcinoma (ESCC) cells, and secrete and express multiple cytokines, thus discovering the pathological mechanism of inflammation to carcinoma transition in the occurrence of ESCC, and providing the experimental evidence for the search of new target in the treatment of ESCC. ESCC cells can highly express the receptor subtype EP2 of PGE2 that can transactivate the EGFR, through which PGE2 is involved in the transition mechanism from inflammation to cancer.

Cerebroprotection by the neuronal PGE2 receptor EP2 after intracerebral hemorrhage in middle-aged mice.

Inflammatory responses mediated by prostaglandins such as PGE2 may contribute to secondary brain injury after intracerebral hemorrhage (ICH). However, the cell-specific signaling by PGE2 receptor EP2 differs depending on whether the neuropathic insult is acute or chronic. Using genetic and pharmacologic approaches, we investigated the role of EP2 receptor in two mouse models of ICH induced by intrastriatal injection of collagenase or autologous arterial whole blood. We used middle-aged male mice to enhance the clinical relevance of the study. EP2 receptor was expressed in neurons but not in astrocytes or microglia after collagenase-induced ICH. Brain injury after collagenase-induced ICH was associated with enhanced cellular and molecular inflammatory responses, oxidative stress, and matrix metalloproteinase (MMP)-2/9 activity. EP2 receptor deletion exacerbated brain injury, brain swelling/edema, neuronal death, and neurobehavioral deficits, whereas EP2 receptor activation by the highly selective agonist AE1-259-01 reversed these outcomes. EP2 receptor deletion also exacerbated brain edema and neurologic deficits in the blood ICH model. These findings support the premise that neuronal EP2 receptor activation by PGE2 protects brain against ICH injury in middle-aged mice through its anti-inflammatory and anti-oxidant effects and anti-MMP-2/9 activity. PGE2/EP2 signaling warrants further investigation for potential use in ICH treatment.

Activation of calcitonin gene-related peptide signaling through the prostaglandin E2-EP1/EP2/EP4 receptor pathway in synovium of knee osteoarthritis patients.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is a 37-amino-acid vasodilatory neuropeptide that binds to receptor activity-modifying protein 1 (RAMP1) and the calcitonin receptor-like receptor (CLR). Clinical and preclinical evidence suggests that CGRP is associated with hip and knee joint pain; however, the regulation mechanisms of CGRP/CGRP receptor signaling in synovial tissue are not fully understood. METHODS: Synovial tissues were harvested from 43 participants with radiographic knee osteoarthritis (OA; unilateral Kellgren/Lawrence (K/L) grades 3-4) during total knee arthroplasty. Correlationships between the mRNA expression levels of CGRP and those of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, and cycloxygenase-2 (COX-2) were evaluated using real-time PCR analysis of total RNA extracted from the collected synovial tissues. To investigate the factors controlling the regulation of CGRP and CGRP receptor expression, cultured synovial cells were stimulated with TNF-α, IL-1ß, IL-6, and prostaglandin E2 (PGE2) and were also treated with PGE2 receptor (EP) agonist. RESULTS: CGRP and COX-2 localized in the synovial lining layer. Expression of COX-2 positively correlated with CGRP mRNA expression in the synovial tissue of OA patients. The gene expression of CGRP and RAMP1 increased significantly in synovial cells exogenously treated with PGE2 compared to untreated control cells. In cultured synovial cells, CGRP gene expression increased significantly following EP4 agonist treatment, whereas RAMP1 gene expression increased significantly in the presence of exogenously added EP1 and EP2 agonists. CONCLUSIONS: PGE2 appears to regulate CGRP/CGRP receptor signaling through the EP receptor in the synovium of knee OA patients.

Study on Physiological Roles of Stimulation of Prostaglandin E2 Receptor Subtype EP2 in Urethral Function in Rats.

OBJECTIVES: We investigated the relaxant effect of stimulation of prostaglandin E2 (PGE2 ) receptor subtype EP2 as well as the involvement of a cyclic AMP (cAMP)-dependent pathway related to stimulation of EP2 receptors in urethral function in rats by evaluating effects of PGE2 and selective EP2 receptor agonist CP-533,536. METHODS: Effects of PGE2 and CP-533,536 on cAMP accumulation were assessed in Chinese hamster ovary (CHO)-K1 cells expressing rat EP2 or EP4 receptors. Relaxant responses to PGE2 and CP-533,536 (0.01-10 µmol/L) in rat urethral tissue pre-contracted with 10 µmol/L phenylephrine were evaluated, and cAMP levels in isolated rat urethral tissue treated with these compounds were determined as well. The effects of PGE2 and CP-533,536 (0.003-0.3 mg/kg intravenously) on urethral perfusion pressure (UPP) in anesthetized rats were also evaluated. RESULTS: PGE2 concentration-dependently increased the accumulation of cAMP in cells expressing rat EP2 (EC50 value = 1.3 nmol/L) and EP4 receptors (EC50 value = 17 nmol/L). While CP-533,536 similarly increased the accumulation of cAMP in cells expressing rat EP2 receptors (EC50 value = 3.0 nmol/L), no such effects were noted in cells expressing rat EP4 receptors up to 10 µmol/L. Both PGE2 and CP-533,536 produced relaxation and increased cAMP levels in urethral tissues in a concentration-dependent manner. PGE2 and CP-533,536 both dose-dependently decreased UPP in anesthetized rats. CONCLUSIONS: Taken together, these results suggest that stimulation of EP2 receptors induces relaxation likely via activation of cAMP-dependent mechanisms in rat urethral tissue, leading to a reduction of UPP.

Doxorubicin resistant cancer cells activate myeloid-derived suppressor cells by releasing PGE2.

Chemotherapies often induce drug-resistance in cancer cells and simultaneously stimulate proliferation and activation of Myeloid-Derived Suppressor Cells (MDSCs) to inhibit anti-tumor T cells, thus result in poor prognosis of patients with breast cancers. To date, the mechanism underlying the expansion of MDSCs in response to chemotherapies is poorly understood. In the present study, we used in vitro cell culture and in vivo animal studies to demonstrate that doxorubicin-resistant breast cancer cells secret significantly more prostaglandin E2 (PGE2) than their parental doxorubicin-sensitive cells. The secreted PGE2 can stimulate expansion and polymerization of MDSCs by directly target to its receptors, EP2/EP4, on the surface of MDSCs, which consequently triggers production of miR-10a through activating PKA signaling. More importantly, activated MDSCs can inhibit CD4(+)CD25(-) T cells as evidenced by reduced proliferation and IFN-γ release. In order to determine the molecular pathway that involves miR-10a mediated activation of MDSCs, biochemical and pharmacological studies were carried out. We found that miR-10a can activate AMPK signaling to promote expansion and activation of MDSCs. Thus, these results reveal, for the first time, a novel role of PGE2/miR-10a/AMPK signaling axis in chemotherapy-induced immune resistance, which might be targeted for treatment of chemotherapy resistant tumors.

Role of the ER stress in prostaglandin E2/E-prostanoid 2 receptor involved TGF-ß1-induced mice mesangial cell injury.

This study aims to investigate the relationship between prostaglandin E2 E-prostanoid 2 receptor (EP2) and Endoplasmic reticulum (ER) stress in transforming growth factor-ß1 (TGF-ß1)-induced mouse glomerular mesangial cells (MCs) injury. We cultured primary WT, EP2(-/-) MCs (EP2 deleted), and adenovirus-EP2-infected WT MCs (EP2 overexpressed). PCR, Western blot, flow cytometry, and immunohistochemical technique were used in in vitro and in vivo experiments. We found that TGF-ß1-induced PGE2 synthesis decreased in EP2-deleted MCs and increased in EP2-overexpressed MCs. EP2 deficiency in these MCs augmented the coupling of TGF-ß1 to ER stress-associated proteins [chaperone glucose-regulated protein 78 (GRP78), transient receptor potential channel 1 (TRPC1), and transient receptor potential channel 4 (TRPC4)], and upregulation of EP2 showed no significant change of GRP78, but augmented the expression of TRPC1, while TRPC4 expression was downregulated in comparison to normal MCs. In addition, EP2 deficiency in MCs augmented TGF-ß1-induced fibronectin (FN), cyclooxygenase-2 (COX2), and CyclinD1 expression. Silencing of EP2 also strengthened TGF-ß1-induced extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Flow Cytometry showed that silencing of EP2 significantly promoted the apoptosis of MCs. In contrast, EP2 overexpression reversed the effects of EP2 deficiency. 8 weeks after 5/6 nephrectomy (Nx), blood urea nitrogen and creatinine concentrations were significantly increased in EP2(-/-) 5/6Nx mice as compared to those of WT 5/6Nx mice. The pathological changes in kidney of EP2(-/-) mice were markedly aggravated compared with WT mice. Immunohistochemical analysis showed significant augment of TRPC4 and ORP150 in the kidney of EP2(-/-) mice compared with WT mice. Considering all the findings, it is suggested that increased expression of EP2 may prevent TGF-ß1-induced MCs damage through ER stress regulatory pathway.

Definition of Prostaglandin E2-EP2 Signals in the Colon Tumor Microenvironment That Amplify Inflammation and Tumor Growth.

Inflammation in the colon contributes significantly to colorectal cancer development. While aspirin reduces the colorectal cancer risk, its action mechanism, especially in inflammation in tumor microenvironment, still remains obscure. Here, we examined this issue by subjecting mice deficient in each prostaglandin (PG) receptor to colitis-associated cancer model. Deficiency of PGE receptor subtype EP2 selectively reduced, and deficiency of EP1 and EP3 enhanced, the tumor formation. EP2 is expressed in infiltrating neutrophils and tumor-associated fibroblasts in stroma, where it regulates expression of inflammation- and growth-related genes in a self-amplification manner. Notably, expression of cytokines such as TNFα and IL6, a chemokine, CXCL1, a PG-producing enzyme, COX-2, and Wnt5A was significantly elevated in tumor lesions of wild-type mice but this elevation was significantly suppressed in EP2-deficient mice. Intriguingly, EP2 stimulation in cultured neutrophils amplified expression of TNFα, IL6, CXCL1, COX-2, and other proinflammatory genes synergistically with TNFα, and EP2 stimulation in cultured fibroblasts induced expression of EP2 itself, COX-2, IL6, and Wnt genes. EP2 expression in infiltrating neutrophils and tumor-associated fibroblasts was also found in clinical specimen of ulcerative colitis-associated colorectal cancer. Bone marrow transfer experiments suggest that EP2 in both cell populations is critical for tumorigenesis. Finally, administration of a selective EP2 antagonist potently suppressed tumorigenesis in this model. Our study has thus revealed that EP2 in neutrophils and tumor-associated fibroblasts promotes colon tumorigenesis by amplifying inflammation and shaping tumor microenvironment, and suggests that EP2 antagonists are promising candidates of aspirin-alternative for chemoprevention of colorectal cancer.

EP2 and EP4 receptors mediate PGE2 induced relaxation in murine colonic circular muscle: pharmacological characterization.

BACKGROUND: Prostaglandin E2 (PGE2) is a regulator of gastrointestinal motility that might be involved in impaired motor function associated to gut inflammation. The aim of the present work is to pharmacologically characterize responses to exogenous and endogenous PGE2 in the mouse colon targeting EP2 and EP4 receptors. METHODS: Wild type (WT) and EP2 receptor knockout (EP2-KO) mice were used to characterize PGE2 and butaprost (EP2 receptor agonist) effects on smooth muscle resting membrane potential and myogenic contractility in circularly oriented colonic preparations. RESULTS: In WT animals, PGE2 and butaprost concentration-dependently inhibited spontaneous contractions and hyperpolarized smooth muscle cells. Combination of both EP2 (PF-04418948 0.1µM) and EP4 receptor antagonists (L-161,982 10µM) was needed to block both electrical and mechanical PGE2 responses. Butaprost inhibitory responses (both electrical and mechanical) were totally abolished by PF-04418948 0.1µM. In EP2-KO mice, PGE2 (but not butaprost) concentration-dependently inhibited spontaneous contractions and hyperpolarized smooth muscle cells. In EP2-KO mice, PGE2 inhibition of spontaneous contractility and hyperpolarization was fully antagonized by L-161,982 10µM. In WT animals, EP2 and EP4 receptor antagonists caused a smooth muscle depolarization and an increase in spontaneous mechanical activity. CONCLUSIONS: PGE2 responses in murine circular colonic layer are mediated by post-junctional EP2 and EP4 receptors. PF-04418948 and L-161,982 are selective EP2 and EP4 receptor antagonists that inhibit PGE2 responses. These antagonists might be useful pharmacological tools to limit prostaglandin effects associated to dismotility in gut inflammatory processes.

Antagonism of the prostaglandin E2 EP1 receptor in MDCK cells increases growth through activation of Akt and the epidermal growth factor receptor.

The actions of prostaglandin E2 (PGE2) in the kidney are mediated by G protein-coupled E-prostanoid (EP) receptors, which affect renal growth and function. This report examines the role of EP receptors in mediating the effects of PGE2 on Madin-Darby canine kidney (MDCK) cell growth. The results indicate that activation of Gs-coupled EP2 and EP4 by PGE2 results in increased growth, while EP1 activation is growth inhibitory. Indeed, two EP1 antagonists (ONO-8711 and SC51089) stimulate, rather than inhibit, MDCK cell growth, an effect that is lost following an EP1 knockdown. Similar observations were made with M1 collecting duct and rabbit kidney proximal tubule cells. ONO-8711 even stimulates growth in the absence of exogenous PGE2, an effect that is prevented by ibuprofen (indicating a dependence upon endogenous PGE2). The involvement of Akt was indicated by the observation that 1) ONO-8711 and SC51089 increase Akt phosphorylation, and 2) MK2206, an Akt inhibitor, prevents the increased growth caused by ONO-8711. The involvement of the EGF receptor (EGFR) was indicated by 1) the increased phosphorylation of the EGFR caused by SC51089 and 2) the loss of the growth-stimulatory effect of ONO-8711 and SC51089 caused by the EGFR kinase inhibitor AG1478. The growth-stimulatory effect of ONO-8711 was lost following an EGFR knockdown, and transduction of MDCK cells with a dominant negative EGFR. These results support the hypothesis that 1) signaling via the EP1 receptor involves Akt as well as the EGFR, and 2), EP1 receptor pharmacology may be employed to prevent the aberrant growth associated with a number of renal diseases.

Vasodilatory effect of 14,15-epoxyeicosatrienoic acid on mesenteric arteries in hypertensive and aged rats.

The objective of this study was to investigate the 14,15-epoxyeicosatrienoic acid (14,15-EET)-induced vasodilatations as well as the underlying signaling pathways in rat mesenteric arteries from young, adult and old normotensive (WKY) and hypertensive rats. Protein expressions for prostaglandin EP(1-4) receptors, large conductance Ca(2+)-activated K(+) (BK(Ca)) channels, and adenylate cyclase (AC) were determined together with 14,15-EET-induced vasodilatations in primary- versus secondary-branches of the mesenteric artery. Responses to 14,15-EET were greater in the smaller secondary- versus primary-branches (and also more sensitive with lower EC50) and were reduced in vessels from old (80 weeks) rats as well as from vessels from the spontaneous hypertensive rats (SHR). Regardless of age or hypertension responses to 14,15-EET were inhibited by the EP2 antagonist AH6809, BK(Ca) channel inhibitor iberiotoxin, or 3',5'-cyclic monophosphate (cAMP)-protein kinase A (PKA) pathway antagonists. These data indicate 14,15-EET-induced vasodilatation is mediated via the activation of EP2 receptors and opening of BK(Ca) channels. The expressions of the EP2 receptor and AC were markedly reduced in vessels from SHR as well as old rats, whereas BK(Ca) expression was reduced in old WKY and SHR, but not adult SHR. Furthermore, expression of the p53 protein, an indicator of cell senescence and apoptosis, was elevated in adult and old SHR as well as in old WKY. In summary, attenuated 14,15-EET-induced vasodilatation in mesenteric arteries from old normotensive WKY as well as adult and old SHR is associated with reduced expression of EP2 receptors and AC.

Molecular cloning and expression analysis of prostaglandin E receptor 2 gene in cashmere goat (Capra hircus) skin during hair follicle development.

As a member of the four subtypes of receptors for prostaglandin E2 (PGE2), prostaglandin E receptor 2 (PTGER2) is in the family of G-protein coupled receptors and has been characterized to be involved in the development and growth of hair follicles. In this study, we cloned and characterized the full-length coding sequence (CDS) of PTGER2 gene from cashmere goat skin. The entire open reading frame (ORF) of PTGER2 gene was 1047 bp and encoded 348 amino acid residues. The deduced protein contained one G-protein coupled receptors family 1 signature, seven transmembrane domains, and other potential sites. Tissue expression analysis showed that PTGER2 gene was expressed strongly in the skin. The general expression tendency of PTGER2 gene at different hair follicle developmental stages in the skin was gradually decreased from anagen to catagen to telogen. After comparing with the expression of BMP4 gene and related reports, we further presume that it seems to have a relationship between the hair follicle cycle and the expression level of PTGER2 gene in cashmere goat skin.

Prostaglandin E2 regulates its own inactivating enzyme, 15-PGDH, by EP2 receptor-mediated cervical cell-specific mechanisms.

CONTEXT: Prostaglandins play important roles in parturition and have been used to induce cervical ripening and labor. Prior to cervical ripening at term, 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is highly expressed in the cervix and metabolizes cyclooxygenase-2-mediated increases in active prostaglandin E2 (PGE2) to inactive 15-keto PGE2. At term, 15-PGDH gene expression decreases and PGE2 accumulates, leading to cervical ripening and labor. Previously, we found that the cervical isoform of microphthalmia-associated transcription factor (MiTF-CX) serves as a progestational transcription factor that represses IL-8 and hypoxia-mediated increases in cyclooxygenase-2. OBJECTIVE: We tested the hypothesis that PGE2 regulates its own inactivation through MiTF-CX. DESIGN: We used human cervical stromal cells to investigate the regulation of 15-PGDH. SETTING: This was a laboratory-based study using cells from clinical tissue samples. MAIN OUTCOME MEASURES: We evaluated the mechanisms by which PGE2 regulates 15-PGDH in human cervical stromal cells. RESULTS: PGE2 repressed MiTF-CX and 15-PGDH, whereas ectopic overexpression of MiTF-CX induced 15-PGDH expression levels. Stabilization of HIF-1α by deferoxamine resulted in concomitant down-regulation of MiTF-CX and 15-PGDH. Ectopic overexpression of MiTF-CX abrogated PGE2- and deferoxamine-mediated loss of MiTF-CX and 15-PGDH. PGE2-induced loss of MiTF-CX and 15-PGDH was mediated through prostaglandin E2 receptor (EP2) receptors (PTGER2), but not cAMP. CONCLUSIONS: The 15-PGDH gene is a MiTF-CX target gene in cervical stromal cells and is down-regulated by PGE2 through EP2 receptors. The findings suggest that EP2 receptor-specific antagonists may be used as an adjunct to present clinical management for the prevention of preterm cervical ripening and preterm labor.

Bovine oviduct epithelial cells downregulate phagocytosis of sperm by neutrophils: prostaglandin E2 as a major physiological regulator.

This study aimed to investigate the presence of polymorphonuclear neutrophils (PMNs) in bovine oviduct fluid under physiological conditions and to determine the possible role of bovine oviduct epithelial cells (BOECs) in the regulation of the phagocytic activity of PMNs for sperm. During the pre-ovulatory stage, PMNs were identified in the bovine oviduct fluid in relatively constant numbers. In our experiments, PMNs were incubated for 4 h with the supernatant of cultured BOECs stimulated for 24 h by LH (10 ng/ml). Phagocytosis was then assayed by co-incubation of these PMNs with sperm treated to induce capacitation. The BOEC supernatant significantly suppressed sperm phagocytosis by PMNs, and the LH-stimulated BOEC supernatant further suppressed phagocytosis. Importantly, in the BOEC culture, LH stimulated the secretion of prostaglandin E2 (PGE2), which dose-dependently (10(-6), 10(-7), and 10(-8) M) suppressed sperm phagocytosis by PMNs. Furthermore, a PGEP2 receptor antagonist significantly abrogated the inhibition of phagocytosis by the LH-stimulated BOEC supernatant. Additionally, using scanning electron microscopy, incubation of PMNs with either PGE2 or LH-stimulated BOEC supernatant before phagocytosis was found to prevent the formation of DNA-based neutrophil extracellular traps for sperm entanglement. The results indicate that sperm are exposed to PMNs in the oviduct and PGE2 released into the oviduct fluid after LH stimulation may play a major role in the suppression of the phagocytic activity of PMNs for sperm via interaction with EP2 receptors. Thus, the bovine oviduct provides a PGE2-rich microenvironment to protect sperm from phagocytosis by PMNs, thereby supporting sperm survival in the oviduct. Free Japanese abstract A Japanese translation of this abstract is freely available at http://www.reproduction-online.org/content/147/2/211/suppl/DC1.

[Expression and function of E prostanoid receptors in urological cancer].

The biological activities of prostaglandin E2 are mediated through their specific receptors, E prostanoid receptors (EPRs). This family comprises 4 subtypes (EP1R-4R), and has been associated with cancer development and progression. In urological cancers, expression of EP2R and EP4R can be significant predictors of survival for renal cell carcinoma (RCC). On the other hand, EP1R, EP2R, and EP4R are known to be associated with carcinogenesis and malignant aggressiveness in prostate cancer. In addition, EP4R has been associated with tumor progression and prognosis in urothelial cancer of the upper urinary tract. There is a general agreement that non-steroidal anti-inflammatory drugs (NSAIDs) can reduce the risk of several malignancies including colorectal cancer. However, NSAIDs often cause gastrointestinal injury and nephropathy. On the other hand, cyclooxygenase (COX)-2-selective inhibitors can reduce the progression of cancer via the suppression of cell proliferation angiogenesis without decreasing adverse reactions. However, COX-2-selective inhibitors might increase the risk of cardiovascular disease, including myocardial infarction. More selective and detailed control of COX-2-mediated signals is thus needed to improve anti-tumor effects and to decrease adverse reactions. EPRs are expected to serve as new therapeutic targets in urological cancer, because they are more selective in malignant phenotypes. Finally, we speculate that some EPRs inhibitors may reduce adverse events and exert more intense effects on urological cancer.