Prostaglandin E2 receptor subtype EP-2 is not involved in the induction of non-pregnant guinea pig uterine contractions associated with terminal pregnancy

Prostaglandin E2 (PGE2) exerts its biological effects through 4 different receptor subtypes, EP-1, EP-2, EP-3, and EP-4. Recently we have demonstrated the importance of the prostaglandin E2 receptor subtype EP-2 in the healing of bone defects and fractures. This discovery led to the identification of CP-533,536, an EP-2 selective agonist, a promising therapeutic alternative for the enhancement of bone healing and the treatment of fractures (J Bone Miner Res 18 (2003) 2033). PGE2 has a myriad of effects throughout the body including the induction of uterine contractions, which results in termination of pregnancies. Our objective in this study was to determine the role of the EP-2 receptor and specifically that of CP-533,536, an EP-2 specific agonist, to induce uterine contractions and terminate pregnancy in guinea pigs, an animal model of human pregnancy. Preliminary experiments confirmed earlier reports that the guinea pig uterus was more sensitive than that of the rat. The guinea pig uterus contains the four PGE2 receptor subtypes, and ex vivo treatment of the uterus with PGE2 as expected causes profound uterine contractions. However, using receptor selective prostaglandin agonists including CP-533,536 we showed that the EP-1 and 3 receptors not the EP-2 receptor is responsible for the induction of uterine contractions of PGE2. Further, CP-533,536 did not antagonize the ability of PGE2 to induce uterine contractions in this model.     

Expression of prostanoid receptors in human lower segment pregnant myometrium

Prostanoids, especially prostaglandin (PG) E(2), are important mediators of uterine relaxation and contractions during gestation and parturition. Inhibitors of PG formation as well as PG analogues are used to modulate uterine tonus. So far, only limited data are available regarding the expression of prostanoid receptors in human pregnant myometrium. In the present study, the expression of the receptors for PGE(2) (EP1, EP2, EP3, EP4), PGF(2alpha) (FP), prostacyclin (IP), and thromboxane A(2) (TP) in human pregnant myometrium was studied by RT-PCR, in situ hybridization and immunohistochemistry. Myometrial tissue was obtained from five women at term and not in labour and from two women who delivered preterm. Tissue specimens were excised from the upper edge of the transverse lower uterine segment incision. In all tissues analysed, EP1, EP2, EP3, EP4, FP, TP and IP receptor mRNA and protein was detected. mRNA expression for PGD(2) (DP) receptor was not detected in the majority of tissue specimens. EP1, EP2, EP4, IP, TP and FP receptor protein was detected on myometrial smooth muscle cells, whereas EP3 receptor protein was only expressed by stromal and endothelial cells. In situ hybridization experiments yielded similar results. The expression of the EP2 receptor mRNA was inversely related to gestational age. We suggest that the contractile effect of PGE(2) at term is probably mediated directly by the EP1 receptor expressed in myometrial smooth muscle cells and indirectly by the EP3 receptor expressed in stromal cells and a decrease in EP2 receptor expression.     

In vitro response of prostaglandin E2 receptor (EP3) in the term pregnant rat uterus and cervix to misoprostol

We examined and compared the in vitro effects of misoprostol (synthetic prostaglandin E1 (PGE1) analogue) on prostaglandin E2 (PGE2) secretion and EP3 receptor mRNA expression in the pregnant rat myometrium and cervix at 19 days gestation. Myometrial and cervical tissue samples were exposed to media with or without misoprostol (50 or 100 pg/ml) and incubated for 15 and 30 min, and 1, 3, 6, 12, and 24 h. Media and tissue samples were collected for quantification of PGE2 and mRNA expression of rEP3alpha and rEP3beta receptor, respectively. PGE2 secretion increased (P < or = 0.05) in the myometrium exposed to 50 and 100 pg/ml misoprostol. Cervical PGE2 secretion increased following exposure to the 100 pg/ml dose only. In the myometrium, 50 and 100 pg/ml misoprostol induced elevations in rEP3alpha and rEP3beta receptor mRNA expression. rEP3alpha and rEP3beta receptor mRNA expression in the cervix was not different from controls. These data demonstrate that the EP3 receptor is differentially expressed in the myometrium and cervix in response to misoprostol. This may account for the ability of misoprostol to stimulate the myometrium when administered for cervical ripening.     

Differential regulation of prostaglandin EP and FP receptors in pregnant sheep myometrium and endometrium during spontaneous term labor.

In the present study, we characterized the mRNA abundance of prostaglandin E(2) receptor subtypes (EP1 and EP3, which stimulate excitatory responses; EP2 and EP4, which stimulate inhibitory responses) and the FP receptor in pregnant sheep myometrium and endometrium in relation to parturition. Myometrial and endometrial poly(A) RNA was extracted from control ewes at 143-147 days gestational age (dGA, n = 6) and from ewes in spontaneous term labor at 145-147 dGA (n = 6), and was subjected to Northern blot analysis for FP, EP1, EP2, EP3, and EP4 mRNA. Myometrial EP3, EP4, and FP mRNA abundance increased during labor (P<0.05); EP2 mRNA did not change. EP1 mRNA was not detectable in the myometrium. Endometrial EP2 and EP4 mRNA remained unchanged during labor. EP3 mRNA was expressed at a very low level, and EP1 and FP mRNA were not detected in endometrium in any animals studied. In conclusion, there is differential expression in myometrium and endometrium of EP subtypes and FP receptor in relation to labor. Increases in EP3 and FP, together with increased prostaglandin production from intrauterine tissues, may lead to the switch in the myometrial contraction pattern that occurs during labor. These differences within and between myometrium and endometrium may result from different anatomical location, such as longitudinal or circular layers of myometrium, or vascular location.     

Prostaglandin e and f receptor expression and myometrial sensitivity at labor onset in the sheep

Prostaglandins (PGs) play a pivotal role in the initiation and progression of term and preterm labor. Uterine activity is stimulated primarily by PGE(2) and PGF(2alpha) acting on prostaglandin E (EP) and prostaglandin F (FP) receptors, respectively. Activation of FP receptors strongly stimulates the myometrium, whereas stimulation of EP receptors may lead to contraction or relaxation, depending on the EP subtype (EP1-4) expression. Thus, the relative expression of FP and EP1-4 may determine the responsiveness to PGE(2) and PGF(2alpha). The aims of this study were to characterize the expression of EP1-4 and FP in intrauterine tissues and placentome, together with myometrial responsiveness to PG, following the onset of dexamethasone-induced preterm and spontaneous term labor. Receptor mRNA expression was measured using quantitative real-time polymerase chain reaction using species-specific primers. There was no increase in myometrial contractile receptor expression at labor onset, nor was there a change in sensitivity to PGE(2) and PGF(2alpha). This suggests expression of these receptors reaches maximal levels by late gestation in sheep. Placental tissue showed a marked increase in EP2 and EP3 receptor expression, the functions of which are unknown at this time. Consistent with previous reports, these results suggest that PG synthesis is the main factor in the regulation of uterine contractility at labor. This is the first study to simultaneously report PG E and F receptor expression in the key gestational tissues of the sheep using species-specific primers at induced-preterm and spontaneous labor onset.     

Prostaglandin E2 Represses Interleukin 1 Beta-Induced Inflammatory Mediator Output from Pregnant Human Myometrial Cells Through the EP2 and EP4 Receptors

Inflammatory mediators, including prostaglandins, cytokines, and chemokines, are strongly implicated in the mechanism of human labor, though their precise roles remain unknown. Here we demonstrate that interleukin 1 beta (IL-1beta) significantly increased the expression and release of interleukin-8 (CXCL8), monocyte chemotactic protein-1 (CCL2), and granulocyte macrophage colony-stimulating factor (CSF2) by primary human myometrial cells. However, this effect was repressed by prostaglandin E(2) (PGE(2)). As PGE(2) can activate four distinct PGE(2) receptors (EP(1), EP(2), EP(3), and EP(4)) to elicit various responses, we sought to define the EP receptor(s) responsible for this repression. Using selective EP receptor agonists and a selective EP(4) antagonist, we show that PGE(2) mediates the repression of IL-1beta-induced release of CXCL8, CCL2, and CSF2 via activation of the EP(2) and EP(4) receptors. The use of siRNA gene-specific knockdown further confirmed a role for both receptors. Real-time RT-PCR demonstrated that EP(2) was the most highly expressed of all four EP receptors at the mRNA level in human myometrial cells, and immunocytochemistry showed that EP(2) protein is abundantly present throughout the cells. Interestingly, PGE(2) does not appear to reduce mRNA expression of CXCL8, CCL2, and CSF2. Our results demonstrate that PGE(2) can elicit anti-inflammatory responses via activation of the EP(2) and EP(4) receptors in lower segment term pregnant human myometrial cells. Further elucidation of the EP receptor-mediated signaling pathways in the pregnant human uterus may be beneficial for optimizing the maintenance of pregnancy, induction of labor or indeed treatment of preterm labor.     

Effect of deletion of the prostaglandin EP2 receptor on the anabolic response to prostaglandin E2 and a selective EP2 receptor agonist

Studies using prostaglandin E receptor (EP) agonists indicate that prostaglandin (PG) E(2) can have anabolic effects through both EP4 and EP2 receptors. We previously found that the anabolic response to a selective EP4 receptor agonist (EP4A, Ono Pharmaceutical) was substantially greater than to a selective EP2 receptor agonist (EP2A) in cultured murine calvarial osteoblastic cells. To further define the role of the EP2 receptor in PG-mediated effects on bone cells, we examined the effects of EP2A and PGE(2) on both calvarial primary osteoblasts (POB) and marrow stromal cells (MSC) cultured from mice with deletion of one (Het) or both (KO) alleles of the EP2 receptor compared to their wild-type (WT) littermates. Deletion of EP2 receptor was confirmed by quantitative real-time PCR, Western blot and immunohistochemistry. The 1 month-old mice used to provide cells in these studies did not show any significant differences in their femurs by static histomorphometry. EP2A was found to enhance osteoblastic differentiation as measured by alkaline phosphatase mRNA expression and activity as well as osteocalcin mRNA expression and mineralization in the WT cell cultures from both marrow and calvariae. The effects were somewhat diminished in cultures from Het mice and abrogated in cultures from KO mice. PGE(2) effects were greater than those of EP2A, particularly in POB cultures and were only moderately diminished in Het and KO cell cultures. We conclude that activation of the EP2 receptor is able to enhance differentiation of osteoblasts, that EP2A is a true selective agonist for this receptor and that PGE(2) has an additional anabolic effect likely mediated by the EP4 receptor.     

Prostaglandin E2 induces cyclooxygenase-2 expression in human non-pigmented ciliary epithelial cells through activation of p38 and p42/44 mitogen-activated protein kinases

Prostaglandins (PGs) have been implicated in lowering intraocular pressure (IOP). A possible role of cyclooxygenase-2 (COX-2) in this process was emphasized by findings showing impaired COX-2 expression in the non-pigmented ciliary epithelium (NPE) of patients with primary open-angle glaucoma. The present study investigates the effect of the major COX-2 product, PGE(2), on the expression of its synthesizing enzyme in human NPE cells (ODM-2). PGE(2) led to an increase of COX-2 mRNA and protein expression, whereas the expression of COX-1 remained unchanged. Upregulation of COX-2 expression by PGE(2) was accompanied by time-dependent phosphorylations of p38 mitogen-activated protein kinase (MAPK) and p42/44 MAPK, and was abrogated by inhibitors of both pathways. Moreover, PGE(2)-induced COX-2 expression was suppressed by the intracellular calcium chelator, BAPTA/AM, and the protein kinase C inhibitor bisindolylmaleimide II, whereas the protein kinase A inhibitor H-89 was inactive in this respect. Induction of COX-2 expression was also elicited by butaprost (EP(2) receptor agonist) and 11-deoxy PGE(1) (EP(2)/EP(4) receptor agonist), but not by EP(1)/EP(3) receptor agonists (17-phenyl-omega-trinor PGE(2), sulprostone). Consistent with these findings, the EP(1)/EP(2) receptor antagonist, AH-6809, and the selective EP(4) receptor antagonist, ONO-AE3-208, significantly reduced PGE(2)-induced COX-2 expression. Collectively, our results demonstrate that PGE(2) at physiologically relevant concentrations induces COX-2 expression in human NPE cells via activation of EP(2)- and EP(4) receptors and phosphorylation of p38 and p42/44 MAPKs. Positive feedback regulation of COX-2 may contribute to the production of outflow-facilitating PGs and consequently to regulation of IOP.     

Prostaglandin E receptor subtypes involved in stimulation of gastroduodenal bicarbonate secretion in rats and mice.

We investigated prostaglandin E (EP) receptor subtypes responsible for the HCO3- stimulatory action of prostaglandin E2 (PGE2) in the gastroduodental mucosa, by examining the effects of various prostanoids with subtype specific EP receptor agonists in rats and those of PGE2 in knockout mice lacking EP1 or EP3 receptors. In rats, gastric HCO3- secretion was stimulated by i.v. administration of PGE2, 17-phenyl PGE2 the selective EP1 agonist as well as sulprostone the EP1 and EP3 agonist, but was not affected by other EP agonists such as butaprost the selective EP2 agonist, ONO-NT-012 the selective EP3 agonist or 11-deoxy PGE1 the EP3 and EP4 agonist. In contrast, the HCO3- secretion in rat duodenums was stimulated by PGE2, sulprostone, ONO-NT-012 as well as 11-deoxy PGE1 but not affected by either 17-phenyl PGE2 or butaprost. The HCO stimulatory effect of sulprostone in the stomach was significantly inhibited by ONO-AE-829, the selective EP1 antagonist. On the other hand, PGE2 applied topically to the mucosa for 10 min caused a dose-dependent increase of HCO3- secretion in both the stomach and duodenum of wild-type mice. The HCO3- stimulatory action of PGE2 in the stomach was also observed dose-dependently in knockout mice lacking EP3-receptors but was absent in EP1-receptor knockout mice, while the stimulatory effect in the duodenum was observed in EP1-receptor knockout mice, similar to wild-type animals, but not in knockout mice lacking EP3-receptors. These results indicate that PGE2 stimulates HCO3- secretion via different EP receptor subtypes in the stomach and duodenum; the former is mediated by EP1-receptors, while the latter mediated by EP3-receptors.     

EP₃ receptors mediate PGE₂-induced hypothalamic paraventricular nucleus excitation and sympathetic activation

Prostaglandin E(2) (PGE(2)), an important mediator of the inflammatory response, acts centrally to elicit sympathetic excitation. PGE(2) acts on at least four E-class prostanoid (EP) receptors known as EP(1), EP(2), EP(3), and EP(4). Since PGE(2) production within the brain is ubiquitous, the different functions of PGE(2) depend on the expression of these prostanoid receptors in specific brain areas. The type(s) and location(s) of the EP receptors that mediate sympathetic responses to central PGE(2) remain unknown. We examined this question using PGE(2), the relatively selective EP receptor agonists misoprostol and sulprostone, and the available selective antagonists for EP(1), EP(3), and EP(4). In urethane-anesthetized rats, intracerebroventricular (ICV) administration of PGE(2), sulprostone or misoprostol increased renal sympathetic nerve activity, blood pressure, and heart rate. These responses were significantly reduced by ICV pretreatment with the EP(3) receptor antagonist; the EP(1) and EP(4) receptor antagonists had little or no effect. ICV PGE(2) or misoprostol increased the discharge of neurons in the hypothalamic paraventricular nucleus (PVN). ICV misoprostol increased the c-Fos immunoreactivity of PVN neurons, an effect that was substantially reduced by the EP(3) receptor antagonist. Real-time PCR detected EP(3) receptor mRNA in PVN, and immunohistochemical studies revealed sparsely distributed EP(3) receptors localized in GABAergic terminals and on a few PVN neurons. Direct bilateral PVN microinjections of PGE(2) or sulprostone elicited sympathoexcitatory responses that were significantly reduced by the EP(3) receptor antagonist. These data suggest that EP(3) receptors mediate the central excitatory effects of PGE(2) on PVN neurons and sympathetic discharge.     

Prostanoid receptor expression by human airway smooth muscle cells and regulation of the secretion of granulocyte colony-stimulating factor

The prostanoid receptors on human airway smooth muscle cells (HASMC) that augment the release by IL-1beta of granulocyte colony-stimulating factor (G-CSF) have been characterized and the signaling pathway elucidated. PCR of HASM cDNA identified products corresponding to EP(2), EP(3), and EP(4) receptor subtypes. These findings were corroborated at the protein level by immunocytochemistry. IL-1beta promoted the elaboration of G-CSF, which was augmented by PGE(2). Cicaprost (IP receptor agonist) was approximately equiactive with PGE(2), whereas PGD(2), PGF(2alpha), and U-46619 (TP receptor agonist) were over 10-fold less potent. Neither SQ 29,548 nor BW A868C (TP and DP(1) receptor antagonists, respectively) attenuated the enhancement of G-CSF release evoking any of the prostanoids studied. With respect to PGE(2), the EP receptor agonists 16,16-dimethyl PGE(2) (nonselective), misoprostol (EP(2)/EP(3) selective), 17-phenyl-omega-trinor PGE(2) (EP(1) selective), ONO-AE1-259, and butaprost (both EP(2) selective) were full agonists at enhancing G-CSF release. AH 6809 (10 microM) and L-161,982 (2 microM), which can be used in HASMC as selective EP(2) and EP(4) receptor antagonists, respectively, failed to displace to the right the PGE(2) concentration-response curve that described the augmented G-CSF release. In contrast, AH 6809 and L-161,982 in combination competitively antagonized PGE(2)-induced G-CSF release. Augmentation of G-CSF release by PGE(2) was mimicked by 8-BrcAMP and abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA). These data demonstrate that PGE(2) facilitates G-CSF secretion from HASMC through a PKA-dependent mechanism by acting through EP(2) and EP(4) prostanoid receptors and that effective antagonism is realized only when both subtypes are blocked concurrently.     

Effects of selective prostaglandins E2 receptor agonists on cultured calvarial murine osteoblastic cells

We compared the direct effects of selective EP4 and EP2 receptor agonists (EP4A and EP2A) with prostaglandin E(2) (PGE(2)) on the differentiation of cultured murine calvarial osteoblastic cells. EP4A increased alkaline phosphatase activity and osteocalcin mRNA levels in these cultures similar to PGE(2). This effect was seen with both "direct plating" immediately after isolating the cells, or "indirect plating" in which the cells were grown to confluence and replated. EP2A had a smaller effect, significant only in "indirect plating" experiments. All three agents decreased the DNA and protein content in indirect plating experiments, but not in direct plating experiments. We conclude that the anabolic effect of PGE(2) in calvarial osteoblastic cell cultures is largely mediated by activation of the EP4 receptor, while activation of the EP2 receptor is less effective.     

Uterine region-dependent differences in responsiveness to prostaglandins in the non-pregnant porcine myometrium

To clarify the uterine region-dependent distribution of prostanoid receptors, we compared the mechanical responses to selective prostanoid receptor agonists (FP, EP3, DP, EP2) and naturally occurring prostaglandins (PGF2alpha PGE2, PGD2) in longitudinal and circular muscles isolated from three different regions (cornu, corpus and cervix) of the non-pregnant porcine uterus. Expression levels of FP receptor and cyclooxygenase (COX-1 and COX-2) in the respective regions were also examined using RT-PCR and Western blotting. The contractile responses to fluprostenol (an FP agonist) and PGF2alpha in both longitudinal and circular muscles were strongest in the cornu but weak in the corpus and cervix. Expression levels of mRNA and protein of FP receptor were highest in the cornu, consistent with the contractile responses. ONO-AE-248 (an EP3 agonist) caused contraction of both muscle layers, but region-related difference in responsiveness was observed only in the longitudinal muscle. ONO-AE1-259 (an EP2 agonist) inhibited spontaneous contraction of the myometrium, and inhibition was conspicuously stronger in the cervix. PGE2 caused contraction (<100 nM, cornu > corpus = cervix) and inhibition (>300 nM, cornu = corpus < or = cervix) of contractility depending on the concentration in both muscle layers. BW245C (a DP agonist) inhibited the spontaneous contraction, and region-dependent different responsiveness was marked in the longitudinal muscle (cervix = corpus > cornu). COX-1 but not COX-2 was detected in the non-pregnant porcine uterus. Expression level of COX-1 was different in the longitudinal muscle (cornu > corpus = cervix) but the same in the circular muscle. SC-560 inhibited the spontaneous contraction of longitudinal muscles in all regions. The results of the present study indicate that there are region-related heterogeneous distributions of contractile (FP and EP3, cornu > cervix) and relaxant (EP2 and DP, cervix > cornu) prostanoid receptors and COX-1 in the porcine uterus. The results also suggest involvement of endogenous PGs in the regulation of spontaneous uterine contractility. Region-related differences in COX-1 and prostanoid receptors might be necessary to produce a gradient of uterine motility decreasing from the cornu to the cervix that manages movement of luminal contents.     

Effect of deletion of the prostaglandin EP4 receptor on stimulation of calcium release from cultured mouse calvariae: impaired responsiveness in heterozygotes

The ability of prostaglandin E2 (PGE2), selective receptor agonists for EP2 and EP4 receptors (EP2A and EP4A) and parathyroid hormone (PTH) to stimulate calcium release from cultured fetal mouse calvariae was compared in wild type (WT) mice and in mice heterozygous (HET) or homozygous (KO) for deletion of the EP4 receptor. Calvariae from 19 day fetal mice were used in order to avoid the problem of high neonatal mortality. Calcium release was increased by PGE2, EP4A or PTH in WT mice, but EP2A had no significant effect. There was a significant decrease in calcium release in response to PGE2, EP4A and PTH in calvariae from HET mice compared to WT mice. The response to PGE2 and EP4A was abrogated and the response to PTH was further diminished in EP4 receptor KO mice. These results suggest that the EP4 receptor may be rate limiting not only for PGE2 stimulated resorption but also for resorption stimulated by other agonists, like PTH that induce PGE2 production.     

Prostaglandin E(2) upregulates cyclooxygenase-2 expression in lipopolysaccharide-stimulated RAW 264.7 macrophages

Prostaglandin E(2) (PGE(2)) has been implicated in the regulation of inflammatory and immunological events. Using RAW 264.7 macrophages, the present study investigates the influence of PGE(2) on the expression of cyclooxygenase-2 (COX-2). Incubation of cells with PGE(2) increased lipopolysaccharide (LPS)-induced COX-2 mRNA levels in a concentration-dependent manner. Upregulation of COX-2 expression by PGE(2) was completely abolished by the specific adenylyl cyclase inhibitor 2',5'-dideoxyadenosine and mimicked by butaprost, a selective agonist of the adenylyl cyclase-coupled PGE(2) receptor subtype 2 (EP(2)), or 11-deoxy PGE(1), an EP(2)/EP(4) receptor agonist. By contrast, the EP(3)/EP(1) receptor agonists 17-phenyl-omega-trinor PGE(2) and sulprostone left LPS-induced COX-2 expression virtually unaltered. Upregulation of LPS-induced COX-2 expression and subsequent PGE(2) synthesis was also observed in the presence of the cell-permeable cAMP analogue dibutyryl cAMP and the adenylyl cyclase activator cholera toxin. Together, our data demonstrate that PGE(2) potentiates COX-2 mRNA expression via an adenylyl cyclase/cAMP-dependent pathway. In conclusion, upregulation of COX-2 expression via an autocrine feed-forward loop may in part contribute to the well-known capacity of PGE(2)/cAMP to modulate inflammatory processes.     

PGE2 exerts its effect on the LPS-induced release of TNF-alpha, ET-1, IL-1alpha, IL-6 and IL-10 via the EP2 and EP4 receptor in rat liver macrophages

Lipopolysaccharide (LPS) induces a release of tumor necrosis factor (TNF)-alpha, endothelin (ET)-1, interleukin (IL)-1alpha, IL-6 and IL-10 in rat liver macrophages (Kupffer cells). Prostaglandin (PG)E2 inhibits the release of the fibrogenic mediators TNF-alpha, ET-1 and IL-1alpha, and enhances the release of the anti-fibrogenic mediators IL-6 and IL-10. This effect of PGE2 is mimicked by specific agonists for the PGE2 receptors EP2 and EP4; whereas, agonists for the PGE2 receptors EP1 and EP3 are inactive. Rat liver macrophages express mRNA encoding the PGE2 receptors EP2 and EP4 but not the PGE2 receptors EP1 and EP3. These data suggest that PGE2 exerts its anti-fibrogenic effect through the EP2 and EP4 receptor by inhibiting the release of the fibrogenic mediators TNF-alpha, ET-1 and IL-1alpha, and by enhancing the release of the anti-fibrogenic mediators IL-6 and IL-10 in liver macrophages.     

Role of prostaglandin E2 receptor subtypes in ovarian follicle growth in the rat in vivo. Correlation with interleukin-8 and neutrophils

This study was conducted to elucidate the role of three of prostaglandin E2 (PGE2) receptor subtype (EP2, EP3, and EP4) agonists in the process of follicular growth. The influence of these agonists on ovarian expression of intimately related factors to follicle development (neutrophils and interleukin-8 (IL-8)) was also investigated. Immature female Wistar rats were injected once with these agonists and killed 48 hours later. Another group of rats were injected pregnant mare serum gonadotrophin. For evaluation of follicle growth, morphometric assessment of antral and ovulatory follicles was performed in serial ovarian sections. The study demonstrated that, EP2 and EP4 agonists showed the maximum follicle counts and diameters versus the control. EP2 and EP4 agonists mimicked PMSG induced follicle growth. Injection of the three agonists induced neutrophil infiltration into theca layer. EP4 agonist showed the most intense ovarian neutrophil accumulation. In addition, dense ovarian IL-8 expression was observed only after EP4 agonist injection. CONCLUSIONS: Our data suggests that: 1) EP2 and EP4 receptors are the key PGE2 receptors engaged in follicle growth. 2) Ovarian IL-8 expression and neutrophil infiltration are chiefly mediated via the EP4 receptor. EP2 and EP4 receptor agonists may be candidates for promising reagents that induce follicle maturation in clinical or agricultural fields. This knowledge could provide numerous targets for manipulation of fertility.     

Identification of prostaglandin E2 receptors mediating perinatal masculinization of adult sex behavior and neuroanatomical correlates

Prostaglandin E2 (PGE2) mediates the organization of male rat sexual behavior and medial preoptic area (MPOA) neuroanatomy during a sensitive perinatal window. PGE2 is up-regulated in response to estradiol, and initiates a two-fold increase in dendritic spines densities on neurons. All the four receptors for PGE2 and EP1-4 are present in developing POA, a critical region controlling male sexual behavior. Previous studies explored that EP receptors are involved in PGE2-induction of neonatal levels of spinophilin protein, a surrogate marker for dendritic spine formation, but did not assess behavioral masculinization. Here, we used two approaches, suppression of EP receptor expression with antisense oligonucleotides and activation of EP receptors with selective agonists, to test which receptors are necessary and sufficient, respectively, for the effects of PGE2 on behavior and neuronal morphology. In female rats, neonatal treatment with antisense oligonucleotides against EP2 or EP4 but not EP1 or EP3 completely prevented the expression of adult behavior organized by PGE2 exposure. The effects of ONO-DI-004, ONO-AE-259-01, ONO-AE-248, and ONO-AE1-329 (EP1-4 agonists respectively) were equivalent to PGE2 treatment, which suggests activating any EP receptor neonatally suffices in masculinizing sex behavior. When given alone, not all EP agonists increased neonatal POA spinophilin levels; yet giving each agonist neonatally increased adult levels. Moreover, adult spinophilin levels significantly correlated with two measures of male sexual behavior. The body of evidence suggests that EP2 and EP4 are both necessary and sufficient for PGE2-induced masculinization of sex behavior, whereas EP1 and EP3 provide redundant roles.     

Prostaglandin E2-EP4 receptor promotes endothelial cell migration via ERK activation and angiogenesis in vivo

Prostaglandin E2 (PGE(2)), a major product of cyclooxygenase, exerts its functions by binding to four G protein-coupled receptors (EP1-4) and has been implicated in modulating angiogenesis. The present study examined the role of the EP4 receptor in regulating endothelial cell proliferation, migration, and tubulogenesis. Primary pulmonary microvascular endothelial cells were isolated from EP4(flox/flox) mice and were rendered null for the EP4 receptor with adenoCre virus. Whereas treatment with PGE(2) or the EP4 selective agonists PGE(1)-OH and ONO-AE1-329 induced migration, tubulogenesis, ERK activation and cAMP production in control adenovirus-transduced endothelial EP4(flox/flox) cells, no effects were seen in adenoCre-transduced EP4(flox/flox) cells. The EP4 agonist-induced endothelial cell migration was inhibited by ERK, but not PKA inhibitors, defining a functional link between PGE(2)-induced endothelial cell migration and EP4-mediated ERK signaling. Finally, PGE(2), as well as PGE(1)-OH and ONO-AE1-329, also promoted angiogenesis in an in vivo sponge assay providing evidence that the EP4 receptor mediates de novo vascularization in vivo.