Prostaglandin E2 Receptor EP4 Research Articles

Prostaglandin EP2 and EP4 receptors modulate expression of the chemokine CCL2 (MCP-1) in response to LPS-induced renal glomerular inflammation

The pro-inflammatory chemokine CCL2 [chemokine (Cys-Cys motif) ligand 2; also known as MCP-1 (monocyte chemotactic protein-1)] is up-regulated in the glomerular compartment during the early phase of LPS (lipopolysaccharide)-induced nephritis. This up-regulation also occurs in cultured MCs (mesangial cells) and is more pronounced in MCs lacking the PGE2 (prostaglandin E2) receptor EP2 or in MCs treated with a prostaglandin EP4 receptor antagonist. To examine a possible feedback mechanism of EP receptor stimulation on CCL2 expression, we used an in vitro model of MCs with down-regulated EP receptor expression. Selectively overexpressing the various EP receptors in these cells then allows the effects on the LPS-induced CCL2 expression to be examined. Cells were stimulated with LPS and CCL2 gene expression was examined and compared with LPS-stimulated, mock-transfected PTGS2 [prostaglandin-endoperoxide synthase 2, also known as COX-2 (cyclo-oxygenase-2)]-positive cells. Overexpression of EP1, as well as EP3, had no effect on LPS-induced Ccl2 mRNA expression. In contrast, overexpression of EP2, as well as EP4, significantly decreased LPS-induced CCL2 expression. These results support the hypothesis that PTGS2-derived prostaglandins, when strongly induced, counter-balance inflammatory processes through the EP2 and EP4 receptors in MCs.

Prostaglandin E2 Receptor EP2, EP3, and EP4 Agonists Inhibit Antigen-Induced Mucus Hypersecretion in the Nasal Epithelium of Sensitized Rats

Prostaglandin (PG) E2 is a potential anti-inflammatory mediator that attenuates airway inflammation. To elucidate the functions of the PGE2 receptors (EP1, EP2, EP3, and EP4) in allergic inflammation, we examined the in vivo effects of EP agonists on mucus hypersecretion and eosinophil infiltration in rat nasal epithelium. We induced hypertrophic and metaplastic changes in goblet cells in nasal epithelium of ovalbumin-sensitized rats by intranasal challenge with ovalbumin. The effects of subcutaneous injections of EP agonists on mucus production and eosinophil infiltration were examined. The EP4 agonist (1 to 100 microg/kg) dose-dependently inhibited ovalbumin-induced mucus production. The EP2 and EP3 agonists (100 microg/kg) also significantly inhibited mucus production. The EP3 agonist inhibited antigen-induced eosinophil infiltration, whereas the EP1 agonist showed no effect. This suppression of mucus production by the EP4 agonist was only effective when the EP4 agonist was given in the effector phase; administration in the induction phase resulted in no effect. These results indicate that PGE2 acts as an anti-inflammatory mediator via the EP receptors of airways in allergic inflammation. Selective EP agonists may provide a new therapeutic strategy for airway mucus hypersecretion.

Selective Inhibition of Prostaglandin E2 Receptors EP2 and EP4 Induces Apoptosis of Human Endometriotic Cells through Suppression of ERK1/2, AKT, NFκB and β-CATENIN Pathways and Activation of Intrinsic Apoptotic Mechanisms

Endometriosis is a benign chronic gynecological disease of reproductive age women characterized by the presence of functional endometrial tissues outside the uterine cavity. It is an estrogen-dependent disease. Current treatment modalities to inhibit biosynthesis and actions of estrogen compromise menstruation, pregnancy, reproductive health of women, and fail to prevent reoccurrence of disease. There is a critical need to identify new specific signaling modules for non-estrogen targeted therapies for endometriosis. In our previous study, we reported that selective inhibition of cyclooxygenase-2 prevented survival, migration and invasion of human endometriotic epithelial and stromal cells which was due to decreased PGE2 production. In this study, we determined mechanisms through which prostaglandin E2 (PGE2) promoted survival of human endometriotic cells. Results of the present study indicate that: (i) PGE2 promotes survival of human endometriotic cells through EP2 and EP4 receptors by activating ERK1/2, AKT, NFκB, and β-catenin signaling pathways; (ii) selective inhibition of EP2 and EP4 suppresses these cell survival pathways and augments interactions between proapoptotic proteins (Bax and Bad) and antiapoptotic proteins (Bcl-2/Bcl-XL), facilitates the release of cytochrome C and thus activates caspase-3/PARP-mediated intrinsic apoptotic pathways; and (iii) these PGE2 signaling components are more abundantly expressed in ectopic endometriosis tissues compared to eutopic endometrial tissues during the menstrual cycle in women. These novel findings may provide an important molecular framework for further evaluation of selective inhibition of EP2 and EP4 as potential therapy, including non-estrogen target, to expand the spectrum of currently available treatment options for endometriosis in women.

Extracellular Ca2+ regulates the stimulus-elicited ATP release from urothelium

Accumulating evidence shows that the epithelial cells in urinary bladder (urothelium) serve as a sensory organ in micturition and/or in nociception pathway by releasing ATP in response to mechanical and/or chemical stimuli. Here, we compared the effects of capsaicin, acetylcholine, and prostaglandin E2 receptor EP1 agonist (ONO-DI-004) on the urothelial ATP release in primary cultured mouse urothelial cells in low Ca2+ medium. All of these chemicals induced a gradual ATP release from urothelium, implying that the downstream Ca2+ release from endoplasmic reticulum could trigger the ATP release. Consistent with this suggestion, blockade of inositol 1,4,5-triphosphate receptor reduced the distention-induced ATP release from urothelial tissues. The distention-induced ATP release was not affected by tetrodotoxin. However, an increase in extracellular Ca2+ diminished both chemical- and distention-induced ATP release from urothelium. Thus raising the extracellular Ca2+ concentration was found to inhibit stimulation-evoked ATP urothelial release.

Selective Inhibition of Prostaglandin E2 Receptors EP2 and EP4 Induces Apoptosis of Human Endometriotic Cells through Suppression of ERK1/2, AKT, NFκB, and β-Catenin Pathways and Activation of Intrinsic Apoptotic Mechanisms

Endometriosis is a benign chronic gynecological disease of reproductive-age women characterized by the presence of functional endometrial tissues outside the uterine cavity. It is an estrogen-dependent disease. Current treatment modalities to inhibit biosynthesis and actions of estrogen compromise menstruation, pregnancy, and the reproductive health of women and fail to prevent reoccurrence of disease. There is a critical need to identify new specific signaling modules for non-estrogen-targeted therapies for endometriosis. In our previous study, we reported that selective inhibition of cyclooxygenase-2 prevented survival, migration, and invasion of human endometriotic epithelial and stromal cells, which was due to decreased prostaglandin E(2) (PGE(2)) production. In this study, we determined mechanisms through which PGE(2) promoted survival of human endometriotic cells. Results of the present study indicate that 1) PGE(2) promotes survival of human endometriotic cells through EP2 and EP4 receptors by activating ERK1/2, AKT, nuclear factor-kappaB, and beta-catenin signaling pathways; 2) selective inhibition of EP2 and EP4 suppresses these cell survival pathways and augments interactions between proapoptotic proteins (Bax and Bad) and antiapoptotic proteins (Bcl-2/Bcl-XL), facilitates the release of cytochrome c, and thus activates caspase-3/poly (ADP-ribose) polymerase-mediated intrinsic apoptotic pathways; and 3) these PGE(2) signaling components are more abundantly expressed in ectopic endometriosis tissues compared with eutopic endometrial tissues during the menstrual cycle in women. These novel findings may provide an important molecular framework for further evaluation of selective inhibition of EP2 and EP4 as potential therapy, including nonestrogen target, to expand the spectrum of currently available treatment options for endometriosis in women.

Human sebocytes express prostaglandin E2 receptors EP2 and EP4 but treatment with prostaglandin E2 does not affect testosterone production

Prostaglandins (PG) play an important role in cutaneous homeostasis. Among other skin cells, human sebocytes express cyclooxygenases and can produce PGE(2). Various prostanoid receptors have been demonstrated in epidermis and hair follicles, while limited data are available regarding their expression in sebaceous glands. In addition, the interaction between PGE(2) and androgenesis remains largely unclear. To examine the expression of PGE(2) receptor (EP) and PGF(2alpha) receptor (FP) in human sebocytes and the influence of PGE(2) or PGF(2alpha) on testosterone production. A reverse transcription-polymerase chain reaction study was used to detect the expression of EP subtypes and FP. A testosterone radioimmunoassay was used to measure the amount of testosterone in the supernatant of cultured SZ95 sebocytes treated with PGE(2) or PGF(2alpha) alone or in the presence of various androgen precursor substrates. SZ95 sebocytes expressed mainly EP2 and EP4 but not EP3 or FP. Testosterone production was not induced by PGE(2) or PGF(2alpha), alone or in the presence of cholesterol. PGE(2) did not affect androgenesis in cultured sebocytes. The expression patterns of prostanoid receptors differ between sebocytes, hair follicles and epidermis. The effects of PGE(2) and PGF(2alpha) on the proliferation, lipogenesis and inflammation of sebocytes appear not to be associated with androgenesis.

Activation of prostaglandin E2 EP1 receptor increases arteriolar tone and blood pressure in mice with type 2 diabetes

Type 2 diabetes mellitus is frequently associated with hypertension, but the underlying mechanisms are not completely understood. We tested the hypothesis that activation of type 1 prostaglandin E(2) (PGE(2)) receptor (EP1) increases skeletal muscle arteriolar tone and blood pressure in mice with type 2 diabetes. In 12-week-old, male db/db mice (with homozygote mutation in leptin receptor), systolic blood pressure was significantly elevated, compared with control heterozygotes. Isolated, pressurized gracilis muscle arterioles ( approximately 90 microm) of db/db mice exhibited an enhanced pressure- and angiotensin II (0.1-10 nM)-induced tone, which was reduced by the selective EP1 receptor antagonist, AH6809 (10 microM), to the level observed in arterioles of control mice. Exogenous application of PGE(2) (10 pM-100 nM) or the selective agonist of the EP1 receptor, 17-phenyl-trinor-PGE(2) (10 pM-100 nM), elicited arteriolar constrictions that were significantly enhanced in db/db mice (max: 31 +/- 4 and 29 +/- 5%), compared with controls (max: 20 +/- 2 and 14 +/- 3%, respectively). In the aorta of db/db mice, an increased protein expression of EP1, but not EP4, receptor was also detected by western immunoblotting. Moreover, we found that oral administration of the EP1 receptor antagonist, AH6809 (10 mg/kg/day, for 4 days), significantly reduced the systolic blood pressure in db/db, but not in control mice. Activation of EP1 receptors increases arteriolar tone, which could contribute to the development of hypertension in the db/db mice.

Expression of prostaglandin E2 receptors in oral squamous cell carcinomas and growth inhibitory effects of an EP3 selective antagonist, ONO-AE3-240

Prostaglandin E2 (PGE2) can stimulate tumor progression by both direct and indirect mechanisms. However, its influence on cell proliferation is still unclear. The present study characterized expression of subtypes of PGE2 receptors in oral squamous cell carcinomas, while also investigating the effects of EP3 and EP4 selective antagonists on oral carcinoma cell lines. EP1, EP2, EP3 and EP4 receptor mRNAs were detected in 4, 5, 10 and 10 of 11 surgical specimens respectively. Application of an EP3 antagonist (ONO-AE3-240) strongly inhibited cell growth in COX-2 and PGE2 high expression cells (Ca9-22) but not in COX-2 and PGE2 low expression cells (HSC4). The antagonist also reduced the production of endogenous PGE2 and induced G0/G1 phase cell arrest. Addition of exogenous PGE2 only partly abrogated the growth inhibition, indicating that the anti-proliferative effect via EP3 receptor signaling was not only due to PGE2-dependent but also PGE2-independent mechanisms. In contrast, an EP4 antagonist (ONO-AE3-208) did not inhibit growth in either of the cancer cell lines. In summary, PGE2 receptor EP3 signaling probably contributes to development of oral carcinomas and use of EP3 antagonist may be a new therapeutic strategy for head and neck cancer.

Impaired cognition, sensorimotor gating, and hippocampal long-term depression in mice lacking the prostaglandin E2 EP2 receptor

Cyclooxygenase-2 (COX-2) is a neuronal immediate early gene that is regulated by N-methyl d aspartate (NMDA) receptor activity. COX-2 enzymatic activity catalyzes the first committed step in prostaglandin synthesis. Recent studies demonstrate an emerging role for the downstream PGE2 EP2 receptor in diverse models of activity-dependent synaptic plasticity and a significant function in models of neurological disease including cerebral ischemia, Familial Alzheimer's disease, and Familial amyotrophic lateral sclerosis. Little is known, however, about the normal function of the EP2 receptor in behavior and cognition. Here we report that deletion of the EP2 receptor leads to significant cognitive deficits in standard tests of fear and social memory. EP2−/− mice also demonstrated impaired prepulse inhibition (PPI) and heightened anxiety, but normal startle reactivity, exploratory behavior, and spatial reference memory. This complex behavioral phenotype of EP2−/− mice was associated with a deficit in long-term depression (LTD) in hippocampus. Our findings suggest that PGE2 signaling via the EP2 receptors plays an important role in cognitive and emotional behaviors that recapitulate some aspects of human psychopathology related to schizophrenia.

Prostaglandin E2 Enhances B-Type Natriuretic Peptide Receptor Expression in Calvarial Osteoblasts through EP1 Subtype of Prostaglandin E2 Receptor

The B-type natriuretic peptide recepto r( NPR-B) is a specific receptor for th eC -type natriuretic peptide (CNP) and the binding of the peptide to NPR-B stimulates the bone formation by osteoblasts. However, the mechanism behind the regulation of NPR-B expression in osteoblasts remains unknown. In this study, we examined the role of prostaglandin E2 (PGE2 )t hrough the PGE2 receptor subtypes, EP1, EP2, EP3 and EP4, in the regulation of NPR-B expression using calvarial osteoblasts from rats of various ages. Reverse Transcription-PCR (RT-PCR) and Western blotting analyses revealed that PGE2 or 17-phenyl-ω-trinor PGE2 ,a n EP1 agonist, increased the expression of NPR-B of calvarial osteoblasts from 25-week-old rats in a time- and dose-dependent manner. The PGE2 -a nd EP1 agonist-induced increase in NPR-B expression was blocked by treating with SC19220, an EP1 antagonist. By contrast, agonists for EP2, EP3, and EP4 failed to affect the NPR-B expression. The basal mRNA level of NPR-B and EP1 continuously decreased with the age of cell donors between 10 to 60 weeks and remained constant over 60 weeks. The degree of EP1 agonist-induced increase in NPR-B mRNA level gradually decreased with age of cell donors between 10 to 60 weeks, and no significant effect of EP1 agonist on the NPR-B mRNA level was observed over 60 weeks. From these results, we concluded that PGE2 acts as a regulator of NPR-B expression through the EP1 receptor in osteoblasts and age-related decrease i nE P1 expression causes a decrease in NPR-B expression.

Prognostic Role of PGE2 Receptor EP2 in Esophageal Squamous Cell Carcinoma

Prostaglandin E2 (PGE2), a major cyclooxygenase-2 (COX-2) product, has been shown to affect numerous tumorigenic processes. PGE2 acts through G-protein-coupled receptors designated as EPs. Recently it has been documented that PGE2 promotes colon cancer cell growth via EP2. However, the expression and the prognostic role of EP2 in esophageal squamous cell carcinoma (ESCC) remained unknown. From January 1995 to January 2001, tissue samples from 226 patients with ESCC who underwent esophagectomies at our institutions were collected and made into tissue core arrays for study. EP2 expression was examined by immunohistochemical staining and confirmed by Western blot. The clinicopathologic data were then analyzed. EP2 overexpression was observed in 43.4% (98/226) of ESCC. Overexpression of EP2 correlated positively with depth of tumor invasion (T status) (P = 0.016) and was associated with worse overall survival (P = 0.047). In patients without regional or distant lymph node metastasis (N0 or M0), EP2 overexpression was associated with worse overall survival (P = 0.033 and P = 0.003, respectively). Using Cox regression analysis, T status, N status, and M status were the independent factors of overall survival, but EP2 expression was not. However, when focusing on patients with T1-3N0M0 status, EP2 expression became an independent factor of overall survival (P = 0.048). Our results show that EP2 overexpression was associated with worse prognosis, and correlated positively with T status in ESCC. Meanwhile, among those patients at earlier stages, EP2 overexpression significantly disclosed patients at high risks for poor prognosis.

Extracorporeal Shock Wave-Mediated Changes in Proliferation, Differentiation, and Gene Expression of Human Osteoblasts

The goal of this study was to determine whether cell proliferation, differentiation, and gene expression of primary human osteoblasts (hOB) are influenced by shock wave application (SWA). Osteoblast cultures were isolated from cancellous bone fragments and treated with 500 impulses of energy flux densities of 0.06 mJ/mm, 0.18 mJ/mm, 0.36 mJ/mm, and 0.50 mJ/mm. Twenty-four hours and 96 hours after SWA cell proliferation, alkaline phosphatase activity, and mineralization were analyzed. The global gene expression profiling was determined 96 hours after SWA employing Affymetrix HG-U133A microarrays. After 24 hours, hOB showed a dose-dependent increase in cell proliferation from 68.7% (at 0.06 mJ/mm, p = 0.002) up to 81.6% (at 0.5 mJ/mm, p = 0.001), which also persisted after 96 hours. Numbers of alkaline phosphatase-positive hOB increased after SWA treatment with peak levels of response between 0.18 mJ/mm and 0.5 mJ/mm after 24 hours. Mineralization was significantly higher in all groups compared with controls. Microarray analyses revealed SWA-induced differential expression of 94 genes involved in physiologic processes, cell homeostasis, and bone formation. Most intriguing was the up-regulation of multiple genes involved in skeletal development and osteoblast differentiation (e.g., PTHrP, prostaglandin E2-receptor EP3, BMP-2 inducible kinase, chordin, cartilage oligomeric matrix protein, matrillin). We showed that shock waves have direct dose-dependent stimulatory effects on proliferation and differentiation of osteoblasts from normal human cancellous bone. We demonstrated that several genes critical for osteoblast differentiation and function are regulated after SWA. Overall, data presented herein will aid further understanding of the osteogenic effect of shock waves and, in addition, will enhance current knowledge of the SWA-mediated gene expression.

Prostaglandin E2 (PGE2) Treatment Rapidly Increases the Fraction of Non-Dividing Hematopoietic Stem Cells (HSCs)

HSCs are pluripotent cells responsible for the establishment and renewal of the entire hematopoietic system. Our group and others have established that osteoblastic cells in the bone marrow microenvironment regulate HSC cell fate decisions. Specifically, Parathyroid hormone (PTH) expands HSCs by activating osteoblasts in the HSC niche. However, the molecular mechanisms for this increase are unknown. PTH increases local production of prostaglandin E2 (PGE2) in osteoblasts by stimulating cyclo-oxygenase 2 (Cox-2). We also recently found that treatment of osteoblastic MC3T3 cells with PTH (10−7 M) rapidly induces PGE2 Synthase expression. Therefore, we hypothesized that PGE2 may act as a mediator of the PTH effect on HSCs. We have shown that in vivo PGE2 treatment caused a 2.75-fold increase in lineage− Sca-1+ c-kit+ (LSK) cells within the bone marrow compared with vehicle treated mice (p=0.0061, n=8/group). Bone marrow mononuclear cells (BMMC) from mice treated with PGE2 also demonstrated superior lymphomyeloid reconstitution in competitive repopulation analyses, suggesting that HSCs are being expanded or modulated to more efficiently reconstitute the hematopoietic system in the recipients. It is known that HSCs that reside in the G0 phase of the cell cycle have increased ability to reconstitute myeloablated recipient mice. Since PGE2 treatment resulted in superior reconstitution, we hypothesized that PGE2 may increase the percentage of HSCs residing in G0. To test this hypothesis, we treated BMMC from male C57b/6 mice with 10−6 M PGE2 or vehicle for 90 minutes. The percentage of cells in G0 vs. G1 was determined by flow-cytometric analysis using the RNA and DNA dyes, Pyronin-Y and Hoechst 33342 respectively. As we predicted, PGE2 treatment increased the percentage of wild-type LSK cells in G0 1.85 fold over vehicle-treated LSK cells (23.63% in vehicle-treated, n=4 vs. 43.7% in PGE2-treated, n=6). Since the PTH-dependent increase in HSCs is Protein Kinase A (PKA) mediated and the PGE2 receptors EP2 and EP4 signal via PKA, we assayed the effect of PGE2 on the percentage of cells in G0 in mice lacking the EP2 receptor (EP2−/− mice). Interestingly, there was no enrichment for HSC in G0 when BMMC from EP2−/− mice were treated with PGE2 (55.25% in vehicle-treated, n=4 vs. 56.06% in PGE2-treated, n=5). These findings suggest that PGE2-dependent regulation of HSC activity may involve increasing the percentage of HSCs that reside in G0 by activation of EP2, thereby augmenting their ability to reconstitute the hematopoietic system of a myeloablated recipient. 5-bromo-2-deoxyuridine (BrdU) incorporation was also used to investigate the effect of PGE2 on cell cycling of HSCs. Male 6–8 week old C57b/6 mice were injected intraperitoneally with 1 mg BrdU and PGE2 (6 mg/kg) or vehicle. After 30, 60, 90 or 120 minutes, mice were sacrificed and BMMC were subjected to flow cytometric analysis for incorporation of BrdU and DNA content in HSCs. As expected for the highly quiescent HSC population, only a small fraction of HSCs incorporated BrdU. After 30 and 60 minutes of treatment, there was no difference in the percentage of cells that incorporated BrdU between vehicle and PGE2-treated mice. However, at the 90 and 120 minute time points, there were significantly less HSCs cycling in the bone marrow from the PGE2 treated mice (12.1% vs. 5.3% at 90 min, n=2 per group; 11.1% vs. 1.8% at 120 min, n=5 per group, p=0.0060), suggesting that fewer PGE2-treated cells were synthesizing DNA. Taken together, the increase in the percentage of HSCs in G0 and the decrease in cycling HSCs after PGE2 treatment indicate that PGE2 could improve engraftment and reconstitution of the hematopoietic system by enriching for HSCs in G0. These results suggest that PGE2 may exert its beneficial effect on bone marrow reconstitution by altering cell cycle dynamics in HSCs. Identification of the molecular events mediating this novel PGE2 action on HSC could provide additional targets for HSC manipulation in clinical situations requiring rapid and efficient bone marrow reconstitution, such as recovery from iatrogenic or pathologic myeloablative injury.

The prostaglandin E2 EP2 receptor accelerates disease progression and inflammation in a model of amyotrophic lateral sclerosis

Inflammation has emerged as an important factor in disease progression in human and transgenic models of amyotrophic lateral sclerosis (ALS). Recent studies demonstrate that the prostaglandin E(2) EP2 receptor is a major regulator of inflammatory oxidative injury in innate immunity. We tested whether EP2 signaling participated in disease pathogenesis in the G93A superoxide dismutase (SOD) model of familial ALS. We examined the phenotype of G93A SOD mice lacking the EP2 receptor and performed immunocytochemistry, quantitative reverse transcriptase polymerase chain reaction, and Western analyses to determine the mechanism of EP2 toxicity in this model. EP2 receptor is significantly induced in G93A SOD mice in astrocytes and microglia in parallel with increases in expression of proinflammatory enzymes and lipid peroxidation. In human ALS, EP2 receptor immunoreactivity was upregulated in astrocytes in ventral spinal cord. In aging G93A SOD mice, genetic deletion of the prostaglandin E(2)EP2 receptor improved motor strength and extended survival. Deletion of the EP2 receptor in G93A SOD mice resulted in significant reductions in levels of proinflammatory effectors, including cyclooxygenase-1, cyclooxygenase-2, inducible nitric oxide synthase, and components of the NADPH oxidase complex. In alternate models of inflammation, including the lipopolysaccharide model of innate immunity and the APPSwe-PS1DeltaE9 model of amyloidosis, deletion of EP2 also reduced expression of proinflammatory genes. These data suggest that prostaglandin E(2) signaling via the EP2 receptor functions in the mutant SOD model and more broadly in inflammatory neurodegeneration to regulate expression of a cassette of proinflammatory genes. Inhibition of EP2 signaling may represent a novel strategy to downregulate the inflammatory response in neurodegenerative disease.

Induction of peritoneal endometriosis in nude mice with use of human immortalized endometriosis epithelial and stromal cells: a potential experimental tool to study molecular pathogenesis of endometriosis in humans

To determine whether a mixed population of immortalized human endometriosis epithelial and stromal cells is able to induce peritoneal endometriosis in nude mice. Prospective experimental study. Human immortalized endometriosis epithelial and stromal cells were xenografted into ovariectomized nude mice. Macroscopically, the number of induced endometriosis-like lesions and their color were determined. Microscopically, histomorphology of endometriosis glands and their structure were analyzed, and comparisons were made with tissue from spontaneous endometriosis in women. College of Veterinary Medicine and Biomedical Sciences, Texas A&M University. Seven ovariectomized nude mice. Minimal invasive procedures were performed to administer estrogen pellets and transplant immortalized human endometriosis epithelial and stromal cells into nude mice. Peritoneal endometriosis-like lesions induced in nude mice were characterized and compared with spontaneous peritoneal endometriosis in women. Xenografts of human immortalized endometriosis epithelial and stromal cells into the peritoneal cavity of the recipient nude mice are able to proliferate, attach, invade, reorganize, and establish peritoneal endometriosis. Endometriosis glands at different stages of growth were present in induced endometriosis-like lesions. Proliferating cell nuclear antigen, metalloproteinase 2, estrogen receptor-alpha, cyclooxygenase-2, and prostaglandin E(2) receptors EP2 and EP4 proteins were expressed in both endometriosis glandular epithelial and stromal cells of the induced endometriosis-like lesions. This xenograft model could be used as a potential experimental tool to understand the molecular and cellular aspects of the pathogenesis of endometriosis in humans.

The Neuroprotective Effect of Prostaglandin E2 EP1 Receptor Inhibition has a Wide Therapeutic Window, is Sustained in Time and is not Sexually Dimorphic

We investigated the preclinical characteristics of the neuroprotective effect of the prostaglandin E2 type 1 receptor (EP1) antagonist SC51089 in models of focal cerebral ischemia produced by occlusion of the mouse middle cerebral artery (MCA). We found that systemic administration of SC51089 (5 to 20 microg/kg; i.p.) reduces the brain injury produced by transient (-50%+/-8%; n=12; P<0.05) or permanent (-39%+/-7%; n=12; P<0.05) MCA occlusion. SC51089 was effective even when administered up to 12 h after ischemia. The protective effect was observed both in male and female mice and was sustained for at least 2 weeks after induction of ischemia. The reduction in injury volume was associated with an improvement in neurological function assessed by the Bederson deficit score, the hanging wire test and the corner test. The data provide proof of principle that EP1 receptor inhibition is a potentially valuable strategy for neuroprotection that deserves further preclinical investigation for therapeutic application in human stroke.

Lipopolysaccharide toxicity to hippocampal intermediate neuronal progenitors is diminished in mice lacking prostaglandin E2 receptor subtype EP1

Innate immune activation is implicated in Alzheimer's and other neurodegenerative diseases, and is mediated in the central nervous system primarily by microglia via prostaglandin E2 (PGE2) signaling. Lipopolysaccharide (LPS) activates innate immunity, and leads to a microglia‐dependent reduction in hippocampal neurogenesis. We examined the effects of unilateral intraventricular LPS injection on hippocampal neuronal progenitor cells (hNPC) in wt mice and in mice lacking PGE2 receptor subtypes EP1 and EP2. Mice received post‐operative bromodeoxyuridine (BrdU) injections and were euthanized 72 hrs post‐surgery. Total Tbr2+ hNPC per section were significantly reduced in wt (−51%) LPS mice (79±15) compared with wt controls (164±28), and in EP2−/− (−32%) mice (97±9) compared with EP2−/− controls (142±21). Surprisingly, LPS EP1−/− mice (187±28) showed a significant increase (+47%) compared with EP1−/− controls (127±16). Total BrdU+ cells increased in LPS wt (+40%) and EP1−/− (+90%), while EP2−/− mice showed no significant difference (+2.1%). There were no significant differences in Tbr2+ hNPC between LPS and vehicle in wt and EP knockouts after 24 hours, between control wt and EP knockouts, or between ipsilateral and contralateral hippocampi. These results implicate PGE2 receptor EP1 activation in the regulation of hNPC cell proliferation and/or survival in response to innate immune activation.

PGE2 receptor EP1 renders dopaminergic neurons selectively vulnerable to low‐level oxidative stress and direct PGE2 neurotoxicity

Oxidative stress and increased cyclooxygenase-2 (COX-2) activity are both implicated in the loss of dopaminergic neurons from the substantia nigra (SN) in idiopathic Parkinson's disease (PD). Prostaglandin E(2) (PGE(2)) is one of the key products of COX-2 activity and PGE(2) production is increased in PD. However, little is known about its role in the selective death of dopaminergic neurons. Previously, we showed that oxidative stress evoked by low concentrations of 6-hydroxydopamine (6-OHDA) was selective for dopaminergic neurons in culture and fully dependent on COX-2 activity. We postulated that this loss was mediated by PGE(2) acting through its receptors, EP1, EP2, EP3, and EP4. Using double-label immunohistochemistry for specific EP receptors and tyrosine hydroxylase (TH), we identified EP1 and EP2 receptors on dopaminergic neurons in rat SN. EP2 receptors were also found in non-dopaminergic neurons of this nucleus, as were EP3 receptors, whereas the EP4 receptor was absent. PGE(2), 16-phenyl tetranor PGE(2) (a stable synthetic analogue), and 17-phenyl trinor PGE(2) (an EP1 receptor-selective agonist) were significantly toxic to dopaminergic cells at nanomolar concentrations; EP2- and EP3-selective agonists were not. We challenged dopaminergic neurons in embryonic rat mesencephalic primary neuronal cultures and tested whether these receptors mediate selective 6-OHDA toxicity. The nonselective EP1-3 receptor antagonist AH-6809 and two selective EP1 antagonists, SC-19220 and SC-51089, completely prevented the 40%-50% loss of dopaminergic neurons caused by exposure to 5 muM 6-OHDA. Together, these results strongly implicate PGE(2) activation of EP1 receptors as a mediator of selective toxicity in this model of dopaminergic cell loss.

CHOP Transcription Factor Mediates IL-8 Signaling in Cystic Fibrosis Bronchial Epithelial Cells

Interleukin (IL)-8 is a potent neutrophil chemoattractant that drives the inflammatory response in cystic fibrosis (CF). Traditional approaches to the pathophysiology of this inflammation have focused on targeting NF-kappaB-dependent signaling and therapy with glucocorticoids. We test the hypothesis that an alternative pathway, independent of NF-kappaB, operates through prostaglandin E2 (PGE-2) receptor EP-2 and stimulates IL-8 chemokine secretion. Using CF bronchial epithelial cells (IB3-1) in vitro, exogenous PGE-2 induces IL-8 release in a dose-dependent manner. These events are associated with elevation in the EP-2 receptors. Inhibition of cyclooxygenase (Cox)-2 with NS-398 was associated with reductions in Cox-2 (2-fold) and IL-6 (1.3-fold) mRNA transcripts, and in IL-8 and PGE-2 chemokine secretion. The inhibition of Cox-2 signaling led to down-regulation of the downstream C/EBP homologous protein (CHOP) transcription factor, resulting in a decrease in IL-8 activation. We confirmed the regulation of IL-8 promoter by CHOP in CF cells using the IL-8 reporter assay. We conclude that PGE-2 stimulates IL-8 production through the CHOP transcription factor in CF cells.

Prostaglandin E2 receptors EP1 and EP4 are up-regulated in rabbit chondrocytes by IL-1β, but not by TNFα

Prostaglandin E2 (PGE2) exerts its actions through the binding of the high affinity EP receptors. We wanted to evaluate the regulation of EP1 and EP4, and the expression of cyclooxygenase (COX)-2, main enzyme responsible for PGE2 synthesis in inflammatory situations, in healthy rabbit chondrocytes stimulated with inflammatory mediators locally increased during osteoarthritis. Articular chondrocytes obtained from healthy rabbits were stimulated with interleukin (IL)-1beta (0.1-100 u/ml) or tumour necrosis factor (TNF)alpha (100 ng/ml). Where indicated, cells were preincubated with non-steroidal antiinflammatory drugs (NSAIDs) (10(-6) M) to inhibit PGE2 synthesis. IL-1beta induced a dose and time-dependent increase in EP1, EP4 and COX-2 expression. However, TNFalpha presence did not induce a significant modification in EP1, EP4 or COX-2 gene expression at any time of study. NSAID presence significantly inhibited PGE2 release but did not modify the EP receptors or COX-2 expression induced by IL-1beta. Our results indicate that EP1 and EP4 receptors, and COX-2 are up-regulated in IL-1beta-stimulated chondrocytes, while no significant modifications are observed in TNFalpha-stimulated cells. NSAIDs were unable to modify the expression of these mediators induced by IL-1beta. Therefore, the increase in PGE2 synthesis, induced by IL-1beta, does not seem to mediate the increase in EP receptor expression, in rabbit chondrocytes.