The Epigenetic Legacy of Maternal Protein Restriction: Renal Ptger1 DNA Methylation Changes in Hypertensive Rat Offspring.

Nutrient imbalances during gestation are a risk factor for hypertension in offspring. Although the effects of prenatal nutritional deficiency on the development of hypertension and cardiovascular diseases in adulthood have been extensively documented, its underlying mechanisms remain poorly understood. In this study, we aimed to elucidate the precise role and functional significance of epigenetic modifications in the pathogenesis of hypertension. To this end, we integrated methylome and transcriptome data to identify potential salt-sensitive hypertension genes using the kidneys of stroke-prone spontaneously hypertensive rat (SHRSP) pups exposed to a low-protein diet throughout their fetal life. Maternal protein restriction during gestation led to a positive correlation between DNA hypermethylation of the renal prostaglandin E receptor 1 (Ptger1) CpG island and high mRNA expression of Ptger1 in offspring, which is consistently conserved. Furthermore, post-weaning low-protein or high-protein diets modified the Ptger1 DNA hypermethylation caused by fetal malnutrition. Here, we show that this epigenetic variation in Ptger1 is linked to disease susceptibility established during fetal stages and could be reprogrammed by manipulating the postnatal diet. Thus, our findings clarify the developmental origins connecting the maternal nutritional environment and potential epigenetic biomarkers for offspring hypertension. These findings shed light on hypertension prevention and prospective therapeutic strategies.

EP1 receptor: Devil in emperors coat.

EP1 receptor belongs to prostanoid receptors and is activated by prostaglandin E2. The receptor performs contrasting functions in central nervous system (CNS) and other tissues. Although the receptor is neurotoxic and proapoptotic in CNS, it has also been reported to act in an antiapoptotic manner by modulating cell survival, proliferation, invasion, and migration in different types of cancers. The receptor mediates its neurotoxic effects by increasing cytosolic Ca2+ levels, leading to the activation of its downstream target, protein kinase C, in different neurological disorders including Alzheimer's disease, Parkinson's disease, stroke, amyotrophic lateral sclerosis, and epilepsy. Antagonists ONO-8713, SC51089, and SC51322 against EP1 receptor ameliorate the neurotoxic effect by attenuating the neuroinflammation. The receptor also shows increased expression in different types of cancers and has been found to activate different signaling pathways, which lead to the development, progression, and metastasis of different cancers. The receptor stimulates the cell survival pathway by phosphorylating the AKT and PTEN (phosphatase and tensin homolog deleted on chromosome 10) signaling pathways. Although there are limited studies about this receptor and not a single clinical trial has been targeting the EP1 receptor for different neurological disorders or cancer, the receptor is appearing as a potential candidate for therapeutic targets. The aim of this article is to review the recent progress in understanding the pathogenic roles of EP1 receptors in different pathological conditions.

Prostaglandin E2 receptor EP1 (PGE2/EP1) deletion promotes glomerular podocyte and endothelial cell injury in hypertensive TTRhRen mice.

Prostaglandin E2 receptor EP1 (PGE2/EP1) promotes diabetic renal injury, and EP1 receptor deletion improves hyperfiltration, albuminuria, and fibrosis. The role of EP1 receptors in hypertensive kidney disease (HKD) remains controversial. We examined the contribution of EP1 receptors to HKD. EP1 null (EP1-/-) mice were bred with hypertensive TTRhRen mice (Htn) to evaluate kidney function and injury at 24 weeks. EP1 deletion had no effect on elevation of systolic blood pressure in Htn mice (HtnEP1-/-) but resulted in pronounced albuminuria and reduced FITC-inulin clearance, compared with Htn or wild-type (WT) mice. Ultrastructural injury to podocytes and glomerular endothelium was prominent in HtnEP1-/- mice; including widened subendothelial space, subendothelial lucent zones and focal lifting of endothelium from basement membrane, with focal subendothelial cell debris. Cortex COX2 mRNA was increased by EP1 deletion. Glomerular EP3 mRNA was reduced by EP1 deletion, and EP4 by Htn and EP1 deletion. In WT mice, PGE2 increased chloride reabsorption via EP1 in isolated perfused thick ascending limb (TAL), but PGE2 or EP1 deletion did not affect vasopressin-mediated chloride reabsorption. In WT and Htn mouse inner medullary collecting duct (IMCD), PGE2 inhibited vasopressin-water transport, but not in EP1-/- or HtnEP1-/- mice. Overall, EP1 mediated TAL and IMCD transport in response to PGE2 is unaltered in Htn, and EP1 is protective in HKD.

Inhibition of the Prostaglandin EP-1 Receptor in Periosteum Progenitor Cells Enhances Osteoblast Differentiation and Fracture Repair.

Fracture healing is a complex and integrated process that involves mesenchymal progenitor cell (MPC) recruitment, proliferation and differentiation that eventually results in bone regeneration. Prostaglandin E2 (PGE2) is an important regulator of bone metabolism and has an anabolic effect on fracture healing. Prior work from our laboratory showed EP1-/- mice have enhanced fracture healing, stronger cortical bones, higher trabecular bone volume and increased in vivo bone formation. We also showed that bone marrow MSCs from EP1-/- mice exhibit increased osteoblastic differentiation in vitro. In this study we investigate the changes in the periosteal derived MPCs (PDMPCs), which are crucial for fracture repair, upon EP1 deletion. EP1-/- PDMPCs exhibit increased numbers of total (CFU-F) and osteoblastic colonies (CFU-O) as well as enhanced osteoblastic and chondrogenic differentiation. Moreover, we tested the possible therapeutic application of a specific EP1 receptor antagonist to accelerate fracture repair. Our findings showed that EP1 antagonist administration to wild type mice in the early stages of repair similarly resulted in enhanced CFU-F, CFU-O, and osteoblast differentiation in PDMPCs and resulted in enhanced fracture callus formation at 10 days post fracture and increased bone volume and improved biomechanical healing of femur fractures at 21 days post fracture.

Role of the PGE2 receptor subtypes EP1, EP2, and EP3 in repetitive traumatic brain injury.

AIMS: The goal was to explore the signaling pathways of PGE2 to investigate therapeutic effects against secondary injuries following TBI. METHODS: Young (4.9 ± 1.0 months) and aged (20.4 ± 1.4 months) male wild type (WT) C57BL/6 and PGE2 EP1, 2, and 3 receptor knockout mice were selected to either receive sham or repetitive concussive head injury. Immunohistochemistry protocols with Iba1 and GFAP were performed to evaluate microgliosis and astrogliosis in the hippocampus, two critical components of neuroinflammation. Passive avoidance test measured memory function associated with the hippocampus. RESULTS: No differences in hippocampal microgliosis were found when aged EP2-/- and EP3-/- mice were compared with aged WT mice. However, the aged EP1-/- mice had 69.2 ± 7.5% less hippocampal microgliosis in the contralateral hemisphere compared with WT aged mice. Compared with aged EP2-/- and EP3-/- , EP1-/- aged mice had 78.9 ± 5.1% and 74.7 ± 6.2% less hippocampal microgliosis in the contralateral hemisphere. Within the EP1-/- mice, aged mice had 90.7 ± 2.7% and 81.1 ± 5.6% less hippocampal microgliosis compared with EP1-/- young mice in the contralateral and ipsilateral hemispheres, respectively. No differences were noted in all groups for astrogliosis. There was a significant difference in latency time within EP1-/- , EP2-/- , and EP3-/- on day 1 and day 2 in aged and young mice. CONCLUSION: These findings demonstrate that the PGE2 EP receptors may be potential therapeutic targets to treat repetitive concussions and other acute brain injuries.

EP1 receptor is involved in prostaglandin E2-induced osteosarcoma growth.

Recent studies showed that the activation of prostaglandin (PG) receptor EP1 promotes cell migration and invasion in different cancers. The aim of this study was to investigate the role of EP1 in the proliferation of osteosarcoma (OS) cells in vitro and in vivo. EP1 mRNA and protein levels were analyzed by real-time RT-PCR and Western blot, respectively in human OS cell lines MG63, OS732, U-2OS, and 143B compared to human fetal osteoblastic hFOB 1.19 cells. MG63 cells were treated with PGE2, EP1 specific agonist 17-PT-PGE2, 17-PT-PGE2 + EP1 specific antagonist SC51089, or DMSO (control). EP1R-siRNA or a non-silencing irrelevant RNA duplex (negative control) were used for the transfection of MG63 cells, followed by PGE2 treatment. Nude mice carrying MG63 xenografts were treated with SC51089 (2 mg/kg/day). MG63 cells/xenografts were analyzed by MTT assay, TUNEL assay, PKC enzyme activity assay, and Western blot (EP1 and apoptotic proteins), and tumor growth/volume was evaluated in mice. EP1 levels were significantly higher in OS cells compared to osteoblasts. PGE2 or 17-PT-PGE2 treatment increased the proliferation and decreased the apoptosis of MG63 cells. Inhibition of EP1 by SC51089 or siRNA markedly decreased the viability of MG63 cells. Similarly, SC51089 treatment significantly inhibited MG63 cell proliferation and promoted apoptosis in vivo. The silencing of EP1 receptor by siRNA or blockade of EP1 signaling by SC51089 activated extrinsic and intrinsic apoptotic pathways both in vivo and in vitro, as evidenced by increased levels of Bax, cyt c, cleaved caspase-3, caspase-8 and caspase-9. EP1 appears to be involved in PGE2-induced proliferative activity of MG63 cells. Antagonizing EP1 may provide a novel therapeutic approach to the treatment of OS.

Maternal Protein Restriction Alters the Renal Ptger1 DNA Methylation State in SHRSP Offspring.

We previously reported that maternal protein restriction (LP) during pregnancy increases salt sensitivity in offspring using the Stroke-Prone Spontaneously Hypertensive Rat (SHRSP). In the present study, we focus on DNA methylation profiles of prostaglandin E receptor 1 gene (ptger1), which is known to be associated with hypertension. We evaluated the ptger1 DNA methylation status via bisulfite sequencing, and analyzed the expression of ptger1-related genes. The results of these analyses showed that, compared to controls, the LP-S offspring exhibited both marked ptger1 hypermethylation, and significantly increased ptger1 expression. Moreover, they also exhibited significantly decreased expression of the downstream gene epithelial Na⁺ channel alpha (enacα). Interestingly, LP offspring that were provided with a standard water drinking supply (W) also exhibited increased ptger1 methylation and expression. Together, these results suggest that maternal protein restriction during pregnancy modulates the renal ptger1 DNA methylation state in SHRSP offspring, and thereby likely mediates ptger1 and enacα gene expression to induce salt sensitivity.

PGE2 upregulates renin through E-prostanoid receptor 1 via PKC/cAMP/CREB pathway in M-1 cells.

During the early phase of ANG II-dependent hypertension, tubular PGE2 is increased. Renin synthesis and secretion in the collecting duct (CD) are upregulated by ANG II, contributing to further intratubular ANG II formation. However, what happens first and whether the triggering mechanism is independent of tubular ANG II remain unknown. PGE2 stimulates renin synthesis in juxtaglomerular cells via E-prostanoid (EP) receptors through the cAMP/cAMP-responsive element-binding (CREB) pathway. EP receptors are also expressed in the CD. Here, we tested the hypothesis that renin is upregulated by PGE2 in CD cells. The M-1 CD cell line expressed EP1, EP3, and EP4 but not EP2. Dose-response experiments, in the presence of ANG II type 1 receptor blockade with candesartan, demonstrated that 10-6 M PGE2 maximally increases renin mRNA (approximately 4-fold) and prorenin/renin protein levels (approximately 2-fold). This response was prevented by micromolar doses of SC-19220 (EP1 antagonist), attenuated by the EP4 antagonist, L-161982, and exacerbated by the highly selective EP3 antagonist, L-798106 (~10-fold increase). To evaluate further the signaling pathway involved, we used the PKC inhibitor calphostin C and transfections with PKCα dominant negative. Both strategies blunted the PGE2-induced increases in cAMP levels, CREB phosphorylation, and augmentation of renin. Knockdown of the EP1 receptor and CREB also prevented renin upregulation. These results indicate that PGE2 increases CD renin expression through the EP1 receptor via the PKC/cAMP/CREB pathway. Therefore, we conclude that during the early stages of ANG II-dependent hypertension, there is augmentation of PGE2 that stimulates renin in the CD, resulting in increased tubular ANG II formation and further stimulation of renin.

Stoichiometric analysis of oligomeric states of three class-A GPCRs, chemokine-CXCR4, dopamine-D2, and prostaglandin-EP1 receptors, on living cells.

G-protein-coupled receptors (GPCRs) form the largest family of transmembrane receptors, and their oligomerization has been suggested to be related to their functions. Despite extensive studies, their oligomeric states are highly controversial. One of the reasons is the overestimation of oligomerization by conventional methods. We recently established a stoichiometric analysis method for precisely determining the oligomeric state of membrane proteins on living cells with the combined use of the coiled-coil labeling method and a spectral imaging technique and showed that the prototypical class-A GPCR ß2 -adrenergic receptor (ß2 AR) did not form functional oligomers. In this study, we expanded our study to three well-studied class-A GPCRs: C-X-C chemokine receptor of stromal cell-derived factor-1α (CXCR4), dopamine receptor D2 short isotype (D2R), and prostaglandin E receptor subtype 1 (EP1R). We found that these receptors did not form constitutive homooligomers. The receptors exhibited calcium signaling upon agonist stimulation as monomers, although CXCR4 and EP1R gradually clustered after fast signaling. We conclude that homooligomerization is not necessary for the signal transductions of these four class-A GPCRs. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

PGE2 Receptor Subtype 1 (EP1) Regulates Mesenchymal Stromal Cell Osteogenic Differentiation by Modulating Cellular Energy Metabolism.

Mesenchymal stromal cells (MSCs) are multipotent progenitors capable of differentiation into osteoblasts and can potentially serve as a source for cell-based therapies for bone repair. Many factors have been shown to regulate MSC differentiation into the osteogenic lineage such as the Cyclooxygenase-2 (COX2)/Prostaglandin E2 (PGE2) signaling pathway that is critical for bone repair. PGE2 binds four different receptors EP1-4. While most studies focus on the role PGE2 receptors EP2 and EP4 in MSC differentiation, our study focuses on the less studied, receptor subtype 1 (EP1) in MSC function. Recent work from our laboratory showed that EP1-/- mice have enhanced fracture healing, stronger cortical bones, higher trabecular bone volume and increased in vivo bone formation, suggesting that EP1 is a negative regulator of bone formation. In this study, the regulation of MSC osteogenic differentiation by EP1 receptor was investigated using EP1 genetic deletion in EP1-/- mice. The data suggest that EP1 receptor functions to maintain MSCs in an undifferentiated state. Loss of the EP1 receptor changes MSC characteristics and permits stem cells to undergo more rapid osteogenic differentiation. Notably, our studies suggest that EP1 receptor regulates MSC differentiation by modulating MSC bioenergetics, preventing the shift to mitochondrial oxidative phosphorylation by maintaining high Hif1α activity. Loss of EP1 results in inactivation of Hif1α, increased oxygen consumption rate and thus increased osteoblast differentiation. J. Cell. Biochem. 118: 4383-4393, 2017. © 2017 Wiley Periodicals, Inc.

Direct growth-inhibitory effects of prostaglandin E2 in pancreatic cancer cells in vitro through an EP4/PKA-mediated mechanism.

BACKGROUND: There is strong evidence linking inflammation and the development of pancreatic ductal adenocarcinoma. Cyclooxygenase-2 (COX-2) and COX-2-derived PGE2 are overexpressed in human and murine pancreatic ductal adenocarcinoma. Several studies have demonstrated an important role of COX-2-derived PGE2 in tumor-stroma interactions; however, the direct growth effects of prostaglandin E2 (PGE2) on pancreatic ductal adenocarcinoma cells is less well defined. Our aim was to investigate the effects of PGE2 on pancreatic ductal adenocarcinoma cell growth and to characterize the underlying mechanisms. METHODS: Human pancreatic ductal adenocarcinoma cell lines, Panc-1 and MIA PaCa-2, were treated with PGE2 in varying doses (0-10 µM). Effects on the phosphorylation of ERK1/2 were evaluated by Western blot. Colony formation was observed for cells treated with PGE2 for 11 days. DNA synthesis was determined by (3H)-thymidine incorporation assay. Gene expression of E-type prostaglandin (EP)2/EP4 receptors and their correlation with survival in patients with pancreatic ductal adenocarcinoma were assessed using the RNA-Seq data set from The Cancer Genome Atlas Research Network. RESULTS: PGE2 decreased the size and number of colonies in Panc-1 but not MIA PaCa-2 cells. In the Panc-1 cells, PGE2 activated PKA/CREB and decreased phosphorylation of ERK1/2, which was reversed by an EP4 receptor antagonist, while an EP2 receptor antagonist had no effect. In contrast, in MIA PaCa-2 cells, PGE2 had no effect on ERK1/2 phosphorylation. Treatment of both Panc-1 and MIA PaCa-2 cells with forskolin/IBMX decreased ERK1/2 phosphorylation. Finally, PGE2 decreased DNA synthesis only in Panc-1 cells, which was reversed by an EP4 receptor antagonist. In human pancreatic ductal adenocarcinoma, high EP2 and low EP4 gene expression was correlated to worse median overall survival (15.6 vs 20.8 months, log-rank P = .017). CONCLUSION: Our study provides evidence that PGE2 can inhibit directly pancreatic ductal adenocarcinoma cell growth through an EP4-mediated mechanism. Together with our gene expression and survival analysis, this observation suggests a protective role of EP4 receptors in human pancreatic ductal adenocarcinoma that expresses E-type prostaglandin receptors.

Copy number variation in ALOX5 and PTGER1 is associated with NSAIDs-induced urticaria and/or angioedema.

OBJECTIVE: Cross-intolerance to NSAIDs is a class of drug hypersensitivity reaction, of which NSAIDs-induced urticaria and/or angioedema (NIUA) are the most frequent clinical entities. They are considered to involve dysregulation of the arachidonic acid pathway; however, this mechanism has not been confirmed for NIUA. In this work, we assessed copy number variations (CNVs) in eight of the main genes involved in the arachidonic acid pathway and their possible genetic association with NIUA. MATERIALS AND METHODS: CNVs in ALOX5, LTC4S, PTGS1, PTGS2, PTGER1, PTGER2, PTGER3, and PTGER4 were analyzed using TaqMan copy number assays. Genotyping was carried out by real-time quantitative PCR. Individual genotypes were assigned using the CopyCaller Software. Statistical analysis was carried out using GraphPad prism 5, PLINK, EPIDAT, and R version 3.1.2. RESULTS AND CONCLUSION: A total of 151 cases and 139 controls were analyzed during the discovery phase and 148 cases and 140 controls were used for replication. CNVs in open reading frames were found for ALOX5, PTGER1, PTGER3, and PTGER4. Statistically significant differences in the CNV frequency between NIUA and controls were found for ALOX5 (Pc=0.017) and PTGER1 (Pc=1.22E-04). This study represents the first analysis showing an association between CNVs in exonic regions of ALOX5 and PTGER1 and NIUA. This suggests a role of CNVs in this pathology that should be explored further.

Validation of EP1 Antibody Clone for Estrogen Receptor Immunohistochemistry for Breast Cancer.

BACKGROUND: SP1 Rabbit monoclonal antibody to estrogen receptor (ER) has long been the standard for determination of ER status in breast cancer but has been replaced by the rabbit EP1 clone. AIM: To validate the EP1 antibody clone for use in determination of breast cancer ER status in a large clinical population against the previous standard SP1. MATERIALS AND METHODS: ER immunohistochemistry was assessed in 523 consecutive cases from a clinical setting using tissue microarrays. RESULTS: The kappa statistic showed that the agreement of ER status between SP1 and EP1 was considered to be almost perfect (kappa=0.97, 95% confidence interval=0.94-1.00). Sensitivity was 99.3%, specificity was 98.6% and overall agreement was 99.2%. CONCLUSION: The EP1 antibody was herein validated regarding its use in breast cancer with almost perfect agreement with the previously used standard SP1 antibody.

Detrimental role of the EP1 prostanoid receptor in blood-brain barrier damage following experimental ischemic stroke.

Cyclooxygenase-2 (COX-2) is activated in response to ischemia and significantly contributes to the neuroinflammatory process. Accumulation of COX-2-derived prostaglandin E2 (PGE2) parallels the substantial increase in stroke-mediated blood-brain barrier (BBB) breakdown. Disruption of the BBB is a serious consequence of ischemic stroke, and is mainly mediated by matrix metalloproteinases (MMPs). This study aimed to investigate the role of PGE2 EP1 receptor in neurovascular injury in stroke. We hypothesized that pharmacological blockade or genetic deletion of EP1 protects against BBB damage and hemorrhagic transformation by decreasing the levels and activity of MMP-3 and MMP-9. We found that post-ischemic treatment with the EP1 antagonist, SC-51089, or EP1 genetic deletion results in a significant reduction in BBB disruption and reduced hemorrhagic transformation in an experimental model of transient focal cerebral ischemia. These neurovascular protective effects of EP1 inactivation are associated with a significant reduction in MMP-9/-3, less peripheral neutrophil infiltration, and a preservation of tight junction proteins (ZO-1 and occludin) composing the BBB. Our study identifies the EP1 signaling pathway as an important link between neuroinflammation and MMP-mediated BBB breakdown in ischemic stroke. Targeting the EP1 receptor could represent a novel approach to diminish the devastating consequences of stroke-induced neurovascular damage.

Responsivity to PGE2 labor induction involves concomitant differential prostaglandin E receptor gene expression in cervix and myometrium.

Prostaglandin E2 (dinoprostone) is largely used for labor induction. However, one-third of patients do not respond to treatment. One cause of this poor response may be associated with changes in regulation of prostaglandin E receptors (EP1-4). In this study, we investigated EP mRNA expression in the uterine cervix and lower uterine segment myometrium for term births. Biopsies were obtained from women with successful (responders) and failed (non-responders) dinoprostone labor induction, while women that underwent spontaneous labor were included as controls. EP1 mRNA was upregulated in the cervical tissue of women who did not respond to dinoprostone induction. In addition, in the myometrium, significantly higher levels of EP3 mRNA were observed in women treated with dinoprostone, independent of their responsiveness. Dinoprostone-responders presented 3.6-fold higher levels of EP3 mRNA expression than the spontaneous labor group. Significantly higher levels of EP3 mRNA in the myometrium of the dinoprostone-treated group indicated that dinoprostone may regulate the EP3 gene on the transcriptional level. These results highlight the relationship between EP gene expression and delivery and indicate that understanding the regulation of prostaglandin E receptors may lead to improved labor induction.

Prostaglandin E2 activates the mTORC1 pathway through an EP4/cAMP/PKA- and EP1/Ca2+-mediated mechanism in the human pancreatic carcinoma cell line PANC-1.

Obesity, a known risk factor for pancreatic cancer, is associated with inflammation and insulin resistance. Proinflammatory prostaglandin E2 (PGE2) and elevated insulin-like growth factor type 1 (IGF-1), related to insulin resistance, are shown to play critical roles in pancreatic cancer progression. We aimed to explore a potential cross talk between PGE2 signaling and the IGF-1/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway in pancreatic cancer, which may be a key to unraveling the obesity-cancer link. In PANC-1 human pancreatic cancer cells, we showed that PGE2 stimulated mTORC1 activity independently of Akt, as evaluated by downstream signaling events. Subsequently, using pharmacological and genetic approaches, we demonstrated that PGE2-induced mTORC1 activation is mediated by the EP4/cAMP/PKA pathway, as well as an EP1/Ca(2+)-dependent pathway. The cooperative roles of the two pathways were supported by the maximal inhibition achieved with the combined pharmacological blockade, and the coexistence of highly expressed EP1 (mediating the Ca(2+) response) and EP2 or EP4 (mediating the cAMP/PKA pathway) in PANC-1 cells and in the prostate cancer line PC-3, which also robustly exhibited PGE2-induced mTORC1 activation, as identified from a screen in various cancer cell lines. Importantly, we showed a reinforcing interaction between PGE2 and IGF-1 on mTORC1 signaling, with an increase in IL-23 production as a cellular outcome. Our data reveal a previously unrecognized mechanism of PGE2-stimulated mTORC1 activation mediated by EP4/cAMP/PKA and EP1/Ca(2+) signaling, which may be of great importance in elucidating the promoting effects of obesity in pancreatic cancer. Ultimately, a precise understanding of these molecular links may provide novel targets for efficacious interventions devoid of adverse effects.

Regulatory mechanism of the gastric hyperemic response following barrier disruption: roles of cyclooxygenase-1, the prostaglandin E2/EP1 receptor and sensory neurons.

We herein reviewed the mechanism underlying the gastric hyperemic response following barrier disruption, with a focus on cyclooxygenase (COX) isozymes, prostaglandin (PG) E2, and capsaicin-sensitive afferent neurons. Mucosal damage was induced by exposing the stomach to 20 mM taurocholate (TC) with 50 mM HCl. The TC treatment disrupted surface epithelial cells, and then increased acid back-diffusion and mucosal blood flow (GMBF) in the stomachs of rats or wild-type mice. This hyperemic response in the rat stomach was inhibited by indomethacin without affecting acid back-diffusion, which resulted in the aggravation of lesions. The effect of indomethacin was mimicked by loxoprofen and the selective COX-1 inhibitor, SC-560, but not by the selective COX-2 inhibitor, celecoxib. The GMBF responses induced by TC were similarly observed in the stomachs of wild-type mice and EP3 receptor knockout mice, but not in mice lacking the EP1 receptor or pretreated with an EP1 antagonist. The increase in the GMBF response associated with acid back-diffusion after the TC treatment was also inhibited by the chemical ablation of capsaicin-sensitive afferent neurons, but not capsazepine, a TRPV1 antagonist. Thus, endogenous PGE2 produced by COX-1 plays a role in the gastric hyperemic response following barrier disruption of the stomach by interacting with capsaicin-sensitive afferent neurons, mainly through EP1 receptors, and facilitating the GMBF response to acid back-diffusion. These findings have also contributed to a deeper understanding of mucosal defensive mechanisms following barrier disruption and the development of new strategies for the treatment of gastrointestinal diseases.

Prostaglandin E2 promotes proliferation of skeletal muscle myoblasts via EP4 receptor activation.

We recently demonstrated that conditioned media (CM) from osteocytes enhances myogenic differentiation of myoblasts, suggesting that signaling from bone may be important for skeletal muscle myogenesis. The effect of CM was closely mimicked by prostaglandin E2 (PGE2), a bioactive lipid mediator in various physiological or pathological conditions. PGE2 is secreted at high levels by osteocytes and such secretion is further enhanced under loading conditions. Although four types of receptors, EP1 to EP4, mediate PGE2 signaling, it is unknown whether these receptors play a role in myogenesis. Therefore, in this study, the expression of EPs in mouse primary myoblasts was characterized, followed by examination of their roles in myoblast proliferation by treating myoblasts with PGE2 or specific agonists. All four PGE2 receptor mRNAs were detectable by quantitative real-time PCR (qPCR), but only PGE2 and EP4 agonist CAY 10598 significantly enhance myoblast proliferation. EP1/EP3 agonist 17-phenyl trinor PGE2 (17-PT PGE2) and EP2 agonist butaprost did not have any significant effects. Moreover, treatment with EP4 antagonist L161,982 dose-dependently inhibited myoblast proliferation. These results were confirmed by cell cycle analysis and the gene expression of cell cycle regulators. Concomitant with the inhibition of myoblast proliferation, treatment with L161,982 significantly increased intracellular reactive oxygen species (ROS) levels. Cotreatment with antioxidant N-acetyl cysteine (NAC) or sodium ascorbate (SA) successfully reversed the inhibition of myoblast proliferation and ROS overproduction caused by L161,982. Therefore, PGE2 signaling via the EP4 receptor regulates myogenesis by promoting myoblast proliferation and blocking this receptor results in increased ROS production in myoblasts.

Loss of the PGE2 receptor EP1 enhances bone acquisition, which protects against age and ovariectomy-induced impairments in bone strength.

PGE2 exerts anabolic and catabolic effects on bone through the discrete actions of four prostanoid receptors (EP1-4). We have previously demonstrated that loss EP1 accelerates fracture repair by enhancing bone formation. In the present study we defined the role of EP1 in bone maintenance and homeostasis during aging and in response to ovariectomy. The femur and L4 vertebrae of wild type (WT) and EP1(-/-) mice were examined at 2-months, 6-months, and 1-year of age, and in WT and EP1(-/-) mice following ovariectomy (OVX) or sham surgery. Bone volume fraction, trabecular architecture and mechanical properties were maintained during aging in EP1(-/-) mice to a greater degree than age-matched WT mice. Moreover, significant increases in bone formation rate (BFR) (+60%) and mineral apposition rate (MAR) (+50%) were observed in EP1(-/-), relative to WT, while no change in osteoclast number and osteoclast surface were observed. Following OVX, loss of EP1 was protective against bone loss in both femur and L4 vertebrae, with increased bone volume/total volume (BV/TV) (+32% in femur) and max load at failure (+10% in femur) relative to WT OVX, likely resulting from the increased bone formation rate that was observed in these mice. Taken together these studies identify inhibition of EP1 as a potential therapeutic approach to suppress bone loss in aged or post-menopausal patients.

Prostaglandin E2 EP1 receptor enhances TGF-ß1-induced mesangial cell injury.

Increasing evidence indicates that transforming growth factor-ß1 (TGF-ß1) is a pivotal mediator in the pathogenesis of renal fibrosis. Mesangial cells (MCs) are important for glomerular function under both physiological and pathological conditions. Studies have found that the expression level of prostaglandin E2 (PGE2) in MCs increases under high glucose conditions, that PGE2 affects the proliferation and hypertrophy of MCs mainly through the EP1 pathway, and that the proliferation of MCs and the accumulation of extracellular matrix are the main events leading to glomerular fibrosis. In this study, we investigated the effects and mechanisms of action of the EP1 receptor, which is induced by transforming growth factor (TGF)-ß1, on the proliferation of mouse MCs, the accumulation of extracellular matrix and the expression of PGE2 synthase. Primary mouse glomerular MCs were isolated from EP1 receptor-deficient mice (EP1-/- mice, in which the EP1 receptor was knocked down) and wild-type (WT) mice (WT MCs). In our preliminary experiments, we found that cell proliferation, as well as the mRNA and protein expression of cyclin D1, proliferating cell nuclear antigen (PCNA), fibronectin (FN), collagen I (ColI), membrane-associated PGE2 synthase-1 (mPGES-1) and cyclooxygenase-2 (COX-2) in the WT MCs were significantly increased following treatment with 10 ng/ml TGF-ß1 for 24 h. Compared with the WT MCs, following the knockdown of the EP1 gene, the TGF-ß1-induced MC injury was markedly suppressed. The aforementioned changes were notably enhanced following treatment with the EP1 agonist, 17-phenyl trinor PGE2 ethyl amide. Additionally, TGF-ß1 induced extracellular signal-regulated kinase (ERK) phosphorylation. We found that the TGF-ß1-induced ERK phosphorylation was alleviated by EP1 knockdown and promoted by EP1 expression. These results suggest that the EP1 receptor plays a role in the proliferation of mouse MCs, in the accumulation of extracellular matrix and in the expression of mPGES-1 induced by TGF-ß1. Its mechanisms of action are possibly related to the reinforcement of ERK phosphorylation.