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BACKGROUND: Group 2 innate lymphoid cells (ILC2s) play a critical role in asthma pathogenesis. Non-steroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (NERD) is associated with reduced signaling via EP2, a receptor for prostaglandin E 2 (PGE 2 ). However, the respective roles for the PGE 2 receptors EP2 and EP4 (both share same downstream signaling) in the regulation of lung ILC2 responses has yet been deciphered. METHODS: The roles of PGE 2 receptors EP2 and EP4 on ILC2-mediated lung inflammation were investigated using genetically modified mouse lines and pharmacological approaches in IL-33-induced lung allergy model. The effects of PGE 2 receptors and downstream signals on ILC2 metabolic activation and effector function were examined using in vitro cell cultures. RESULTS: Deficiency of EP2 rather than EP4 augments IL-33-induced mouse lung ILC2 responses and eosinophilic inflammation in vivo. In contrast, exogenous agonism of EP4 and EP2 or inhibition of phosphodiesterase markedly restricts IL-33-induced lung ILC2 responses. Mechanistically, PGE 2 directly suppresses IL-33-dependent ILC2 activation through the EP2/EP4-cAMP pathway, which downregulates STAT5 and MYC pathway gene expression and ILC2 energy metabolism. Blocking glycolysis diminishes IL-33-dependent ILC2 responses in mice where endogenous PG synthesis or EP2 signaling is blocked but not in mice with intact PGE 2 -EP2 signaling. CONCLUSION: We have defined a mechanism for optimal suppression of mouse lung ILC2 responses by endogenous PGE 2 -EP2 signaling which underpins the clinical findings of defective EP2 signaling in patients with NERD. Our findings also indicate that exogenously targeting the PGE 2 -EP4-cAMP and energy metabolic pathways may provide novel opportunities for treating the ILC2-initiated lung inflammation in asthma and NERD.

Gulf War I veterans who were victims of depleted uranium (DU) "friendly-fire" incidents have undergone longitudinal health surveillance since 1994. During the spring of 2019, 36 members of the cohort were evaluated with a monitoring protocol including exposure assessment for total and isotopic uranium concentrations in urine and a comprehensive review of health outcomes, including measures of bone metabolism and bone mineral density (BMD) determination. Elevated urine U concentrations were observed in cohort members with retained depleted uranium (DU) shrapnel fragments. In addition, a measure of bone resorption, N-telopeptide, showed a statistically significant increase in those in the high DU subgroup, a finding consistent with a statistically significant decrease in bone mass also observed in this high DU subgroup compared to the low DU subgroup. After more than 25 y since first exposure to DU, an aging cohort of military veterans continues to show few U-related health effects in known target organs of U toxicity. The new finding of impaired BMD in the high DU subgroup has now been detected in two consecutive surveillance visits. While this is a biologically plausible uranium effect, it is not reflected in other measures of bone metabolism in the full cohort, which have largely been within normal limits. However, ongoing accrual of the U burden from fragment absorption over time and the effect of aging further impairing BMD suggest the need for future surveillance assessments of this cohort.

Group 2 innate lymphoid cells (ILC2s) play a critical role in asthma pathogenesis. Non-steroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (NERD) is associated with reduced signaling via EP2, a receptor for prostaglandin EThe roles of PGEDeficiency of EP2 rather than EP4 augments IL-33-induced mouse lung ILC2 responses and eosinophilic inflammation in vivo. In contrast, exogenous agonism of EP4 and EP2 or inhibition of phosphodiesterase markedly restricts IL-33-induced lung ILC2 responses. Mechanistically, PGEWe have defined a mechanism for optimal suppression of mouse lung ILC2 responses by endogenous PGE

Rationale: Vascular remodeling, including smooth muscle cell hypertrophy and proliferation, is the key pathological feature of pulmonary arterial hypertension (PAH). Prostaglandin I2 analogs (beraprost, iloprost, and treprostinil) are effective in the treatment of PAH. Of note, the clinically favorable effects of treprostinil in severe PAH may be attributable to concomitant activation of DP1 (D prostanoid receptor subtype 1).Objectives: To study the role of DP1 in the progression of PAH and its underlying mechanism.Methods: DP1 levels were examined in pulmonary arteries of patients and animals with PAH. Multiple genetic and pharmacologic approaches were used to investigate DP1-mediated signaling in PAH.Measurements and Main Results: DP1 expression was downregulated in hypoxia-treated pulmonary artery smooth muscle cells and in pulmonary arteries from rodent PAH models and patients with idiopathic PAH. DP1 deletion exacerbated pulmonary artery remodeling in hypoxia-induced PAH, whereas pharmacological activation or forced expression of the DP1 receptor had the opposite effect in different rodent models. DP1 deficiency promoted pulmonary artery smooth muscle cell hypertrophy and proliferation in response to hypoxia via induction of mTORC1 (mammalian target of rapamycin complex 1) activity. Rapamycin, an inhibitor of mTORC1, alleviated the hypoxia-induced exacerbation of PAH in DP1-knockout mice. DP1 activation facilitated raptor dissociation from mTORC1 and suppressed mTORC1 activity through PKA (protein kinase A)-dependent phosphorylation of raptor at Ser791. Moreover, treprostinil treatment blocked the progression of hypoxia-induced PAH in mice in part by targeting the DP1 receptor.Conclusions: DP1 activation attenuates hypoxia-induced pulmonary artery remodeling and PAH through PKA-mediated dissociation of raptor from mTORC1. These results suggest that the DP1 receptor may serve as a therapeutic target for the management of PAH.

Exposure to retained metal fragments from war-related injuries can result in increased systemic metal concentrations, thereby posing potential health risks to target organs far from the site of injury. Given the large number of veterans who have retained fragments and the lack of clear guidance on how to medically manage these individuals, the Department of Veterans Affairs (VA) convened a meeting of chelation experts and clinicians who care for embedded fragment patients to discuss current practices and provide medical management guidance. Based on this group's clinical expertise and review of published literature, the evidence presented suggests that, at least in the case of lead fragments, short-term chelation therapy may be beneficial for embedded fragment patients experiencing acute symptoms associated with metal toxicity; however, in the absence of clinical symptoms or significantly elevated blood lead concentrations (greater than 80 µg/dL), chelation therapy may offer little to no benefit for individuals with retained fragments and pose greater risks due to remobilization of metals stored in bone and other soft tissues. The combination of periodic biomonitoring to assess metal body burden, longitudinal fragment imaging, and selective fragment removal when metal concentrations approach critical injury thresholds offers a more conservative management approach to caring for patients with embedded fragments.

The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts1. Intestinal stem cells are closely associated with a diverse but poorly characterized network of mesenchymal cell types2,3. However, whether the physiological mesenchymal microenvironment of mutant stem cells affects tumour initiation remains unknown. Here we provide in vivo evidence that the mesenchymal niche controls tumour initiation in trans. By characterizing the heterogeneity of the intestinal mesenchyme using single-cell RNA-sequencing analysis, we identified a population of rare pericryptal Ptgs2-expressing fibroblasts that constitutively process arachidonic acid into highly labile prostaglandin E2 (PGE2). Specific ablation of Ptgs2 in fibroblasts was sufficient to prevent tumour initiation in two different models of sporadic, autochthonous tumorigenesis. Mechanistically, single-cell RNA-sequencing analyses of a mesenchymal niche model showed that fibroblast-derived PGE2 drives the expansion οf a population of Sca-1+ reserve-like stem cells. These express a strong regenerative/tumorigenic program, driven by the Hippo pathway effector Yap. In vivo, Yap is indispensable for Sca-1+ cell expansion and early tumour initiation and displays a nuclear localization in both mouse and human adenomas. Using organoid experiments, we identified a molecular mechanism whereby PGE2 promotes Yap dephosphorylation, nuclear translocation and transcriptional activity by signalling through the receptor Ptger4. Epithelial-specific ablation of Ptger4 misdirected the regenerative reprogramming of stem cells and prevented Sca-1+ cell expansion and sporadic tumour initiation in mutant mice, thereby demonstrating the robust paracrine control of tumour-initiating stem cells by PGE2-Ptger4. Analyses of patient-derived organoids established that PGE2-PTGER4 also regulates stem-cell function in humans. Our study demonstrates that initiation of colorectal cancer is orchestrated by the mesenchymal niche and reveals a mechanism by which rare pericryptal Ptgs2-expressing fibroblasts exert paracrine control over tumour-initiating stem cells via the druggable PGE2-Ptger4-Yap signalling axis.

Background: Group 2 innate lymphoid cells (ILC2s) play a critical role in asthma pathogenesis. Non-steroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (NERD) is associated with reduced signaling via EP2, a receptor for prostaglandin E (PGE ). However, the respective roles for the PGE receptors EP2 and EP4 (both share same downstream signaling) in the regulation of lung ILC2 responses has yet been deciphered. Methods: The roles of PGE receptors EP2 and EP4 on ILC2-mediated lung inflammation were investigated using genetically modified mouse lines and pharmacological approaches in IL-33- and Alternaria alternata (A.A.)-induced lung allergy models. The effects of PGE receptors and downstream signals on ILC2 metabolic activation and effector function were examined using in vitro cell cultures. Results: Deficiency of EP2 rather than EP4 augments IL-33-induced lung ILC2 responses and eosinophilic inflammation in vivo. In contrast, exogenous agonism of EP4 but not EP2 markedly restricts IL-33- and Alternaria alternata-induced lung ILC2 responses and eosinophilic inflammation. Mechanistically, PGE directly suppresses IL-33-dependent ILC2 activation through the EP2/EP4-cAMP pathway, which downregulates STAT5 and MYC pathway gene expression and ILC2 energy metabolism. Blocking glycolysis diminishes IL-33-dependent ILC2 responses in mice lacking endogenous PG synthesis but not in PG-competent mice. Conclusion: We have defined a mechanism for optimal suppression of lung ILC2 responses by endogenous PGE -EP2 signaling which underpins the clinical findings of defective EP2 signaling in patients with NERD. Our findings also indicate that exogenously targeting the PGE -EP4-cAMP and energy metabolic pathways may provide novel opportunities for treating ILC2-initiated lung inflammation in asthma and NERD.

Group 2 innate lymphoid cells (ILC2s) play a critical role in asthma pathogenesis. Non-steroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (NERD) is associated with reduced signaling via EP2, a receptor for prostaglandin E2 (PGE2). However, the respective roles for the PGE2 receptors EP2 and EP4 (both share same downstream signaling) in the regulation of lung ILC2 responses has yet been deciphered. Here, we find that deficiency of EP2 rather than EP4 augments IL-33-induced lung ILC2 responses and eosinophilic inflammation in vivo. In contrast, exogenous agonism of EP4 but not EP2 markedly restricts IL-33- and Alternaria alternata-induced lung ILC2 responses and eosinophilic inflammation. Mechanistically, PGE2 directly suppresses IL-33-dependent ILC2 activation through the EP2/EP4-cAMP pathway, which downregulates STAT5 and MYC pathway gene expression and ILC2 energy metabolism. Blocking glycolysis diminishes IL-33-dependent ILC2 responses in mice lacking endogenous PG synthesis but not in PG-competent mice. Together, we have defined a mechanism for optimal suppression of lung ILC2 responses by endogenous PGE2-EP2 signaling which underpins the clinical findings of defective EP2 signaling in patients with NERD. Our findings also indicate that exogenously targeting the PGE2-EP4-cAMP and energy metabolic pathways may provide novel opportunities for treating ILC2-initiated lung inflammation in asthma and NERD.Graphical abstractSchematic of potential roles for activation of EP2 and EP4 by endogenous versus exogenous ligands in regulation of lung ILC2 immune responses. Endogenous PGE2 in the lung preferentially activates EP2 rather than EP4 to inhibit ILC2 responses and eosinophilic inflammation, and ablation of EP2 enhances lung ILC2 responses. Conversely, lung ILC2 responses are not altered by EP4 deficiency. However, they are markedly inhibited by EP4 agonism but not EP2 agonism. Mechanistically, PGE2-EP2/EP4 signaling activates the cAMP pathway which inhibits ILC2 energy metabolism, possibly through interruption of NF-κB (reported in Nagashima H, et al. Immunity 2019;51:682-695) and STAT5 signaling, leading to decline of ILC2 survival, proliferation and type 2 cytokine production.

Objective Type 2 diabetes is characterized by hyperglycemia and inflammation. Prostaglandin E2, which signals through four G protein-coupled receptors (EP1-4), is a mediator of inflammation and is upregulated in diabetes. We have shown previously that EP3 receptor blockade promotes β-cell proliferation and survival in isolated mouse and human islets ex vivo. Here, we analyzed whether systemic EP3 blockade could enhance β-cell mass and identity in the setting of type 2 diabetes using mice with a spontaneous mutation in the leptin receptor (Leprdb). Methods Four- or six-week-old, db/+, and db/db male mice were treated with an EP3 antagonist daily for two weeks. Pancreata were analyzed for α-cell and β-cell proliferation and β-cell mass. Islets were isolated for transcriptomic analysis. Selected gene expression changes were validated by immunolabeling of the pancreatic tissue sections. Results EP3 blockade increased β-cell mass in db/db mice through enhanced β-cell proliferation. Importantly, there were no effects on α-cell proliferation. EP3 blockade reversed the changes in islet gene expression associated with the db/db phenotype and restored the islet architecture. Expression of the GLP-1 receptor was slightly increased by EP3 antagonist treatment in db/db mice. In addition, the transcription factor nuclear factor E2-related factor 2 (Nrf2) and downstream targets were increased in islets from db/db mice in response to treatment with an EP3 antagonist. The markers of oxidative stress were decreased. Conclusions The current study suggests that EP3 blockade promotes β-cell mass expansion in db/db mice. The beneficial effects of EP3 blockade may be mediated through Nrf2, which has recently emerged as a key mediator in the protection against cellular oxidative damage., Highlights • Systemic blockade of EP3 in vivo in db/db mice increase beta-cell proliferation and mass. • EP3 blockade has no effect on alpha-cell proliferation. • EP3 blockade restores islet architecture and beta-cell identity genes in db/db mice. • Beta cell GLP-1 receptor expression is increased by EP3 blockade. • EP3 blockade activates the Nrf2 anti-oxidant pathway.

Background: Blood pressure often rises with aging, but exact mechanisms are still not completely understood. With aging, the level of proinflammatory cytokines increases in T lymphocytes. Prostaglandin D 2 , a proresolution mediator, suppresses Type 1 T helper (Th1) cytokines through D-prostanoid receptor 1 (DP1). In this study, we aimed to investigate the role of the prostaglandin D 2 /DP1 axis in T cells on age-related hypertension. Methods: To clarify the physiological and pathophysiological roles of DP1 in T cells with aging, peripheral blood samples were collected from young and older male participants, and CD4 + T cells were sorted for gene expression, prostaglandin production, and Western blot assays. Mice blood pressure was quantified by invasive telemetric monitor. Results: The prostaglandin D 2 /DP1 axis was downregulated in CD4 + T cells from older humans and aged mice. DP1 deletion in CD4 + T cells augmented age-related hypertension in aged male mice by enhancing Th1 cytokine secretion, vascular remodeling, CD4 + T cells infiltration, and superoxide production in vasculature and kidneys. Conversely, forced expression of exogenous DP1 in T cells retarded age-associated hypertension in mice by reducing Th1 cytokine secretion. Tumor necrosis factor α neutralization or interferon γ deletion ameliorated the age-related hypertension in DP1 deletion in CD4 + T cells mice. Mechanistically, DP1 inhibited Th1 activity via the PKA (protein kinase A)/p-Sp1 (phosphorylated specificity protein 1)/neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) pathway–mediated T-box-expressed-in-T-cells (T-bet) ubiquitination. T-bet deletion or forced NEDD4L expression in CD4 + T cells attenuated age-related hypertension in CD4 + T cell–specific DP1-deficient mice. DP1 receptor activation by BW245C prevented age-associated blood pressure elevation and reduced vascular/renal superoxide production in male mice. Conclusions: The prostaglandin D 2 /DP1 axis suppresses age-related Th1 activation and subsequent hypertensive response in male mice through increase of NEDD4L–mediated T-bet degradation by ubiquitination. Therefore, the T cell DP1 receptor may be an attractive therapeutic target for age-related hypertension.

We recently identified a pathway underlying immune activation in hypertension. Proteins oxidatively modified by reactive isoLG (isolevuglandin) accumulate in dendritic cells (DCs). PGE 2 (Prostaglandin E2) has been implicated in the inflammation associated with hypertension. We hypothesized that PGE 2 via its EP (E prostanoid) 3 receptor contributes to DC activation in hypertension. EP3 −/− mice and wild-type littermates were exposed to sequential hypertensive stimuli involving an initial 2-week exposure to the nitric oxide synthase inhibitor N ω -nitro-L-arginine methyl ester hydrochloride in drinking water, followed by a 2-week washout period, and a subsequent 4% high-salt diet for 3 weeks. In wild-type mice, this protocol increased systolic pressure from 123±2 to 148±8 mm Hg ( P −/− mice. Similar protective effects were also observed in wild-type mice that received intracerebroventricular injection of a lentiviral vector encoding shRNA targeting the EP3 receptor. Further, in vitro experiments indicated that PGE 2 also acts directly on DCs via its EP1 receptors to stimulate intracellular isoLG formation. Together, these findings provide new insight into how EP receptors in both the central nervous system and peripherally on DCs promote inflammation in salt-induced hypertension.

PGE2 inhibits Tregs and promotes intestinal inflammation through actions on mononuclear phagocytes and the gut microbiota., The gut microbiota fundamentally regulates intestinal homeostasis and disease partially through mechanisms that involve modulation of regulatory T cells (Tregs), yet how the microbiota-Treg cross-talk is physiologically controlled is incompletely defined. Here, we report that prostaglandin E2 (PGE2), a well-known mediator of inflammation, inhibits mucosal Tregs in a manner depending on the gut microbiota. PGE2 through its receptor EP4 diminishes Treg-favorable commensal microbiota. Transfer of the gut microbiota that was modified by PGE2-EP4 signaling modulates mucosal Treg responses and exacerbates intestinal inflammation. Mechanistically, PGE2-modified microbiota regulates intestinal mononuclear phagocytes and type I interferon signaling. Depletion of mononuclear phagocytes or deficiency of type I interferon receptor diminishes PGE2-dependent Treg inhibition. Together, our findings provide emergent evidence that PGE2-mediated disruption of microbiota-Treg communication fosters intestinal inflammation.

Niacin, as an antidyslipidemic drug, elicits a strong flushing response by release of prostaglandin (PG) D2. However, whether niacin is beneficial for inflammatory bowel disease (IBD) remains unclear. Here, we observed niacin administration‐enhanced PGD2 production in colon tissues in dextran sulfate sodium (DSS)‐challenged mice, and protected mice against DSS or 2,4,6‐trinitrobenzene sulfonic acid (TNBS)‐induced colitis in D prostanoid receptor 1 (DP1)‐dependent manner. Specific ablation of DP1 receptor in vascular endothelial cells, colonic epithelium, and myeloid cells augmented DSS/TNBS‐induced colitis in mice through increasing vascular permeability, promoting apoptosis of epithelial cells, and stimulating pro‐inflammatory cytokine secretion of macrophages, respectively. Niacin treatment improved vascular permeability, reduced apoptotic epithelial cells, promoted epithelial cell update, and suppressed pro‐inflammatory gene expression of macrophages. Moreover, treatment with niacin‐containing retention enema effectively promoted UC clinical remission and mucosal healing in patients with moderately active disease. Therefore, niacin displayed multiple beneficial effects on DSS/TNBS‐induced colitis in mice by activation of PGD2/DP1 axis. The potential efficacy of niacin in management of IBD warrants further investigation.

Motilin receptors (provisional nomenclature) are activated by motilin, a 22 amino-acid peptide derived from a precursor (MLN, P12872), which may also generate a motilin-associated peptide. Activation of these receptors by endogenous motilin released from endocrine cells within the mucosa of the duodenum during fasting, induces propulsive phase III movements, part of the gastric migrating motor complex, and promotes the sensation of hunger. Drugs and other non-peptide compounds which activate the motilin receptor may generate a more long-lasting ability to increase cholinergic activity within the upper gut, to promote gastrointestinal motility; this activity is suggested to be responsible for the gastrointestinal prokinetic effects of certain macrolide antibiotics (often called motilides; e.g. erythromycin), although for many of these molecules the evidence is sparse. Relatively high doses of these compounds may induce vomiting and in humans, nausea.

Prostanoid receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Prostanoid Receptors [661]) are activated by the endogenous ligands prostaglandins PGD2, PGE1, PGE2 , PGF2α, PGH2, prostacyclin [PGI2] and thromboxane A2. Differences and similarities between human and rodent prostanoid receptor orthologues, and their specific roles in pathophysiologic conditions are reviewed in [423]. Measurement of the potency of PGI2 and thromboxane A2 is hampered by their instability in physiological salt solution; they are often replaced by cicaprost and U46619, respectively, in receptor characterization studies.

The gut microbiota fundamentally regulates intestinal homeostasis and disease partially through mechanisms that involve modulation of regulatory T cells (Tregs), yet how the microbiota-Treg crosstalk is physiologically controlled is incompletely defined. Here, we report that prostaglandin E2 (PGE2), a well-known mediator of inflammation, inhibits mucosal Tregs in a manner depending on the gut microbiota. PGE2 through its receptor EP4 diminishes Treg-favorable commensal microbiota. Transfer of the gut microbiota that was modified by PGE2-EP4 signaling modulates mucosal Treg responses and exacerbates intestinal inflammation. Mechanistically, PGE2-modified microbiota regulates intestinal mononuclear phagocytes and type I interferon signaling. Depletion of mononuclear phagocytes or deficiency of type I interferon receptor contracts PGE2-dependent Treg inhibition. Taken together, our findings provide emergent evidence that PGE2-mediated disruption of microbiota-Treg communication fosters intestinal inflammation.

Type 2 diabetes (T2D) is a chronic condition characterized by hyperglycemia and inflammation. This condition arises in response to a failure to increase β-cell mass in the face of insulin resistance. Prostaglandin E2 (PGE2), which signals through four receptors (EP1-4), is a mediator of inflammation that has been shown to be upregulated in diabetes. Previous data from our lab has shown that EP3 receptor blockade promotes β-cell proliferation and survival in isolated mouse and human islets ex vivo. In the current study, six week-old db/db mice were treated with the EP3 antagonist DG-041 (20 mg/kg) daily for two weeks. Pancreata were excised and analyzed using immunohistochemistry. We found that treatment with DG-041 leads to improved β-cell mass (2.8 ± 1.5 mg) compared to mice treated with vehicle alone (1.0 ± 0.7 mg). Additionally, we also observed increased β-cell proliferation in DG-041-treated mice (2.2 ± 0.6% Ki67+/Ins+ β-cells) compared to vehicle-treated mice (1.6 ± 0.3%). Further analysis demonstrated that the β- to α-cell ratio was altered in mice treated with DG-041 (2.13:1) compared to vehicle-treated mice (1.78:1). We also immunolabeled for MafA and Nkx6.1, as loss of these transcription factors has been correlated with β-cell dysfunction and diabetes progression in db/db mice and humans. DG-041-treated mice had a higher percentage of MafA positive β-cells (50.3 ± 3.8%) compared to vehicle-treated mice (40.7 ± 3.8%) while there were similar numbers of Nkx6.1 positive β-cells in DG-041-treated and vehicle-treated groups (81.3 ± 4.7% and 81 ± 5.1%, respectively). The current results provide evidence that EP3 receptor blockade promotes β-cell mass expansion and proliferation while preserving homeostatic endocrine islet composition. Disclosure A.A. Christensen: None. S.R. Andrei: None. J.C. Dunn: None. V.F. Ricciardi: None. W.A. Pace: None. R.M. Breyer: None. M. Gannon: None. Funding U.S. Department of Veterans Affairs (1I01BX003744-01); National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK120626-01A1)

Objective— Deletion of mPGES-1 (microsomal prostaglandin E synthase-1)—an anti-inflammatory target alternative to COX (cyclooxygenase)-2—attenuates injury-induced neointima formation in mice. This is attributable to the augmented levels of PGI 2 (prostacyclin)—a known restraint of the vascular response to injury, acting via IP (I prostanoid receptor). To examine the role of mPGES-1–derived PGE 2 (prostaglandin E 2 ) in vascular remodeling without the IP. Approach and Results— Mice deficient in both IP and mPGES-1 (DKO [double knockout] and littermate controls [IP KO (knockout)]) were subjected to angioplasty wire injury. Compared with the deletion of IP alone, coincident deletion of IP and mPGES-1 increased neointima formation, without affecting media area. Early pathological changes include impaired reendothelialization and increased leukocyte invasion in neointima. Endothelial cells (ECs), but not vascular smooth muscle cells, isolated from DKOs exhibited impaired cell proliferation. Activation of EP (E prostanoid receptor) 4 (and EP2, to a lesser extent), but not of EP1 or EP3, promoted EC proliferation. EP4 antagonism inhibited proliferation of mPGES-1–competent ECs, but not of mPGES-1–deficient ECs, which showed suppressed PGE 2 production. EP4 activation inhibited leukocyte adhesion to ECs in vitro, promoted reendothelialization, and limited neointima formation post-injury in the mouse. Endothelium-restricted deletion of EP4 in mice suppressed reendothelialization, increased neointimal leukocytes, and exacerbated neointimal formation. Conclusions— Removal of the IP receptors unmasks a protective role of mPGES-1–derived PGE 2 in limiting injury-induced vascular hyperplasia. EP4, in the endothelial compartment, is essential to promote reendothelialization and restrain neointimal formation after injury. Activating EP4 bears therapeutic potential to prevent restenosis after percutaneous coronary intervention.

Objective: Hyperglycemia and systemic inflammation, hallmarks of Type 2 Diabetes (T2D), can induce the production of the inflammatory signaling molecule Prostaglandin E2 (PGE2) in islets. The effects of PGE2 are mediated by its four receptors, E-Prostanoid Receptors 1-4 (EP1-4). EP3 and EP4 play opposing roles in many cell types due to signaling through different G proteins, Gi and GS, respectively. We previously found that EP3 and EP4 expression are reciprocally regulated by activation of the FoxM1 transcription factor, which promotes β-cell proliferation and survival. Our goal was to determine if EP3 and EP4 regulate β-cell proliferation and survival and, if so, to elucidate the downstream signaling mechanisms. Methods: β-cell proliferation was assessed in mouse and human islets ex vivo treated with selective agonists and antagonists for EP3 (sulprostone and DG-041, respectively) and EP4 (CAY10598 and L-161,982, respectively). β-cell survival was measured in mouse and human islets treated with the EP3- and EP4-selective ligands in conjunction with a cytokine cocktail to induce cell death. Changes in gene expression and protein phosphorylation were analyzed in response to modulation of EP3 and EP4 activity in mouse islets. Results: Blockade of EP3 enhanced β-cell proliferation in young, but not old, mouse islets in part through phospholipase C (PLC)-γ1 activity. Blocking EP3 also increased human β-cell proliferation. EP4 modulation had no effect on ex vivo proliferation alone. However, blockade of EP3 in combination with activation of EP4 enhanced human, but not mouse, β-cell proliferation. In both mouse and human islets, EP3 blockade or EP4 activation enhanced β-cell survival in the presence of cytokines. EP4 acts in a protein kinase A (PKA)-dependent manner to increase mouse β-cell survival. In addition, the positive effects of FoxM1 activation on β-cell survival are inhibited by EP3 and dependent on EP4 signaling. Conclusions: Our results identify EP3 and EP4 as novel regulators of β-cell proliferation and survival in mouse and human islets ex vivo. Author Video: Author Video Watch what authors say about their articles Keywords: Pancreatic β-cell, Prostaglandin E2, Proliferation, Cell death

Colonic epithelial cells comprise the mucosal barrier, and their dysfunction promotes microbial invasion from the gut lumen and induces the development of intestinal inflammation. The EP4 receptor is known to mediate the protective effect of prostaglandin (PG) E2 in the gastrointestinal tract; however, the exact role of epithelial EP4 in intestinal pathophysiology remains unknown. In the present study, we aimed to investigate the role of epithelial EP4 in maintaining colonic homeostasis by characterizing the intestinal epithelial cell-specific EP4 knockout (EP4 cKO) mice. Mice harboring the epithelial EP4 deletion showed significantly lower colonic crypt depth and lower numbers of secretory cell lineages, as well as impaired epithelial cells in the colon. Interestingly, EP4-deficient colon epithelia showed a higher number of apoptotic cells. Consistent with the defect in mucosal barrier function of colonic epithelia and secretory cell lineages, EP4 cKO colon stroma showed enhanced immune cell infiltration, which was accompanied by increased production of inflammatory cytokines. Furthermore, EP4-deficient colons were susceptible to dextran sulfate sodium (DSS)-induced colitis. Our study is the first to demonstrate that epithelial EP4 loss resulted in potential “inflammatory” status under physiological conditions. These findings provided insights into the crucial role of epithelial PGE2/EP4 axis in maintaining intestinal homeostasis.

Prostanoid receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Prostanoid Receptors [644]) are activated by the endogenous ligands prostaglandins PGD2, PGE1, PGE2 , PGF2α, PGH2, prostacyclin [PGI2] and thromboxane A2. Measurement of the potency of PGI2 and thromboxane A2 is hampered by their instability in physiological salt solution; they are often replaced by cicaprost and U46619, respectively, in receptor characterization studies.

Background/aims The prostaglandin E2 (PGE2) EP3 receptor has a multifaceted role in metabolism. Drugs targeting EP3 have been proposed as therapeutics for diabetes; however, studies utilizing global EP3 knockout mice suggest that EP3 blockade increases obesity and insulin resistance. The present studies attempt to determine the effect of acute EP3 antagonist treatment on the diabetic phenotype. Methods DG-041 was confirmed to be a high affinity antagonist at the mouse EP3 receptor by competition radioligand binding and by blockade of EP3-mediated responses. DG-041 pharmacokinetic studies were performed to determine the most efficacious route of administration. Male C57BL/6 × BALB/c (CB6F1) mice were fed diets containing 10%, 45%, or 60% calories from fat to induce obesity. Changes to the metabolic phenotype in these mice were evaluated after one week treatment with DG-041. Results Subcutaneous injections of DG-041 at 20 mg/kg blocked the sulprostone-evoked rise in mean arterial pressure confirming the efficacy of this administration regime. Seven day treatment with DG-041 had minimal effect on body composition or glycemic control. DG-041 administration caused a reduction in skeletal muscle triglyceride content while showing a trend toward increased hepatic triglycerides. Conclusion Short term EP3 administration of DG-041 produced effective blockade of the EP3 receptor and decreased skeletal muscle triglyceride content but had no significant effects on the diabetic phenotype.

Type 2 diabetes (T2D) is characterized by chronic hyperglycemia and low-grade inflammation. Failure to expand β-cell mass in response to insulin resistance can lead to T2D. The hyperglycemia and chronic inflammation observed in T2D is associated with enhanced levels of prostaglandin E2 (PGE2) synthesis in pancreatic islets. Downstream effects of PGE2 signaling are carried out by four E-Prostanoid (EP) G-protein coupled receptors, EP1-4. Recent investigations by our lab demonstrated that EP3 and EP4 play opposing roles in pancreatic islets whereby EP3 activation inhibits β-cell proliferation and survival while EP4 activation stimulates β-cell mass expansion and reduces cytokine-induced cell death ex vivo. Preliminary data suggests that db/db mice (a model of T2D) treated with the EP3 antagonist, DG-041, exhibit enhanced levels of β-cell proliferation when compared to their vehicle-treated counterparts (3.04 ± 0.6% Ki67+/Ins+ β-cells and 1.67 ± 0.2%, respectively). Although differences in β-cell death between the vehicle- (0.09 ± 0.1% TUNEL+ β-cells) and DG-041-treated cohorts (0.23 ± 0.1%) did not reach statistical significance, treatment with the EP3 antagonist ultimately culminated in improved β-cell mass in db/db mice (1.57 ± 0.2 mg) compared to vehicle-treated controls (0.94 ± 0.2 mg). Additionally, preliminary results suggest that the EP3 antagonist-induced increase in β-cell proliferation in nondiabetic human cadaver donors occurs, in part, through PLCγ-1 whereby pretreatment of human islets with U-73122 (PLCγ-1 inhibitor) prior to DG-041 addition resulted in decreased β-cell proliferation (1.67 ± 0.2 fold of vehicle-treated control) when compared to the EP3 antagonist alone (2.30 ± 0.3 fold). Taken together, these results provide valuable foundational insight into the potential role of EP3 in the modulation of β-cell mass expansion. Disclosure S.R. Andrei: None. J.C. Dunn: None. A.A. Christensen: None. V.F. Ricciardi: None. W.A. Pace: None. R.M. Breyer: None. M. Gannon: None.

Prostaglandin E2 (PGE2), a cyclooxygenase metabolite that generally acts as a systemic vasodepressor, has been shown to have vasopressor effects under certain physiologic conditions. Previous studies have demonstrated that PGE2 receptor signaling modulates angiotensin II (Ang II)-induced hypertension, but the interaction of these two systems in the regulation of vascular reactivity is incompletely characterized. We hypothesized that Ang II, a principal effector of the renin-angiotensin-aldosterone system, potentiates PGE2-mediated vasoconstriction. Here we demonstrate that pre-treatment of arterial rings with 1 nM Ang II potentiated PGE2-evoked constriction in a concentration dependent manner (AUC−Ang II 2.778 ± 2.091, AUC+Ang II 22.830 ± 8.560, ***P

Background: Prostaglandin E2 receptor subtype 3 (EP3), a Gi protein-coupled receptor activated by prostaglandin E2, plays a particular role in cardioprotection. This study aimed to investigate the impact of EP3 deletion on cardiac remodeling and further elucidate the related involvement of possible signaling pathways. Methods and Results: The animals used were EP3 receptor knockout (EP3KO) mice and wild-type (WT) litter mate controls at 16-18 weeks old. The high-resolution echocardiography and weight index indicated that eccentric cardiac hypertrophy might occur in EP3KO mice, which were having worse cardiac function than WT litter mates. Isolated adult myocytes from EP3KO hearts showed spontaneous lengthening. Cardiac fibrosis was observed in EP3KO mice through Masson trichrome staining. The elevated messenger RNA (mRNA) level in matrix genes and the reduced mRNA, protein, and activity levels of matrix metalloproteinase 2 (MMP-2) indicated an increased synthesis and suppressed degradation of matrix collagen in EP3KO mice. The phosphorylation level of extracellular signal-regulated kinase (ERK) 1/2 protein was reduced in the cardiac tissue of EP3KO mice, accompanied by no significant change in the protein level of total ERK1/2, total p38, phospho-p38, glycogen synthase kinase-3β (GSK3β), phospho-GSK3β, and calcineurin (CaN) as well as CaN activity. Conclusion: EP3 knockout in cardiac tissues could induce eccentric cardiac hypertrophy and cardiac fibrosis at 16-18 weeks old. These effects of EP3 knockout might be regulated through inactivating MAPK/ERK pathway and affecting the MMP-2 expression. Overall, PGE2-EP3 is necessary to maintain the normal growth and development of the heart.

The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts

Pulmonary arterial hypertension (PAH) is commonly associated with chronic hypoxemia in disorders such as chronic obstructive pulmonary disease (COPD). Prostacyclin analogs are widely used in the management of PAH patients; however, clinical efficacy and long-term tolerability of some prostacyclin analogs may be compromised by concomitant activation of the E-prostanoid 3 (EP3) receptor. Here, we found that EP3 expression is upregulated in pulmonary arterial smooth muscle cells (PASMCs) and human distal pulmonary arteries (PAs) in response to hypoxia. Either pharmacological inhibition of EP3 or Ep3 deletion attenuated both hypoxia and monocrotaline-induced pulmonary hypertension and restrained extracellular matrix accumulation in PAs in rodent models. In a murine PAH model, Ep3 deletion in SMCs, but not endothelial cells, retarded PA medial thickness. Knockdown of EP3α and EP3β, but not EP3γ, isoforms diminished hypoxia-induced TGF-β1 activation. Expression of either EP3α or EP3β in EP3-deficient PASMCs restored TGF-β1 activation in response to hypoxia. EP3α/β activation in PASMCs increased RhoA-dependent membrane type 1 extracellular matrix metalloproteinase (MMP) translocation to the cell surface, subsequently activating pro–MMP-2 and promoting TGF-β1 signaling. Activation or disruption of EP3 did not influence PASMC proliferation. Together, our results indicate that EP3 activation facilitates hypoxia-induced vascular remodeling and pulmonary hypertension in mice and suggest EP3 inhibition as a potential therapeutic strategy for pulmonary hypertension.

OBJECTIVE: Deletion of microsomal (m) prostaglandin (PG) E synthase (S)-1, an anti-inflammatory target alternative to cyclooxygenase-2, attenuates injury-induced neointima formation in mice. This is attributable to the augmented levels of PGI(2), a known restraint of the vascular response to injury, acting via I prostanoid receptor (IP). To examine the role of mPGES-1 derived PGE(2) in vascular remodeling without the IP. APPROACH AND RESULTS: Mice deficient in both IP and mPGES-1 (double knockout, DKO) and littermate controls (IP KO) were subjected to angioplasty-wire injury. Compared with the deletion of IP alone, coincident deletion of IP and mPGES-1 increased neointima formation, without affecting media area. Early pathological changes include impaired reendothelialization and increased leukocyte invasion in neointima. Endothelial cells (ECs), but not vascular smooth muscle cells, isolated from DKOs exhibited impaired cell proliferation. Activation of PGE(2) receptor (EP)4 (and EP2, to a lesser extent), but not of EP1 or EP3, promoted EC proliferation. EP4 antagonism inhibited proliferation of mPGES-1-competent ECs, but not of mPGES-1-deficient ECs, which showed suppressed PGE(2) production. EP4 activation inhibited leukocyte adhesion to ECs in vitro, promoted reendothelialization and limited neointima formation post injury in the mouse. Endothelium-restricted deletion of EP4 in mice suppressed reendothelialization, increased neointimal leukocytes, and exacerbated neointimal formation. CONCLUSIONS: Removal of the IP receptors unmasks a protective role of mPGES-1 derived PGE(2) in limiting injury-induced vascular hyperplasia. EP4, in the endothelial compartment, is essential to promote reendothelialization and restrain neointimal formation after injury. Activating EP4 bears therapeutic potential to prevent restenosis after percutaneous coronary intervention.

Rationale: Vascular smooth muscle cell (VSMC) migration and proliferation are the hallmarks of restenosis pathogenesis after angioplasty. Cyclooxygenase (COX)-derived prostaglandin (PG) E 2 is implicated in the vascular remodeling response to injury. However, its precise molecular role remains unknown. Objective: This study investigates the impact of COX-2–derived PGE 2 on neointima formation after injury. Methods and Results: Vascular remodeling was induced by wire injury in femoral arteries of mice. Both neointima formation and the restenosis ratio were diminished in COX-2 knockout mice as compared with controls, whereas these parameters were enhanced in COX-1>COX-2 mice, in which COX-1 is governed by COX-2 regulatory elements. PG profile analysis revealed that the reduced PGE 2 by COX-2 deficiency, but not PGI 2 , could be rescued by COX-1 replacement, indicating COX-2–derived PGE 2 enhanced neointima formation. Through multiple approaches, the EP3 receptor was identified to mediate the VSMC migration response to various stimuli. Disruption of EP3 impaired VSMC polarity for directional migration by decreasing small GTPase activity and restricted vascular neointimal hyperplasia, whereas overexpression of EP3α and EP3β aggravated neointima formation. Inhibition or deletion of EP3α/β, a Gαi protein–coupled receptor, activated the cAMP/protein kinase A pathway and decreased activation of RhoA in VSMCs. PGE 2 could stimulate phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase3β signaling in VSMCs through Gβγ subunits on EP3α/β activation. Ablation of EP3 suppressed phosphatidylinositol 3-kinase signaling and reduced GTPase activity in VSMCs and altered cell polarity and directional migration. Conclusions: COX-2–derived PGE 2 facilitated the neointimal hyperplasia response to injury through EP3α/β-mediated cAMP/protein kinase A and phosphatidylinositol 3-kinase pathways, indicating EP3 inhibition may be a promising therapeutic strategy for percutaneous transluminal coronary angioplasty.

Niacin is a well established drug used to lower cholesterol and prevent cardiovascular disease events. However, niacin also causes cutaneous flushing side effects due to release of the proresolution mediator prostaglandin D2 (PGD2). Recent randomized clinical trials have demonstrated that addition of niacin with laropiprant [a PGD2 receptor subtype 1 (DP1) blocker] to statin-based therapies does not significantly decrease the risk of cardiovascular disease events, but increases the risk of serious adverse events. Here, we tested whether, and how, niacin beneficial effects on myocardial ischemia require the activation of the PGD2/DP1 axis. Myocardial infarction (MI) was reproduced by ligation of the left anterior descending branch of the coronary artery in mice. We found that niacin increased PGD2 release in macrophages and shifted macrophages to M2 polarization both in vitro and in vivo by activation of DP1 and accelerated inflammation resolution in zymosan-induced peritonitis in mice. Moreover, niacin treatment facilitated wound healing and improved cardiac function after MI through DP1-mediated M2 bias and timely resolution of inflammation in infarcted hearts. In addition, we found that niacin intake also stimulated M2 polarization of peripheral monocytes in humans. Collectively, niacin promoted cardiac functional recovery after ischemic myocardial infarction through DP1-mediated M2 polarization and timely resolution of inflammation in hearts. These results indicated that DP1 inhibition may attenuate the cardiovascular benefits of niacin.

Prostaglandins (PGs) are signaling lipids derived from arachidonic acid (AA), which is metabolized by cyclooxygenase (COX)-1 or 2 and class-specific synthases to generate PGD2, PGE2, PGF2α, PGI2 (prostacyclin), and thromboxane A2. PGs signal through G-protein coupled receptors (GPCRs) and are important modulators of an array of physiological functions, including systemic inflammation and insulin secretion from pancreatic islets. The role of PGs in β-cell function has been an active area of interest, beginning in the 1970s. Early studies demonstrated that PGE2 inhibits glucose-stimulated insulin secretion (GSIS), although more recent studies have questioned this inhibitory action of PGE2. The PGE2 receptor EP3 and one of the G-proteins that couples to EP3, GαZ, have been identified as negative regulators of β-cell proliferation and survival. Conversely, PGI2 and its receptor, IP, play a positive role in the β-cell by enhancing GSIS and preserving β-cell mass in response to the β-cell toxin streptozotocin (STZ). In comparison to PGE2 and PGI2, little is known about the function of the remaining PGs within islets. In this review, we discuss the roles of PGs, particularly PGE2 and PGI2, PG receptors, and downstream signaling events that alter β-cell function and regulation of β-cell mass.

Publisher Summary This chapter characterizes the processes of neuroinflammation and related oxidative injury and discusses alterations associated with developmental neurotoxicity. Neuroinflammation is characterized by a host of cellular and molecular changes within the brain. It investigates the effectiveness of drugs currently proposed to inhibit COX, and to suppress innate immune response and/or oxidative damage. The data furnished here imply that α-tocopherol offers broad protection in the cerebrum from oxidative damage. The increased state of neuroinflammation makes the aged brain more vulnerable to the disruptive effects of both intrinsic and extrinsic factors such as disease, infection, toxicants or stress. The exact pattern of cellular and molecular changes depends largely on the type and duration of the inflammatory challenge experienced by the organism. Neuroinflammation can result from classical injuries such as direct insult to the brain that occurs with trauma, encephalitis or ischemia, or from insults such as toxins or infection that follow exposure to bacteria. It is becoming increasingly evident that neuroinflammation and associated oxidative damage play a crucial role in the development and progression of brain diseases. Glia, and in particular microglia, are central to mediating the effects of neuroinflammation.

Among the four prostaglandin E2 receptors, EP3 receptor is the one most abundantly expressed in white adipose tissue (WAT). The mouse EP3 gene gives rise to three isoforms, namely EP3α, EP3β, and EP3γ, which differ only at their C-terminal tails. To date, functions of EP3 receptor and its isoforms in WAT remain incompletely characterized. In this study, we found that the expression of all EP3 isoforms were downregulated in WAT of both db/db and high-fat diet-induced obese mice. Genetic ablation of three EP3 receptor isoforms (EP3-/- mice) or EP3α and EP3γ isoforms with EP3β intact (EP3β mice) led to an obese phenotype with increased food intake, decreased motor activity, reduced insulin sensitivity, and elevated serum triglycerides. Since the differentiation of preadipocytes and mouse embryonic fibroblasts to adipocytes was markedly facilitated by either pharmacological blockade or genetic deletion/inhibition of EP3 receptor via the cAMP/PKA/PPARγ pathway, increased adipogenesis may contribute to obesity in EP3-/- and EP3β mice. Moreover, both EP3-/- and EP3β mice had increased lipolysis in WAT mainly due to the activated cAMP/PKA/hormone-sensitive lipase pathway. Taken together, our findings suggest that EP3 receptor and its α and γ isoforms are involved in both adipogenesis and lipolysis and influence food intake, serum lipid levels, and insulin sensitivity.

We recently identified a pathway underlying immune activation in hypertension. Proteins oxidatively modified by reactive γ-ketoaldehydes (isoketals) accumulate in dendritic cells (DCs). These are immunogenic and lead to subsequent T lymphocytes activation. The local signals that stimulate DCs to accumulate isoketal adducts remain undefined. Prostaglandin E 2 (PGE 2 ) has been implicated in the inflammation associated with hypertension. We hypothesized that PGE 2 via its EP3 receptor contributes to DC activation in hypertension. EP3 -/- mice and wild type (WT) littermates were exposed to sequential hypertensive stimuli involving an initial 2-week exposure to the NOS inhibitor L-NAME (LN) in drinking water, a 2 week washout period, and a subsequent 4% high salt diet (HS) for 3 weeks. In WT mice, this protocol increased systolic pressure from 123±2 to 148±8 mmHg (p+ and CD8 + effector memory T cells by 2 to 3 fold. This was associated with a striking accumulation of isoketal protein adducts in splenic DCs. However, the increases in blood pressure, renal T cell infiltration and DC isoketal formation were completely prevented in EP3 -/- mice. We further hypothesized that EP3 receptors contribute to oxidative stress production in the kidney. As measured by dihydroethidium with confocal microscopy, the LNHS protocol induced marked increases in superoxide production in WT mice, but not in EP3 -/- mice. To examine the direct effects of PGE 2 , splenic DCs were incubated with PGE 2 in vitro for 24 hours. PGE 2 dose-dependently increased isoketal-adduct formation in DCs (vehicle: 8.8±5.1% vs. 50 nM PGE 2 : 41.4±11.7%, p2 in DC activation in hypertension. In vivo, PGE 2 has a predominant effect on EP3 receptors to enhance renal vascular ROS production, which likely leads to isoketal-adduct formation and accumulation in DCs. PGE 2 also acts directly on DCs via its EP1 receptors to stimulate intracellular isoketal formation. Together, these findings provide additional information as to how PGE 2 modulates inflammation in hypertension.

Type 1 diabetes mellitus (T1DM) is associated with cardiovascular complications induced by atherosclerosis. Prostaglandin E2 (PGE2) is often raised in states of inflammation, including diabetes, and regulates inflammatory processes. In myeloid cells, a key cell type in atherosclerosis, PGE2 acts predominately through its Prostaglandin E Receptor 4 (EP4; Ptger4) to modulate inflammation. The effect of PGE2-mediated EP4 signaling specifically in myeloid cells on atherosclerosis in the presence and absence of diabetes is unknown. Because diabetes promotes atherosclerosis through increased arterial myeloid cell accumulation, we generated a myeloid cell-targeted EP4-deficient mouse model (EP4M-/-) of T1DM-accelerated atherogenesis to investigate the relationship between myeloid cell EP4, inflammatory phenotypes of myeloid cells, and atherogenesis. Diabetic mice exhibited elevated plasma PGE metabolite levels and elevated Ptger4 mRNA in macrophages, as compared with non-diabetic littermates. PGE2 increased Il6, Il1b, Il23 and Ccr7 mRNA while reducing Tnfa mRNA through EP4 in isolated myeloid cells. Consistently, the stimulatory effect of diabetes on peritoneal macrophage Il6 was mediated by PGE2-EP4, while PGE2-EP4 suppressed the effect of diabetes on Tnfa in these cells. In addition, diabetes exerted effects independent of myeloid cell EP4, including a reduction in macrophage Ccr7 levels and increased early atherogenesis characterized by relative lesional macrophage accumulation. These studies suggest that this mouse model of T1DM is associated with increased myeloid cell PGE2-EP4 signaling, which is required for the stimulatory effect of diabetes on IL-6, markedly blunts the effect of diabetes on TNF-α and does not modulate diabetes-accelerated atherogenesis.

Objective— The present studies aimed at elucidating the role of prostaglandin E 2 receptor subtype 3 (E-prostanoid [EP] 3) in regulating blood pressure. Methods and Results— Mice bearing a genetic disruption of the EP3 gene (EP 3 −/− ) exhibited reduced baseline mean arterial pressure monitored by both tail-cuff and carotid arterial catheterization. The pressor responses induced by EP3 agonists M&B28767 and sulprostone were markedly attenuated in EP3 −/− mice, whereas the reduction of blood pressure induced by prostaglandin E 2 was comparable in both genotypes. Vasopressor effect of acute or chronic infusion of angiotensin II (Ang II) was attenuated in EP3 −/− mice. Ang II-induced vasoconstriction in mesenteric arteries decreased in EP3 −/− group. In mesenteric arteries from wild-type mice, Ang II-induced vasoconstriction was inhibited by EP3 selective antagonist DG-041 or L798106. The expression of Arhgef-1 is attenuated in EP3 deficient mesenteric arteries. EP3 antagonist DG-041 diminished Ang II-induced phosphorylation of myosin light chain 20 and myosin phosphatase target subunit 1 in isolated mesenteric arteries. Furthermore, in vascular smooth muscle cells, Ang II–induced intracellular Ca 2+ increase was potentiated by EP3 agonist sulprostone but inhibited by DG-041. Conclusion— Activation of the EP3 receptor raises baseline blood pressure and contributes to Ang II–dependent hypertension at least partially via enhancing Ca 2+ sensitivity and intracellular calcium concentration in vascular smooth muscle cells. Selective targeting of the EP3 receptor may represent a potential therapeutic target for the treatment of hypertension.

Prostaglandin E 2 is a major prostanoid found in the kidney and vasculature contributing to the regulation of blood pressure. The prostaglandin E 2 receptor EP1 has been shown to contribute to hypertension by mediating angiotensin II-dependent vasoconstriction, although its precise role is incompletely characterized. Disruption of the EP1 receptor in C57BL/6J mice reduced the incidence of mortality during severe hypertension induced by uninephrectomy, deoxycorticosterone acetate, and angiotensin II. Mortality was dependent on all components of the model. Death was a result of aortic aneurysm rupture or occurred after development of anasarca, each of which was reduced in EP1−/− mice. Mean arterial pressure was increased in treated EP1+/+ and EP1−/− mice; however, this elevation was significantly lower in EP1−/− mice. Blood pressure reduction via administration of hydralazine phenocopied EP1−/− mice. Thus, reduction in blood pressure by disruption of EP1 reduced incidence of mortality and decreased organ damage, suggesting that EP1 receptor blockade may be a viable target for antihypertensive therapy.

Unlike the majority of G protein-coupled receptors, the prostaglandin E(2) (PGE(2)) E-prostanoid 3 (EP3) receptor binds agonist with high affinity that is insensitive to the presence of guanosine 5[prime]-O-(3-thio)triphosphate (GTPγS). We report the identification of mutations that confer GTPγS sensitivity to agonist binding. Seven point mutations were introduced into the conserved motif in the second extracellular loop (ECII) of EP3, resulting in acquisition of GTP-sensitive agonist binding. One receptor mutation W203A was studied in detail. Loss of agonist binding was observed on intact human embryonic kidney 293 cells expressing the W203A receptor, conditions where high GTP levels are present; however, high affinity binding [(3)H]PGE(2) was observed in broken cell preparations washed free of GTP. The [(3)H]PGE(2) binding of W203A in broken cell membrane fractions was inhibited by addition of GTPγS (IC(50) 21 ± 1.8 nM). Taken together, these results suggest that the wild-type EP3 receptor displays unusual characteristics of the complex coupled equilibria between agonist-receptor and receptor-G protein interaction. Moreover, mutation of ECII can alter this coupled equilibrium from GTP-insensitive agonist binding to more conventional GTP-sensitive binding. This suggests that for the mutant receptors, ECII plays a critical role in linking the agonist bound receptor conformation to the G protein nucleotide bound state.

There is significant evidence for a central role of inflammation in the development of Alzheimer disease (AD). Epidemiological studies indicate that chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD in healthy aging populations. As NSAIDs inhibit the enzymatic activity of the inflammatory cyclooxygenases COX-1 and COX-2, these findings suggest that downstream prostaglandin signaling pathways function in the preclinical development of AD. Here, we investigate the function of prostaglandin E(2) (PGE(2) ) signaling through its EP3 receptor in the neuroinflammatory response to Aβ peptide.The function of PGE(2) signaling through its EP3 receptor was examined in vivo in a model of subacute neuroinflammation induced by administration of Aβ(42) peptides. Our findings were then confirmed in young adult APPSwe-PS1ΔE9 transgenic mice.Deletion of the PGE(2) EP3 receptor in a model of Aβ(42) peptide-induced neuroinflammation reduced proinflammatory gene expression, cytokine production, and oxidative stress. In the APPSwe-PS1ΔE9 model of familial AD, deletion of the EP3 receptor blocked induction of proinflammatory gene and protein expression and lipid peroxidation. In addition, levels of Aβ peptides were significantly decreased, as were β-secretase and β C-terminal fragment levels, suggesting that generation of Aβ peptides may be increased as a result of proinflammatory EP3 signaling. Finally, deletion of EP3 receptor significantly reversed the decline in presynaptic proteins seen in APPSwe-PS1ΔE9 mice.Our findings identify the PGE(2) EP3 receptor as a novel proinflammatory, proamyloidogenic, and synaptotoxic signaling pathway, and suggest a role for COX-PGE(2) -EP3 signaling in the development of AD.