EP4 Signaling Research Articles

Prostaglandin PGE2 Receptor EP4 Regulates Microglial Phagocytosis and Increases Susceptibility to Diet-Induced Obesity.

In rodents, susceptibility to diet-induced obesity requires microglial activation, but the molecular components of this pathway remain incompletely defined. Prostaglandin PGE2 levels increase in the mediobasal hypothalamus during high-fat-diet (HFD) feeding, and the PGE2 receptor EP4 regulates microglial activation state and phagocytic activity, suggesting a potential role for microglial EP4 signaling in obesity pathogenesis. To test the role of microglial EP4 in energy balance regulation, we analyzed the metabolic phenotype in a microglia-specific EP4 knockout (MG-EP4 KO) mouse model. Microglial EP4 deletion markedly reduced weight gain and food intake in response to HFD feeding. Corresponding with this lean phenotype, insulin sensitivity was also improved in HFD-fed MG-EP4 KO mice, though glucose tolerance remained surprisingly unaffected. Mechanistically, EP4-deficient microglia showed an attenuated phagocytic state marked by reduced CD68 expression and fewer contacts with pro-opiomelanocortin (POMC) neuron processes. These cellular changes observed in the MG-EP4 KO mice corresponded with an increased density of POMC neurites extending into the paraventricular nucleus (PVN). These findings reveal that microglial EP4 signaling promotes body weight gain and insulin resistance during HFD feeding. Furthermore, the data suggest that curbing microglial phagocytic function may preserve POMC cytoarchitecture and PVN input to limit overconsumption during diet-induced obesity.

Metabolic regulation by prostaglandin E2 impairs lung group 2 innate lymphoid cell responses.

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. The roles of PGE2 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 PGE2 receptors and downstream signals on ILC2 metabolic activation and effector function were examined using in vitro cell cultures. 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, 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 where endogenous PG synthesis or EP2 signaling is blocked but not in mice with intact PGE2 -EP2 signaling. We have defined a mechanism for optimal suppression of mouse 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 the ILC2-initiated lung inflammation in asthma and NERD.

Effects of selective inhibition of prostaglandin E2 receptors EP2 and EP4 on the miRNA profile in endometriosis

Endometriosis is an estrogen-dependent, progesterone-resistant, chronic inflammatory gynecological disease of reproductive-age women. Two major clinical symptoms of endometriosis are chronic pelvic pain and infertility, which profoundly affect the quality of life in women. Current hormonal therapies to induce a hypoestrogenic state are unsuccessful because of undesirable side effects, reproductive health concerns, and failure to prevent disease recurrence. Prostaglandin E2 (PGE2) plays an important role in the survival and growth of endometriotic lesions. MicroRNAs (miRNAs) are small, noncoding RNAs that control gene expressions through multiple mechanisms and have important roles in the pathogenesis of endometriosis. The objective of the present study is to determine the effects of pharmacological inhibition of PGE2 receptors, EP2 and EP4, on miRNA profile in endometriosis. The novel results collectively indicate that inhibition of PGE2-EP2/EP4 signaling regulated several miRNA clusters associated with cell adhesion, migration, invasion, survival and growth in cell-specific and the chromosome-specific manner and reverses the epigenetic silencing of proapoptotic miRNAs 15a and 34c in the human endometriotic epithelial and stromal cells and experimental endometriotic lesions. Thus, selective inhibition of EP2/EP4 receptors could emerge as a potential nonsteroidal therapy for endometriosis.

Abstract 3139: Disrupting prostaglandin E2 signaling to reverse treatment resistance in cancer

Abstract Pancreatic adenocarcinoma (PDA) accounts for 3% of all cancers in the USA and 7% of all cancer deaths partly because of its resistance to immunotherapy. PDA manifests itself as a desmoplastic, myeloid cell-rich tumor with a notable paucity of T cells. Pathway analysis of the T cell low, immunotherapy resistant mouse PDA cell lines has identified the prostaglandin E2 (PGE2) synthetic pathway as one of the most upregulated ones. We have previously shown that the deletion or pharmacological inhibition of COX-2, the rate-limiting PGE2 synthesis enzyme, slows the tumor growth and synergizes with immunotherapy to further suppress PDA progression. The current study investigates how the disruption of PGE2 signaling through its receptors, EP4 and EP2, can affect PDA. We use EP receptor genetic and pharmacological inhibition models - we disrupt EP signaling on tumor cells only or systemically, test the effects in T cell low, intermediate, and high PDA tumors, alone or in combination with chemo and immunotherapy. EP4 receptor blocker combined with the agonistic CD40 and anti-PD-1 antibodies significantly increased tumor-free survival of the T cell intermediate PDA implanted tumor-bearing (TB) mice (57% tumor-free in treated vs 0% in the control group). The same combination therapy tended to increase the survival in autochthonous tumor-bearing KrasG12D and p53R172H transgenic mice (p=0.08). CRISPR knockout (KO) of the EP4 in the T cell low cell line abolished the growth of subcutaneous (SQ) tumors and significantly slowed the progression of orthotopically implanted tumors (p=0.009 EP4 KO vs control). Treatment of the SQ T cell low TB mice with EP2/EP4 blockers in combination with either anti-PD-1 or chemotherapy slowed the tumor growth compared to the controls (p=0.002 and p=0.004, respectively) and increased the survival (p=0.007 and p=0.002, respectively). A week of combination treatment resulted in changes in the myeloid cell compartment of the TME - an increase in the proportion of M1 macrophages and type 1 conventional dendritic cells with no significant changes in the T cell compartment. The study demonstrates an alternative approach to targeting the PGE2 pathway in tumors and aims to develop a multiprong treatment regimen to reverse the therapy resistance in PDA. Normal 0 false false false EN-US X-NONE X-NONE/* Style Definitions */table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;} <![endif]–> Citation Format: Nune Markosyan, Liz Quinones, Charu Aurora, Nikhil Joshi, Ilkyu Kim, Ben Z. Stanger, Robert H. Vonderheide. Disrupting prostaglandin E2 signaling to reverse treatment resistance in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3139.

PGE2-EP2/EP4 signaling elicits immunosuppression by driving the mregDC-Treg axis in inflammatory tumor microenvironment.

Active inflammation generally promotes immune activation. However, in the tumor microenvironment (TME), active inflammation occurs in parallel with immunosuppression, and both contribute to tumor growth. Why inflammation does not lead to immune activation in TME remains unclear. In this study, using the immune checkpoint inhibitor-insensitive mouse cancer model and single-cell RNA sequencing, we show that PGE2-EP2/EP4 signaling simultaneously promotes active inflammation by inducing expression of the NF-κB genes in myeloid cells and elicits immunosuppression by driving the mregDC (mature DC enriched in immunoregulatory molecules)-Treg (regulatory Tcell) axis for Treg recruitment and activation in the tumor. Importantly, the EP2/EP4 expression level is strongly correlated with the gene signatures ofboth active inflammation and the mregDC-Treg axis and has significant prognosis value in various human cancers. Thus, PGE2-EP2/EP4 signaling functions as the key regulatory node linking active inflammation and immunosuppression in TME, which can be targeted by EP2 and EP4 antagonists for cancer therapeutics.

PGE receptor EP4 signaling increased hyaluronan‐vesican complex in abdominal aortic aneurysm

Chronic inflammation and dysregulation of extracellular matrices are hallmarks of abdominal aortic aneurysm (AAA) pathophysiology. We have demonstrated that PGE receptor EP4 signaling in vascular smooth muscle cells (VSMCs) has a pivotal role in AAA formation via immune infiltrates and impairment of elastic fiber repair. However, EP4 signaling‐mediated changes in extracellular matrices have not been fully understood, and expressions of the chondroitin sulfate proteoglycan versican are controversial. Versican has multiple binding partners (e.g., hyaluronan and fibrillin‐1) and modulates VSMC phenotypes. We, therefore, aimed to clarify an expression change in versican in AAA.We administrated angiotensin‐II (AngII) to VSMC‐specific EP4 overexpression mice (EP4‐Tg) to induce AAA. The expression level of versican V0/V1 was gradually increased in the tunica media of EP4‐Tg mice 4 days after AngII administration, but not in Non‐Tg mice. This increase in versican V0/V1 became prominent 14 days after AngII administration in EP4‐Tg mice. Protein expression of versican V0/V1 was increased in the tunica media of human AAA tissues. EP4 stimulation in VSMCs derived from EP4‐Tg mice increased levels of versican mRNAs (20.0±1.4‐fold, n=5, p<0.01) and versican V0/V1 proteins (4.1±0.7‐fold, n=5, p<0.01), while administration of EP1/3 or EP2 agonist did not. The EP4‐mediated increase in versican mRNAs was completely suppressed by Actinomycin D. Versican V0/V1 are cleaved by a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS‐1) and ADAMTS‐5. The expression levels of ADAMTS‐1, ADAMTS‐5, and cleaved versican protein expressions were not changed in EP4‐Tg VSMCs. The expression level of Hyaluronan synthase1 (Has1) mRNA was also increased in EP4‐Tg VSMCs (66.9±10.1‐fold, n=5, p<0.01). Western blotting revealed that versican V0/V1 proteins of approximately 200‐400 kDa in size in the supernatants of EP4‐Tg VSMCs stimulated with an EP4 agonist were increased by hyaluronan digestion with hyaluronidase (6.3±1.5‐fold, n=6, p<0.01). Transmission electron microscopic analysis demonstrated that elastin contractile units between elastic laminae and VSMCs were disrupted in EP4‐Tg mice administrated with 10 days of AngII, but not in Non‐Tg mice.These data suggested that EP4 signaling in VSMCs increased hyaluronan‐versican V0/V1 complex. Versican V0/V1 proteins were increased in the early stage of a mouse AAA model. It has been suggested that versican has inhibitory roles in elastic fiber formation. EP4‐mediated hyaluronan‐versican V0/V1 complex may exacerbate AAA via disruption of elastin contractile units.

A Novel Small Molecular Prostaglandin Receptor EP4 Antagonist, L001, Suppresses Pancreatic Cancer Metastasis.

Metastatic pancreatic cancer remains a major clinical challenge, emphasizing the urgent need for the exploitation of novel therapeutic approaches with superior response. In this study, we demonstrate that the aberrant activation of prostaglandin E2 (PGE2) receptor 4 (EP4) is a pro-metastatic signal in pancreatic cancer. To explore the therapeutic role of EP4 signaling, we developed a potent and selective EP4 antagonist L001 with single-nanomolar activity using a panel of cell functional assays. EP4 antagonism by L001 effectively repressed PGE2-elicited cell migration and the invasion of pancreatic cancer cells in a dose-dependent manner. Importantly, L001 alone or combined with the chemotherapy drug gemcitabine exhibited remarkably anti-metastasis activity in a pancreatic cancer hepatic metastasis model with excellent tolerability and safety. Mechanistically, EP4 blockade by L001 abrogated Yes-associated protein 1 (YAP)-driven pro-metastatic factor expression in pancreatic cancer cells. The suppression of YAP’s activity was also observed upon L001 treatment in vivo. Together, these findings support the notions that EP4–YAP signaling axis is a vital pro-metastatic pathway in pancreatic cancer and that EP4 inhibition with L001 may deliver a therapeutic benefit for patients with metastatic pancreatic cancer.

Activation of regulatory T cell through prostaglandin E<sub>2</sub>-EP4 signaling

Activation of regulatory T cell through prostaglandin E<sub>2</sub>-EP4 signaling

Yersinia pseudotuberculosis YopJ Limits Macrophage Response by Downregulating COX-2-Mediated Biosynthesis of PGE2 in a MAPK/ERK-Dependent Manner.

ABSTRACTProstaglandin E2 (PGE2) is an essential immunomodulatory lipid released by cells in response to infection with many bacteria, yet its function in macrophage-mediated bacterial clearance is poorly understood. Yersinia overall inhibits the inflammatory circuit, but its effect on PGE2 production is unknown. We hypothesized that one of the Yersinia effector proteins is responsible for the inhibition of PGE2 biosynthesis. We identified that yopB-deficient Y. enterocolitica and Y. pseudotuberculosis deficient in the secretion of virulence proteins via a type 3 secretion system (T3SS) failed to inhibit PGE2 biosynthesis in macrophages. Consistently, COX-2-mediated PGE2 biosynthesis is upregulated in cells treated with heat-killed or T3SS-deficient Y. pseudotuberculosis but diminished in the presence of a MAPK/ERK inhibitor. Mutants expressing catalytically inactive YopJ induce similar levels of PGE2 as heat-killed or ΔyopB Y. pseudotuberculosis, reversed by YopJ complementation. Shotgun proteomics discovered host pathways regulated in a YopJ-mediated manner, including pathways regulating PGE2 synthesis and oxidative phosphorylation. Consequently, this study identified that YopJ-mediated inhibition of MAPK signal transduction serves as a mechanism targeting PGE2, an alternative means of inflammasome inhibition by Yersinia. Finally, we showed that EP4 signaling supports macrophage function in clearing intracellular bacteria. In summary, our unique contribution was to determine a bacterial virulence factor that targets COX-2 transcription, thereby enhancing the intracellular survival of yersiniae. Future studies should investigate whether PGE2 or its stable synthetic derivatives could serve as a potential therapeutic molecule to improve the outcomes of specific bacterial infections. Since other pathogens encode YopJ homologs, this mechanism is expected to be present in other infections.IMPORTANCE PGE2 is a critical immunomodulatory lipid, but its role in bacterial infection and pathogen clearance is poorly understood. We previously demonstrated that PGE2 leads to macrophage polarization toward the M1 phenotype and stimulates inflammasome activation in infected macrophages. Finally, we also discovered that PGE2 improved the clearance of Y. enterocolitica. The fact that Y. enterocolitica hampers PGE2 secretion in a type 3 secretion system (T3SS)-dependent manner and because PGE2 appears to assist macrophage in the clearance of this bacterium indicates that targeting of the eicosanoid pathway by Yersinia might be an adaption used to counteract host defenses. Our study identified a mechanism used by Yersinia that obstructs PGE2 biosynthesis in human macrophages. We showed that Y. pseudotuberculosis interferes with PGE2 biosynthesis by using one of its T3SS effectors, YopJ. Specifically, YopJ targets the host COX-2 enzyme responsible for PGE2 biosynthesis, which happens in a MAPK/ER-dependent manner. Moreover, in a shotgun proteomics study, we also discovered other pathways that catalytically active YopJ targets in the infected macrophages. YopJ was revealed to play a role in limiting host LPS responses, including repression of EGR1 and JUN proteins, which control transcriptional activation of proinflammatory cytokine production such as interleukin-1β. Since YopJ has homologs in other bacterial species, there are likely other pathogens that target and inhibit PGE2 biosynthesis. In summary, our study’s unique contribution was to determine a bacterial virulence factor that targets COX-2 transcription. Future studies should investigate whether PGE2 or its stable synthetic derivatives could serve as a potential therapeutic target.

Prostaglandin E2 Regulates Bipotent Monocyte-Dendritic Progenitor Cell Lineage-Commitment.

The factors/mechanisms regulating multipotent or bipotent hematopoietic progenitor cells lineage-commitment are not well understood. In this study, we found that prostaglandin E2 (PGE2) is a crucial physiological regulator of lineage choice for the bipotential monocyte-dendritic progenitor cell (MDP). Inhibition of endogenous PGE2 biosynthesis in mice by the dual cyclooxygenase inhibitor, indomethacin, enhances bone marrow and spleen monocyte (MO) differentiation and reduces dendritic cell (DC) differentiation. Ex vivo treatment of purified MDP with indomethacin preferentially increases MO development at the expense of DC generation, whereas addition of exogenous PGE2 reverses the indomethacin-mediated alteration in MDP differentiation potential. Treatment of MDP with selective EP receptor agonists demonstrated that EP1 signaling promotes MDP differentiation into DC at the expense of MO generation. Conversely, EP1 receptor knockout mice showed reduced DC and increased MO differentiation. Mechanistic studies revealed that PGE2 increases expression of the tyrosine kinase receptor Flt3 on MDP and increases the DC-lineage-related transcription factor PU.1, while reducing expression of M-CSFR and the MO-lineage-related transcription factor MafB. These data indicate that PGE2-EP1 signaling plays a critical role in MDP lineage commitment and DC and MO differentiation.

Autonomic Nerves Regulate Gene Expression Profiles in The Developing Ductus Arteriosus

Background The ductus arteriosus (DA), a bypass vessel between the aorta and main pulmonary artery, is essential for fetal circulation, and immediately closes after birth. Prostaglandin E2 plays important roles in both DA patency and closure via its receptor EP4. During the fetal periods, EP4 signaling dilates the DA through inhibition of myosin light chain kinase and promotes luminal narrowing, i.e., intimal thickening, for complete closure of the DA after birth when placental PGE2 is dramatically decreased. Previous studies have demonstrated that EP4 is prominently expressed in the developing DA and EP4-deficient mice exhibits patent DA after birth, suggesting that EP4 expression in the DA is critical for adaptation from fetal and adult circulatory system. However, the molecular mechanism of specific expression of EP4 in the DA is unknown. It has been reported that sympathetic nerves are distributed around the DA. Therefore, we hypothesized that sympathetic nerves regulated EP4 expression in the DA. In this study, we aimed to investigate the molecular mechanism of EP4 expression in the DA. Methods We generated EP4 reporter mice with EP4-IRES-nlsLacZ. EP4 reporter mice were stained with X-gal, and then tissue sections were prepared. We examined neurite outgrowth by immunohistochemistry with an antibody against polysialylated neural cell adhesion molecule (PSA-NCAM). Distribution of autonomic nerve terminals were detected by immunohistochemistry with an antibody against tyrosine hydroxylase. Results X-gal staining showed that no EP4 expression was observed in the DA at embryonic day 13 (E13). At E14, EP4 expression was observed in the smooth muscle layer of the DA, which was stronger in the outer layer (Figure 1). At E18.5 (full-term), EP4 expression level was significantly higher than pre-term mice and EP4 was uniformly expressed throughout the tunica media of the DA. At postnatal day 2, EP4 expression was decreased throughout the smooth muscle layer of the DA. No visible EP4 expression was observed in the smooth muscle layers of adjacent arteries. i.e., the aorta and pulmonary arteries, and endothelial cells of the DA and adjacent arteries. PSA-NCAM-positive neurites invaded into the adventitia toward the smooth muscle layer of the DA at E13, in which EP4 was not yet expressed. Neurite outgrowth became prominent in the tunica media in the DA at E14 (Figure 2). At full-term, neurites were observed throughout the smooth muscle layer of the DA, which was consistent with the area of the EP4 expression. Neurite outgrowth into the tunica media was not observed in the adjacent arteries. Furthermore, tyrosine hydroxylase was observed in the outer layer of the tunica media and the adventitia in the DA, indicating the presence of autonomic nerve terminals in the DA. Conclusion Sympathetic nerves may regulate specific expression of EP4 in the DA. These data suggest the novel epigenetic regulation of autonomic nerve-mediated vascular development.

TNF-α impairs EP4 signaling through the association of TRAF2-GRK2 in primary fibroblast-like synoviocytes.

Our previous study showed that chronic treatment with tumor necrosis factor-α (TNF-α) decreased cAMP concentration in fibroblast-like synoviocytes (FLSs) of collagen-induced arthritis (CIA) rats. In this study we investigated how TNF-α impairs cAMP homeostasis, particularly clarifying the potential downstream molecules of TNF-α and prostaglandin receptor 4 (EP4) signaling that would interact with each other. Using a cAMP FRET biosensor PM-ICUE3, we demonstrated that TNF-α (20 ng/mL) blocked ONO-4819-triggered EP4 signaling, but not Butaprost-triggered EP2 signaling in normal rat FLSs. We showed that TNF-α (0.02-20 ng/mL) dose-dependently reduced EP4 membrane distribution in normal rat FLS. TNF-α significantly increased TNF receptor 2 (TNFR2) expression and stimulated proliferation in human FLS (hFLS) via ecruiting TNF receptor-associated factor 2 (TRAF2) to cell membrane. More interestingly, we revealed that TRAF2 interacted with G protein-coupled receptor kinase (GRK2) in the cytoplasm of primary hFLS and helped to bring GRK2 to cell membrane in response of TNF-α stimulation, the complex of TRAF2 and GRK2 then separated on the membrane, and translocated GRK2 induced the desensitization and internalization of EP4, leading to reduced production of intracellular cAMP. Silencing of TRAF2 by siRNA substantially diminished TRAF2-GRK2 interaction, blocked the translocation of GRK2, and resulted in upregulated expression of membrane EP4 and intracellular cAMP. In CIA rats, administration of paroxetine to inhibit GRK2 effectively improved the symptoms and clinic parameters with significantly reduced joint synovium inflammation and bone destruction. These results elucidate a novel form of cross-talk between TNFR (a cytokine receptor) and EP4 (a typical G protein-coupled receptor) signaling pathways. The interaction between TRAF2 and GRK2 may become a potential new drug target for the treatment of inflammatory diseases.

Characterization of the Signaling Modalities of Prostaglandin E2 Receptors EP2 and EP4 Reveals Crosstalk and a Role for Microtubules.

Prostaglandin E2 (PGE2) is a lipid mediator that modulates the function of myeloid immune cells such as macrophages and dendritic cells (DCs) through the activation of the G protein-coupled receptors EP2 and EP4. While both EP2 and EP4 signaling leads to an elevation of intracellular cyclic adenosine monophosphate (cAMP) levels through the stimulating Gαs protein, EP4 also couples to the inhibitory Gαi protein to decrease the production of cAMP. The receptor-specific contributions to downstream immune modulatory functions are still poorly defined. Here, we employed quantitative imaging methods to characterize the early EP2 and EP4 signaling events in myeloid cells and their contribution to the dissolution of adhesion structures called podosomes, which is a first and essential step in DC maturation. We first show that podosome loss in DCs is primarily mediated by EP4. Next, we demonstrate that EP2 and EP4 signaling leads to distinct cAMP production profiles, with EP4 inducing a transient cAMP response and EP2 inducing a sustained cAMP response only at high PGE2 levels. We further find that simultaneous EP2 and EP4 stimulation attenuates cAMP production, suggesting a reciprocal control of EP2 and EP4 signaling. Finally, we demonstrate that efficient signaling of both EP2 and EP4 relies on an intact microtubule network. Together, these results enhance our understanding of early EP2 and EP4 signaling in myeloid cells. Considering that modulation of PGE2 signaling is regarded as an important therapeutic possibility in anti-tumor immunotherapy, our findings may facilitate the development of efficient and specific immune modulators of PGE2 receptors.

Activation of EP4 receptor limits transition of acute to chronic heart failure in lipoxygenase deficient mice.

Aim: Immune responsive 12/15 lipoxygenase (12/15LOX)-orchestrate biosynthesis of essential inflammation-resolution mediators during acute inflammatory response in post-myocardial infarction (MI). Lack of 12/15LOX dampens proinflammatory mediator 12-(S)-hydroxyeicosatetraenoic acid (12-(S)-HETE), improves post-MI survival, through the biosynthesis of endogenous mediators epoxyeicosatrienoic acids (EETs; cypoxins) to resolve post-MI inflammation. However, the mechanism that amplifies cypoxins-directed cardiac repair in acute heart failure (AHF) and chronic HF (CHF) remains of interest in MI-directed renal inflammation. Therefore, we determined the role of EETs in macrophage-specific receptor activation in facilitating cardiac repair in 12/15LOX deficient mice experiencing HF.Methods and Results: Risk-free young adult (8 -12 week-old) male C57BL/6J wild-type mice (WT; n = 43) and 12/15LOX-/- mice (n = 31) were subjected to permanent coronary artery ligation and monitored at day (d)1, d5 (as acute HF), and d28 to d56 (8 weeks; chronic HF) post-surgery maintaining no-MI mice that served as d0 naïve controls. Left ventricle (LV) infarcted area of 12/15LOX-/- mice displayed an increase in expression of prostanoid receptor EP4 along with monocyte chemoattractant protein-1 CCL2 in AHF and CHF. The transcriptome analysis of isolated leukocytes (macrophages/neutrophils) from infarcted LV revealed a higher expression of EP4 on reparative macrophages expressing MRC-1 in 12/15LOX-/- mice. Deletion of 12/15LOX differentially modulated the miRNA levels, downregulating miR-23a-3p (~20 fold; p < 0.05) and upregulating miR-125a-5p (~160 fold; p < 0.05) in AHF which promoted polarization of the macrophages towards reparative phenotype. Furthermore, 12/15LOX deletion markedly attenuated renal inflammation with reduced levels of NGAL and KIM-1 and apoptotic markers in the kidney during CHF.Conclusion: In risk-free mice during physiological cardiac repair, absence of 12/15LOX promoted reparative macrophages with marked activation of EP4 signaling thereby improving post-MI survival and limiting renal inflammation in acute and advanced HF. The future studies are warranted to advance the role of EETs in macrophage receptor biology.

Crosstalk between the COX2-PGE2-EP4 signaling pathway and primary cilia in osteoblasts after mechanical stimulation.

Primary cilia have been found to function as mechanosensors in low-magnitude high-frequency vibration (LMHFV)-induced osteogenesis. The PGE2 also regulates bone homeostasis and mechanical osteogenesis through its receptor EP4 signaling, but its involvement in LMHFV-induced or in primary cilia-induced osteogenesis has not been investigated. We hypothesized that LMHFV stimulates osteoblast (OB) differentiation by activating the COX2-PGE2-EP pathway in a manner dependent on primary cilia and that primary cilia are also affected by the PGE2 pathway. In this study, through western blot analysis, RNAinterference, enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, and cytochemical staining, we observed that COX2, mPGES-1,and PGE2 levels were markedly elevated in cells treated with LMHFV and were greatly decreased in LMHFV-treated cells following IFT88 silencing. EP4 expression was significantly increased in OBs following LMHFV treatment, but IFT88 silencing significantly blocked this increase. EP4 localized to the bases of primary cilia. LMHFV reduced the length and abundance of primary cilia, but the cells could self-repair their primary cilia after mechanical damage. EP4 antagonism significantly blocked the LMHFV-induced increase in IFT88 expression and blocked the recovery of primary cilia length and the proportion of cells with primary cilia. In addition, COX2 or EP4 antagonism disrupted LMHFV-induced osteogenesis. These results demonstrate the integration of and crosstalk between primary cilia and the COX2-PGE2-EP4 signaling pathway under mechanical stimulation.

1759-P: Prostaglandin PGE2 Receptor EP4 Signaling Deficiency in Microglia Reduces Weight Gain with Transient Glucose Improvements in Diet-Induced Obesity

High fat diet (HFD) triggers a microglial inflammatory response in the mediobasal hypothalamus that is associated with weight gain and glucose dysregulation. Microglia depletion or selective disruption of the NFkB signaling pathway in microglia during HFD prevents the inflammatory response, and reduces food intake and weight gain. Here, we investigated the role of microglial prostaglandin PGE2 receptor 4 (EP4) signaling on the inflammatory response to HFD and metabolic outcomes. Compared to their wildtype counterparts, microglia-specific EP4 knockout (MG EP4-KO) mice on HFD showed reduced weight gain and improved glucose tolerance at 5 weeks. However, the two groups did not differ in glucose tolerance at 12 weeks, despite the MG EP4-KO mice maintaining lower body weight. This decoupling of body weight and glucose impairment indicates a progressive deterioration of glucose homeostasis associated with EP4 deficiency in microglia. Overall, these findings unravel a complex and dynamic interplay between EP4 signaling in microglia, weight gain and glucose imbalance in HFD. Disclosure A. Niraula: None. R. Fasnacht: None. K.M. Ness: None. J. Frey: None. M.D. Dorfman: None. J. Thaler: None. Funding National Institutes of Health (R01DK119754)

Prostaglandin E2 receptor 3 (EP3) signaling promotes migration of cervical cancer via urokinase-type plasminogen activator receptor (uPAR)

PurposeCervical cancer metastasis results in poor prognosis and increased mortality, which is not separated from inflammatory reactions accumulated by prostaglandin E2 (PGE2). As a specific G-protein coupled PGE2 receptor, EP3 is demonstrated as a negative prognosticator of cervical malignancy. Now, we aimed to investigate the pathological mechanism of EP3 in modulating cervical cancer carcinogenesis.MethodsBioinformatics analysis was used to identify PAI-1 and uPAR correlations with EP3 expression, as well as the prognosis of cervical cancer patients. In vitro analyses were carried out to investigate the role of EP3 on cervical cancer proliferation and migration.ResultsIn vitro studies showed that sulprostone (an EP3 agonist) enhanced the proliferation and migration of cervical cancer cells, whereas silencing of EP3 inhibited their proliferation and migration. Furthermore, EP3 knockdown increased the expression of plasminogen activator inhibitor type 1 (PAI-1), urokinase-type plasminogen activator receptor (uPAR), and phosphorylated extracellular signal-regulated kinases 1/2 (p-ERK1/2), but decreased p53 expression. Bioinformatics analysis showed that both PAI-1 and uPAR were correlated with EP3 expression, as well as the prognosis of cervical cancer patients. The survival analysis further showed that uPAR overexpression (IRS≥2) was correlated with a lower overall survival rate of cervical cancer patients with advanced stages (FIGO III-IV).ConclusionThese results indicated that EP3 signaling pathway might facilitate the migration of cervical cancer cells through modulating uPAR expression. Therefore, EP3 and uPAR could represent novel therapeutic targets in the treatment of cervical cancer in advantaged stages.

Concerted EP2 and EP4 Receptor Signaling Stimulates Autocrine Prostaglandin E2 Activation in Human Podocytes.

Glomerular hyperfiltration is an important mechanism in the development of albuminuria. During hyperfiltration, podocytes are exposed to increased fluid flow shear stress (FFSS) in Bowman’s space. Elevated Prostaglandin E2 (PGE2) synthesis and upregulated cyclooxygenase 2 (Cox2) are associated with podocyte injury by FFSS. We aimed to elucidate a PGE2 autocrine/paracrine pathway in human podocytes (hPC). We developed a modified liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) protocol to quantify cellular PGE2, 15-keto-PGE2, and 13,14-dihydro-15-keto-PGE2 levels. hPC were treated with PGE2 with or without separate or combined blockade of prostaglandin E receptors (EP), EP2, and EP4. Furthermore, the effect of FFSS on COX2, PTGER2, and PTGER4 expression in hPC was quantified. In hPC, stimulation with PGE2 led to an EP2- and EP4-dependent increase in cyclic adenosine monophosphate (cAMP) and COX2, and induced cellular PGE2. PTGER4 was downregulated after PGE2 stimulation in hPC. In the corresponding LC/ESI-MS/MS in vivo analysis at the tissue level, increased PGE2 and 15-keto-PGE2 levels were observed in isolated glomeruli obtained from a well-established rat model with glomerular hyperfiltration, the Munich Wistar Frömter rat. COX2 and PTGER2 were upregulated by FFSS. Our data thus support an autocrine/paracrine COX2/PGE2 pathway in hPC linked to concerted EP2 and EP4 signaling.

Cyclooxygenase-2 dependent prostaglandin E2 signaling through the EP3 receptor is required for cell-mediated immunity to Listeria monocytogenes

Abstract The bacterial pathogen Listeria monocytogenes (Lm) is being developed as a cancer immunotherapeutic platform due to its ability to elicit robust CD8+ T cell responses. Though the role of cytokines in Lm-stimulated immunity is well studied, the role of eicosanoids, lipid modulators of inflammation, is unclear. We previously demonstrated that cyclooxygenase-2 (COX-2) dependent prostaglandin E2 (PGE2) is essential for optimal T-cell priming. Antigen specific T cells and protective immunity were decreased following either treatment with the COX-2 inhibitor celecoxib or immunization of mice deficient in microsomal PGE2 synthase-1. Analysis of splenic eicosanoid levels revealed an upregulation of PGE2 twelve hours post immunization and administration of exogenous PGE2 at this time restored immunity during COX-2 inhibition. To understand how PGE2 impacts T-cell priming we assessed the role of the receptors EP1–4 in mediating this response using small molecule agonists and antagonists. EP3 blockade impaired immune responses while the use of an EP3 specific agonist restored immunity during COX-2 inhibition, suggesting that PGE2 signaling through EP3 is critical for T-cell priming. Conversely, consistent with its predicted inverse role in cAMP signaling, EP4 blockade enhanced responses, providing a mechanism by which to improve Listeria-based immunotherapeutics. Ongoing studies with floxed COX-2, EP3 and EP4 mice will illuminate the cell types producing and responding to PGE2. In conclusion, PGE2 signaling through EP3 is necessary Lm-stimulated immunity while EP4 signaling is detrimental. Further understanding these signaling pathways and where they function will be key to optimizing Lm as therapeutic platform.

EP3 signaling in dendritic cells promotes liver repair by inducing IL-13-mediated macrophage differentiation in mice.

Macrophage plasticity is essential for liver wound healing; however, the mechanisms underlying macrophage phenotype switching are largely unknown. Dendritic cells (DCs) are critical initiators of innate immune responses; as such, they orchestrate inflammation following hepatic injury. Here, we subjected EP3-deficient (Ptger3-/- ) and wild-type (WT) mice to hepatic ischemia-reperfusion (I/R) and demonstrate that signaling via the prostaglandin E (PGE) receptor EP3 in DCs regulates macrophage plasticity during liver repair. Compared with WT mice, Ptger3-/- mice showed delayed liver repair accompanied by reduced expression of hepatic growth factors and accumulation of Ly6Clow reparative macrophages and monocyte-derived DCs (moDCs). MoDCs were recruited to the boundary between damaged and undamaged liver tissue in an EP3-dependent manner. Adoptive transfer of moDCs from Ptger3-/- mice resulted in impaired repair, along with increased numbers of Ly6Chigh inflammatory macrophages. Bone marrow macrophages (BMMs) up-regulated expression of genes related to a reparative macrophage phenotype when co-cultured with moDCs; this phenomenon was dependent on EP3 signaling. In the presence of an EP3 agonist, interleukin (IL)-13 derived from moDCs drove BMMs to increase expression of genes characteristic of a reparative macrophage phenotype. The results suggest that EP3 signaling in moDCs facilitates liver repair by inducing IL-13-mediated switching of macrophage phenotype from pro-inflammatory to pro-reparative.