EP4 Agonist Research Articles

Prostaglandin E2 switches from a stimulator to an inhibitor of cell migration after epithelial-to-mesenchymal transition

Epithelial–mesenchymal transition (EMT) is critical for embryonic development, and this process is recapitulated in adults during wound healing, tissue regeneration, fibrosis and cancer progression. Cell migration is believed to play a key role in both normal wound repair and in abnormal tissue remodeling. Prostaglandin E2 (PGE2) inhibits fibroblast chemotaxis, but stimulates chemotaxis in airway epithelial cells. The current study was designed to explore the role of PGE2 and its four receptors on airway epithelial cell migration following EMT using both the Boyden blindwell chamber chemotaxis assay and the wound closure assay. EMT in human bronchial epithelial cells (HBECs) was induced by TGF-β1 and a mixture of cytokines (IL-1β, TNF-α, and IFN-γ). PGE2 and selective agonists for all four EP receptors stimulated chemotaxis and wound closure in HBECs. Following EMT, the EP1 and EP3 agonists were without effect, while the EP2 and EP4 agonists inhibited chemotaxis as did PGE2. The effects of the EP2 and EP4 receptors on HBEC and EMT cell migration were further confirmed by blocking the expected signaling pathways. Taken together, these results demonstrate that PGE2 switches from a stimulator to an inhibitor of cell migration following EMT of airway epithelial cells and that this inhibition is mediated by an altered effect of EP2 and EP4 signaling and an apparent loss of the stimulatory effects of EP1 and EP3. Change in the PGE2 modulation of chemotaxis may play a role in repair following injury.

Prostaglandin E2 EP1 receptor enhances TGF-β1-induced mesangial cell injury.

Increasing evidence indicates that transforming growth factor-β1 (TGF-β1) is a pivotal mediator in the pathogenesis of renal fibrosis. Mesangial cells (MCs) are important for glomerular function under both physiological and pathological conditions. Studies have found that the expression level of prostaglandin E2 (PGE2) in MCs increases under high glucose conditions, that PGE2 affects the proliferation and hypertrophy of MCs mainly through the EP1 pathway, and that the proliferation of MCs and the accumulation of extracellular matrix are the main events leading to glomerular fibrosis. In this study, we investigated the effects and mechanisms of action of the EP1 receptor, which is induced by transforming growth factor (TGF)-β1, on the proliferation of mouse MCs, the accumulation of extracellular matrix and the expression of PGE2 synthase. Primary mouse glomerular MCs were isolated from EP1 receptor-deficient mice (EP1-/- mice, in which the EP1 receptor was knocked down) and wild-type (WT) mice (WT MCs). In our preliminary experiments, we found that cell proliferation, as well as the mRNA and protein expression of cyclin D1, proliferating cell nuclear antigen (PCNA), fibronectin (FN), collagen I (ColI), membrane-associated PGE2 synthase-1 (mPGES-1) and cyclooxygenase-2 (COX-2) in the WT MCs were significantly increased following treatment with 10 ng/ml TGF-β1 for 24 h. Compared with the WT MCs, following the knockdown of the EP1 gene, the TGF-β1-induced MC injury was markedly suppressed. The aforementioned changes were notably enhanced following treatment with the EP1 agonist, 17-phenyl trinor PGE2 ethyl amide. Additionally, TGF-β1 induced extracellular signal-regulated kinase (ERK) phosphorylation. We found that the TGF-β1-induced ERK phosphorylation was alleviated by EP1 knockdown and promoted by EP1 expression. These results suggest that the EP1 receptor plays a role in the proliferation of mouse MCs, in the accumulation of extracellular matrix and in the expression of mPGES-1 induced by TGF-β1. Its mechanisms of action are possibly related to the reinforcement of ERK phosphorylation.

Prostaglandin E2 stimulates β1-integrin expression in hepatocellular carcinoma through the EP1 receptor/PKC/NF-κB pathway.

Prostaglandin E2 (PGE2) has been implicated in cell invasion in hepatocellular carcinoma (HCC), via increased β1-integrin expression and cell migration; however, the mechanism remains unclear. PGE2 exerts its effects via four subtypes of the E prostanoid receptor (EP receptor 1–4). The present study investigated the effect of EP1 receptor activation on β1-integrin expression and cell migration in HCC. Cell migration increased by 60% in cells treated with 17-PT-PGE2 (EP1 agonist), which was suppressed by pretreatment with a β1-integrin polyclonal antibody. PGE2 increased β1-integrin expression by approximately 2-fold. EP1 receptor transfection or treatment with 17-PT-PGE2 mimicked the effect of PGE2 treatment. EP1 siRNA blocked PGE2-mediated β1-integrin expression. 17-PT-PGE2 treatment induced PKC and NF-κB activation; PKC and NF-κB inhibitors suppressed 17-PT-PGE2-mediated β1-integrin expression. FoxC2, a β1-integrin transcription factor, was also upregulated by 17-PT-PGE2. NF-κB inhibitor suppressed 17-PT-PGE2-mediated FoxC2 upregulation. Immunohistochemistry showed p65, FoxC2, EP1 receptor and β1-integrin were all highly expressed in the HCC cases. This study suggested that PGE2 upregulates β1-integrin expression and cell migration in HCC cells by activating the PKC/NF-κB signaling pathway. Targeting PGE2/EP1/PKC/NF-κB/FoxC2/β1-integrin pathway may represent a new therapeutic strategy for the prevention and treatment of this cancer.

Prostanoid induces premetastatic niche in regional lymph nodes.

The lymphatic system is an important route for cancer dissemination, and lymph node metastasis (LNM) serves as a critical prognostic determinant in cancer patients. We investigated the contribution of COX-2-derived prostaglandin E2 (PGE2) in the formation of a premetastatic niche and LNM. A murine model of Lewis lung carcinoma (LLC) cell metastasis revealed that COX-2 is expressed in DCs from the early stage in the lymph node subcapsular regions, and COX-2 inhibition markedly suppressed mediastinal LNM. Stromal cell-derived factor-1 (SDF-1) was elevated in DCs before LLC cell infiltration to the lymph nodes, and a COX-2 inhibitor, an SDF-1 antagonist, and a CXCR4 neutralizing antibody all reduced LNM. Moreover, LNM was reduced in mice lacking the PGE2 receptor EP3, and stimulation of cultured DCs with an EP3 agonist increased SDF-1 production. Compared with WT CD11c+ DCs, injection of EP3-deficient CD11c+ DCs dramatically reduced accumulation of SDF-1+CD11c+ DCs in regional LNs and LNM in LLC-injected mice. Accumulation of Tregs and lymph node lymphangiogenesis, which may influence the fate of metastasized tumor cells, was also COX-2/EP3-dependent. These results indicate that DCs induce a premetastatic niche during LNM via COX-2/EP3-dependent induction of SDF-1 and suggest that inhibition of this signaling axis may be an effective strategy to suppress premetastatic niche formation and LNM.

Downregulation of the proangiogenic prostaglandin E receptor EP3 and reduced angiogenesis in a mouse model of diabetes mellitus

Vascular complications such as foot ulcers are a hallmark of diabetes mellitus (DM), although the molecular mechanisms that underlie vascular dysfunction remain unclear. Herein, we show that angiogenesis, which is indispensable to the healing of ulcers, is suppressed in polyurethane sponge implants in mice with DM and reduced proangiogenic signaling. DM was induced in male C57BL/6 mice by intraperitoneal injection of streptozotocin (100mg/kg). Polyurethane sponge disks were implanted into subcutaneous tissues on the backs of mice, and angiogenesis and expression of related factors were analyzed in sponge granulation tissues. Densities of platelet endothelial cell adhesion molecule-1 (PECAM-1)-positive vascular structures and PECAM-1 expression in sponge granulation tissues were increased over time in control mice and reduced in diabetic mice. The reductions in diabetic mice were accompanied by reduced expression of inducible cyclo-oxygenase-2 and microsomal prostaglandin E synthase-1. The prostaglandin E receptor subtype EP3 was downregulated in sponge granulation tissues in diabetic mice, whereas EP1, EP2, and EP4 were not. The expression of the proangiogenic growth factor vascular endothelial growth factor (VEGF)-A and the chemokine stromal cell-derived factor-1 (SDF-1) were both reduced in diabetic mice. Treatment of diabetic mice with a selective agonist of EP3, ONO-AE 248 (30 nmol/site/day, topical injection), reversed suppression of angiogenesis in diabetic mice. These results indicate that proangiogenic EP3 signaling is suppressed in diabetic mice with reduced expression of VEGF and SDF-1. Stimulation of EP3 signaling restored angiogenesis in a sponge implant model in mice with DM. This suggests that topical application of an EP3 agonist could be a novel strategy to treat foot ulcers in patients with DM.

Abstract LB-67: Breast cancer-associated lymphangiogenesis: roles of PGE2 and EP4 receptor on lymphatic endothelial cells

Abstract Background: Lymphatic metastasis is a common event in breast cancer, facilitated by tumor-associated lymphangiogenesis, molecular mechanisms of which remain poorly defined. We had earlier shown that endogenous PGE2 resulting from elevated COX-2 expression by breast cancer cells promotes tumor progression and metastasis by multiple mechanisms: inactivation of host immune cells, stimulation of tumor cell migration, tumor-associated angiogenesis and lymphangiogenesis. The latter was due to upregulation of VEGF-C or -D secretion due to activation of EP4 receptors on breast cancer cells and cancer-infiltrating macrophages. Objectives: To examine whether PGE2 in the tumor micro-environment directly promotes lymphangiogenesis by stimulating EP4 receptors on lymphatic endothelial cells (LEC).Approaches: In vitro studies utilized a rat mesenteric lymphatic endothelial cell line (RMLEC) and a COX-2 expressing, VEGF-C/-D producing murine breast cancer cell line C3L5; in vivo studies measured lymphangiogenesis in nude mice. Results RMLEC expressed all EP receptors and responded to PGE2, an EP4 agonist (EP4a) PGE1-OH, or C3L5 cell conditioned media (C3L5-CM) by increased proliferation, migration and accelerated tube formation on growth factor-reduced matrigel. Tube formation on matrigel alone was completely abrogated in the presence of COX 1/2 inhibitor indomethacin (10uM), COX-2 inhibitor (COX-2i) NS-398 (15uM), and a selective EP4 antagonist (EP4A) RQ15986 (2.5 nM), indicating the roles of COX-2 and EP4. Addition of PGE2, EP4a, or C3L5-CM individually in presence of the COX-2i, or EP4A, partially restored the tube formation, reinforcing the role of EP4 on RMLEC. RMLEC grown in the presence of PGE2 showed upregulation of COX2, EP4, VEGF-C and VEGF-D mRNAs. Knocking down EP4 with shRNA in RMLEC abrogated tube formation on matrigel in the absence or presence of PGE-2, EP4a, or C3L5-CM. EP4 silenced RMLEC also became unresponsive to these agents in stimulating PGE2, VEGF-C, and -D production. Finally in a directed in vivo lymphangiogenesis assay (DIVLA) using implants of angioreactors including PGE-2, EP4a or VEGF-C in the dorsal flanks of nude mice, we simultaneously measured lymphangiogenesis and angiogenesis using dual immunostaining of the tissue contents for Lyve-1 and cd31, or prox-1 and cd31. Results demonstrated the lymphangiogenic as well as angiogenic capacity of PGE2 and EP4a in vivo by recruitment of new vessels. Conclusions: Tumor or host-derived PGE2 stimulates lymphangiogenesis, at least in part, by activating EP4 on the LEC. Combined with earlier studies we show that EP4 is a common therapeutic target on tumor and host cells (macrophages and LEC) in abrogating multiple events in breast cancer progression. This may spare cardio-protective prostanoids like PGI2, inhibited by COX-2 inhibitors Citation Format: Peeyush K. Lala, Gannareddy V. Girish, Xiping Xin, Mousumi Majumder, Elena Tutunea-Fatan, Pinki Nandi. Breast cancer-associated lymphangiogenesis: roles of PGE2 and EP4 receptor on lymphatic endothelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-67. doi:10.1158/1538-7445.AM2014-LB-67

Abstract 5217: The role of miR-526b in COX-2 mediated breast cancer progression via EP4 signaling

Abstract A minor tumour cell subset, known as stem-like cells (SLCs), appears to defy conventional therapy, causing relapse. Thus, identification of SLC-specific therapeutic targets and markers is urgently needed. Our laboratory has established that aberrant expression of COX-2 promotes breast cancer progression via multiple mechanisms including SLC induction owing to activation of the PGE receptor EP4 which signals via the canonical cAMP/PKA, and non-canonical PI3K-AKT pathways. Differential gene and microRNA (miR) expression micro arrays in human breast cancer cell lines stably transfected to over-express COX-2 revealed up-regulation of miR-526b. We sought to determine if miR-526b is induced by EP4 activation and whether it promotes breast cancer progression, including SLC stimulation. COX-2-ve human breast cancer cell lines MCF-7 (ER+, HER-2-) and SKBR-3 (ER-, HER-2+) were stably transfected to over-express miR-526b (MCF7-526b and SKBR3-526b), and functional assays were performed. The cells exhibited enhanced proliferation, migration and invasiveness, and increased tumoursphere-forming efficiency on ultra-low attachment plates (in vitro surrogate of SLC phenotype), compared to empty vector-transfected and parental controls. These results indicate that miR-526b is a COX-2 induced oncogenic microRNA. In order to assess the roles of miR-526b in vivo, MCF7-526b, SKBR3-526b, or empty vector control cells were injected into the tail vein of NOD/SCID/GUSB-null female mice. Both miR-526b over-expressing cell lines revealed a significantly increased ability to form proliferative lung colonies at 4 weeks identified with HLA staining and EdU proliferation marker. To examine the role of EP4 activation in miR-526b up-regulation, MCF-7 cells cultured as monolayers or tumourspheres were treated with PGE2 or an EP4 agonist, PGE1OH. Both ligands induced a significant over-expression of miR-526b in both culture conditions. Conversely, MCF7-COX-2 cells treated with either an EP4 specific antagonist (ONO-AE3-208) or a COX-2 inhibitor (NS-398) displayed significant reduction in miR-526b expression, as quantified with qPCR. To investigate the role of PI3K-AKT pathway of EP4 signaling in miR-526b expression, PI3K was stimulated with PGE2 or PGE1OH in MCF7 cells, and inhibited with LY294002 or Wortmannin in MCF7-COX-2 cells. MiR-526b expression levels quantified with qPCR revealed significant increases and declines, respectively, with PI3K-AKT pathway stimulators and inhibitors applied to the above cell lines. Together these results suggest an important association of miR-526b with EP4-induced SLC stimulation and breast cancer progression, and the possibility that miR-526b may be used as a biomarker for monitoring patients and personalizing therapy. (Supported by the CBCF, Ontario chapter and the OICR funds to PKL. EL holds a CBCF and CIHR-STP Graduate Fellowship and MM a TBCRU and CIHR-STP Post Doctoral fellowship). Citation Format: Erin O. Landman, Mousumi Majumder, Ling Liu, Peeyush K. Lala. The role of miR-526b in COX-2 mediated breast cancer progression via EP4 signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5217. doi:10.1158/1538-7445.AM2014-5217

Abstract 3905: Stem like cells in human breast cancer: EP4 as a therapeutic target

Abstract Background: A tumor cell subset known as stem like cells (SLC) defy conventional therapies, requiring identification of SLC-specific therapeutic targets. We established that elevated COX-2 expression by breast cancer cells promotes tumor progression via inactivation of host immune cells, enhancement of cancer cell migration and invasiveness, and promotion of tumor-associated angiogenesis and lymphangiogenesis primarily by activation of the PGE-2 receptor EP4 on tumor and host cells. Cardiovascular side effects of long term use of COX-2 inhibitors argued for an alternative target, potentially EP4. Objectives/approaches: We asked whether COX-2 or EP4 additionally stimulates SLC in breast cancer. COX-2 and HER-2, often co-expressed in human breast cancer, are both major determinants of cancer progression. To define the roles of COX-2 in HER2 +ve and -ve cells, COX-2 cDNA was stably transfected into COX-2-low, HER-2-ve, ER+ve, non-metastatic MCF-7 and COX-2-ve, ER-ve, HER-2-high SKBR-3 human breast cancer cell lines to derive MCF-7-COX-2 and SKBR-3-COX-2 cells which were tested for changes in phenotype and functions in vitro and in vivo. Results: Both COX-2 over-expressing lines showed (1) EMT (low E-Cadherin and high Vimentin, Twist and N-Cadherin), (2) higher proliferation/ migration/ invasion, (3) upregulation of EP4 and angiogenic/ lymphangiogenic factors VEGF-A/C/D, (4) markedly increased SLC contents indicated by clonogenic spheroid formation for successive generations in vitro, increased ALDH activity and co-expression of COX-2 and multiple SLC markers in spheroids. (5) COX-2 expression markedly increased during spheroid formation in COX-2 disparate cell lines, indicating a selection of COX-2 expressing cells. (6) EP4 activation of COX-2 low cell lines with PGE2 or EP4 agonists markedly increased spheroid-forming efficiency. (7) Functional changes listed above (2-5) were blocked with COX-2 inhibitors or an EP4 antagonist (EP4A), indicating dependence on COX-2 and EP4 activity. (8) MCF-7-COX-2 cells showed dramatically increased lung colony forming ability and growth at 4-6 wks in NOD/SCID/GUSB null mice (identified by GUSB staining), which were blocked with EP4A treatment or EP4 knockdown; (9) MCF-7-COX-2 (as low as 5000) cells showed increased orthotopic tumorigenicity in NOD/SCID/IL-2Rγ deficient mice on transplantation for successive generations. (10) Finally, expression of COX-2 and EP4 were positively correlated with the SLC marker ALDH in human breast cancer tissues, and associated with poor patient survival. Conclusion/ Significance: EP4 represents a novel therapeutic target to eradicate SLCs in breast cancer. EP4 antagonists may spare cardiovascular side effects of COX-2 inhibitors, primarily attributed to cardio protective effects of PGI2. (Supported by the CBCF, Ontario Chapter and OICR grants to PKL, TBCRU and CIHR-STP fellowships to MM, CBCF fellowship to EL and a gift of EP4A, RQ-15986 by RaQualia Pharma). Citation Format: Mousumi Majumder, Xiping Xin, Ling Liu, Gillian Bell, Erin Landman, Mauricio Rodriguez- Torres, Lynne-Marie Postovit, David Hess, Peeyush K. Lala. Stem like cells in human breast cancer: EP4 as a therapeutic target. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3905. doi:10.1158/1538-7445.AM2014-3905

PGE2 reduces MMP-14 and increases plasminogen activator inhibitor-1 in cardiac fibroblasts

Prostaglandin E2 (PGE2) is elevated during cardiac injury and we have previously shown that mice lacking the PGE2 EP4 receptor display dilated cardiomyopathy (DCM) with increased expression of the membrane type matrix metalloproteinase, MMP-14. We thus hypothesized that PGE2 regulates expression of MMP-14 and also affects fibroblast migration. Primary cultures of neonatal rat ventricular fibroblasts (NVFs) were used to test the effects of PGE2. Gene and protein expression was assessed by real time RT-PCR and Western blot, MMP activity was determined by zymography and migration of NVF was assessed by motility in a transwell system. PGE2 reduced expression of MMP-14 and these effects were antagonized by an EP4 antagonist. An EP4 agonist mimicked the effect of PGE2. PGE2 also increased mRNA and protein levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of MMP activation. However, PGE2-stimulation of PAI-1 was mediated by the EP1/EP3 receptor and not EP4. Migration of NVF was assessed by motility in a transwell system. Treatment of NVFs with PGE2 reduced the number of cells migrating toward 10% FCS. Treatment with the EP2 agonist also reduced migration but did not affect MMP-14 expression or PAI-1. Our results suggest that PGE2 utilizes different receptors and mechanisms to ultimately decrease MMP expression and NVF migration.

Multiple roles of the PGE2 -EP receptor signal in vascular permeability.

PGE2 is a major prostanoid that regulates inflammation by stimulating EP1-4 receptors. However, how PGE2 induces an initial inflammatory response to vascular hyper-permeability remains unknown. Here we investigated the role of the PGE2 -EP receptor signal in modulating vascular permeability both in vivo and in vitro. We used a modified Miles assay and intravital microscopy to examine vascular permeability in vivo. Endothelial barrier property was assessed by measuring transendothelial electrical resistance (TER) in vitro. Local administration of PGE2 , an EP2 or EP4 receptor agonist into FVB/NJcl mouse ear skin caused vascular leakage, indicated by dye extravasation. Intravital microscopy and laser Doppler blood-flow imaging revealed that these treatments dilated peripheral vessels and increased local blood flow. Pretreatment with the vasoconstrictor phenylephrine inhibited the PGE2 -induced blood flow increase and vascular leakage. In contrast to the EP2 and EP4 receptor agonists, administration of an EP3 receptor agonist suppressed vascular leakage without altering vascular diameter or blood flow. In isolated HUVECs, the EP3 receptor agonist elevated TER and blocked thrombin-induced dextran passage. Inhibiting PKA restored the hypo-permeability induced by the EP3 receptor agonist. Activation of the PGE2 -EP2 or -EP4 receptor signal induces vasodilatation in mural cells, resulting in increased local blood flow and hyper-permeability. In contrast, activation of the PGE2 -EP3 receptor signal induces a cAMP-dependent enhancement of the endothelial barrier, leading to hypo-permeability. We provide the first evidence that endothelial cells and mural cells cooperate to modulate vascular permeability.

Downregulation of COX-2 and CYP 4A signaling by isoliquiritigenin inhibits human breast cancer metastasis through preventing anoikis resistance, migration and invasion

Flavonoids exert extensive in vitro anti-invasive and in vivo anti-metastatic activities. Anoikis resistance occurs at multiple key stages of the metastatic cascade. Here, we demonstrate that isoliquiritigenin (ISL), a flavonoid from Glycyrrhiza glabra, inhibits human breast cancer metastasis by preventing anoikis resistance, migration and invasion through downregulating cyclooxygenase (COX)-2 and cytochrome P450 (CYP) 4A signaling. ISL induced anoikis in MDA-MB-231 and BT-549 human breast cancer cells as evidenced by flow cytometry and the detection of caspase cleavage. Moreover, ISL inhibited the mRNA expression of phospholipase A2, COX-2 and CYP 4A and decreased the secretion of prostaglandin E2 (PGE2) and 20-hydroxyeicosatetraenoic acid (20-HETE) in detached MDA-MB-231 cells. In addition, it decreased the levels of phospho-PI3K (Tyr458), phospho-PDK (Ser241) and phospho-Akt (Thr308). Conversely, the exogenous addition of PGE2, WIT003 (a 20-HETE analog) and an EP4 agonist (CAY10580) or overexpression of constitutively active Akt reversed ISL-induced anoikis. ISL exerted the in vitro anti-migratory and anti-invasive activities, whereas the addition of PGE2, WIT003 and CAY10580 or overexpression of constitutively active Akt reversed the in vitro anti-migratory and anti-invasive activities of ISL in MDA-MB-231 cells. Notably, ISL inhibited the in vivo lung metastasis of MDA-MB-231 cells, together with decreased intratumoral levels of PGE2, 20-HETE and phospho-Akt (Thr308). In conclusion, ISL inhibits breast cancer metastasis by preventing anoikis resistance, migration and invasion via downregulating COX-2 and CYP 4A signaling. It suggests that ISL could be a promising multi-target agent for preventing breast cancer metastasis, and anoikis could represent a novel mechanism through which flavonoids may exert the anti-metastatic activities.

Prostaglandin E-prostanoid4 receptor mediates angiotensin II-induced (pro)renin receptor expression in the rat renal medulla.

Angiotensin II (Ang II) stimulates (pro)renin receptor (PRR) expression in the renal collecting duct, triggering the local renin response in the distal nephron. Our recent study provided evidence for involvement of cyclooxygenase-2-prostaglandin E2 pathway in Ang II-dependent stimulation of PRR expression in the collecting duct. Here, we tested the role of E-prostanoid (EP) subtypes acting downstream of cyclooxygenase-2 in this phenomenon. In primary rat inner medullary collecting duct cells, Ang II treatment for 12 hours induced a 1.8-fold increase in the full-length PRR protein expression. To assess the contribution of EP receptor, the cell was pretreated with specific EP receptor antagonists: SC-51382 (for EP1), L-798106 (for EP3), L-161982 (for EP4), and ONO-AE3-208 (ONO, a structurally distinct EP4 antagonist). The upregulation of PRR expression by Ang II was consistently abolished by L-161982 and ONO and partially suppressed by SC-51382 but was unaffected by L-798106. The PRR expression was also significantly elevated by the EP4 agonist CAY10598 in the absence of Ang II. Sprague-Dawley rats were subsequently infused for 1 or 2 weeks with vehicle, Ang II alone, or in combination with ONO. Ang II infusion induced parallel increases in renal medullary PRR protein and renal medullary and urinary renin activity and total renin content, all of which were blunted by ONO. Both tail cuff plethysmography and telemetry demonstrated attenuation of Ang II hypertension by ONO. Overall, these results have established a crucial role of the EP4 receptor in mediating the upregulation of renal medullary PRR expression and renin activity during Ang II hypertension.

Cyclooxygenase 2 inhibitor celecoxib inhibits glutamate release by attenuating the PGE2/EP2 pathway in rat cerebral cortex endings.

The excitotoxicity caused by excessive glutamate is a critical element in the neuropathology of acute and chronic brain disorders. Therefore, inhibition of glutamate release is a potentially valuable therapeutic strategy for treating these diseases. In this study, we investigated the effect of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor that reduces the level of prostaglandin E2 (PGE2), on endogenous glutamate release in rat cerebral cortex nerve terminals (synaptosomes). Celecoxib substantially inhibited the release of glutamate induced by the K(+) channel blocker 4-aminopyridine (4-AP), and this phenomenon was prevented by chelating the extracellular Ca(2+) ions and by the vesicular transporter inhibitor bafilomycin A1. Celecoxib inhibited a 4-AP-induced increase in cytosolic-free Ca(2+) concentration, and the celecoxib-mediated inhibition of glutamate release was prevented by the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC. However, celecoxib did not alter 4-AP-mediated depolarization and Na(+) influx. In addition, this glutamate release-inhibiting effect of celecoxib was mediated through the PGE2 subtype 2 receptor (EP2) because it was not observed in the presence of butaprost (an EP2 agonist) or PF04418948 [1-(4-fluorobenzoyl)-3-[[6-methoxy-2-naphthalenyl)methyl]-3-azetidinecarboxylic acid; an EP2 antagonist]. The celecoxib effect on 4-AP-induced glutamate release was prevented by the inhibition or activation of protein kinase A (PKA), and celecoxib decreased the 4-AP-induced phosphorylation of PKA. We also determined that COX-2 and the EP2 receptor are present in presynaptic terminals because they are colocalized with synaptophysin, a presynaptic marker. These results collectively indicate that celecoxib inhibits glutamate release from nerve terminals by reducing voltage-dependent Ca(2+) entry through a signaling cascade involving EP2 and PKA.

Prostaglandin E2‐Dependent Blockade of Actomyosin and Stress Fibre Formation Is Mediated Through S1379 Phosphorylation of ROCK2

Prostaglandin E2 is a pleiotropic bioactive lipid that controls cytoskeletal alterations, although the precise G-protein coupled EP receptor signalling mechanisms remain ill defined. We adopted a phosphoproteomic approach to characterize post-receptor downstream signalling substrates using antibodies that selectively recognize and immunoprecipitate phosphorylated substrates of a number of kinases. Using human synovial fibroblasts in monolayer cell culture, PGE2 induced rapid and sustained changes in cellular morphology and reduction in cytoplasmic volume that were associated with disassembly of the phalloidin-stained stress fibres as judged by light and confocal microscopy. Furthermore, PGE2 induced a rapid dephosphorylation of myosin light chain II (MLC) at S19 under basal or cytokine-induced conditions that was linked to an activation of myosin light chain phosphatase. The use of specific synthetic EP agonists suggested that the response was mediated by EP2 receptors, as other EP agonists did not manifest the same effect on MLC phosphorylation. In addition, PGE2 induced sustained Y118 dephosphorylation of phospho-paxillin and loss of focal adhesions as observed by confocal microscopy and Western analysis. Phosphoproteomic analysis of PGE2 /GPCR/PKA phosphosubstrates identified a unique, non-redundant, phosphorylated (>30-fold) site on rho-associated coiled coil-containing kinase 2 (ROCK2) at S1379. Analysis of ROCK2 mutant behaviour (e.g. S1379A) in overexpression studies revealed that PGE2 -dependent phosphorylation of ROCK2 resulted in the inhibition of the kinase, since induced MLC phosphorylation was no longer blocked by PGE2 nor could PGE2 induce disassembly of stress fibres. Thus, PGE2 -dependent blockade of actomyosin fibre formation, characteristic of myofibroblasts, may be mediated through specific ROCK2 S1379 phosphorylation.

Role of prostaglandins in spinal transmission of the exercise pressor reflex in decerebrated rats

Previous studies found that prostaglandins in skeletal muscle play a role in evoking the exercise pressor reflex; however the role played by prostaglandins in the spinal transmission of the reflex is not known. We determined, therefore, whether or not spinal blockade of cyclooxygenase (COX) activity and/or spinal blockade of endoperoxide (EP) 2 or 4 receptors attenuated the exercise pressor reflex in decerebrated rats. We first established that intrathecal doses of a non-specific COX inhibitor Ketorolac (100μg in 10μl), a COX-2-specific inhibitor Celecoxib (100μg in 10μl), an EP2 antagonist PF-04418948 (10μg in 10μl), and an EP4 antagonist L-161,982 (4μg in 10μl) effectively attenuated the pressor responses to intrathecal injections of arachidonic acid (100μg in 10μl), EP2 agonist Butaprost (4ng in 10μl), and EP4 agonist TCS 2510 (6.25μg in 2.5μl), respectively. Once effective doses were established, we statically contracted the hind limb before and after intrathecal injections of Ketorolac, Celecoxib, the EP2 antagonist and the EP4 antagonist. We found that Ketorolac significantly attenuated the pressor response to static contraction (before Ketorolac: 23±5mmHg, after Ketorolac 14±5mmHg; p<0.05) whereas Celecoxib had no effect. We also found that 8μg of L-161,982, but not 4μg of L-161,982, significantly attenuated the pressor response to static contraction (before L-161,982: 21±4mmHg, after L-161,982 12±3mmHg; p<0.05), whereas PF-04418948 (10μg) had no effect. We conclude that spinal COX-1, but not COX-2, plays a role in evoking the exercise pressor reflex, and that the spinal prostaglandins produced by this enzyme are most likely activating spinal EP4 receptors, but not EP2 receptors.

ALTERED LIPID MEDIATORS AND RECEPTORS OF RESOLUTION IN THE ENTORHINAL CORTEX OF ALZHEIMER'S DISEASE

Alzheimer’ disease pathogenesis. Primary murine microglia that lack prostaglandin E 2 receptor subtype 2 (EP2) have a dual phenotype of decreased innate immune-mediated neurotoxicity and increased Ab peptide phagocytosis, features that have been replicated in vivo. Here we tested the hypothesis that expression and activity of scavenger receptor CD36 influences EP2-regulated A b phagocytosis. Methods: Primary microglia were isolated from brains of newborn C57BL/6 mice. CD36 mRNA and protein levels were assessed by quantitative real-time PCR and flow cytometry, respectively.Ab 42 phagocytosis by primary microglia was determined by incubation with pHrodo-labeled Ab 42 followed by flow cytometry. CD36 mRNA levels were measured in hippocampus of mice following intracerebroventricular (ICV) injection of PIC, TLR3 activator. Results: Treatment of microglia with the EP2 agonist Butaprost reduced CD36 expression, as measured by mRNA and cell surface protein levels, and inhibited microglial A b 42 phagocytosis. Consistent with these findings, preventing EP2 activation by both pharmacological and genetic approaches increased microglial CD36 expression as well as A b 42 phagocytosis. Activation of the innate immune response by TLR activators, such as TLR3, TLR4 and TLR7, similarly reduced CD36 expression and phagocytic capacity in primary microglia. These effects were reduced by at least half by an EP2 antagonist, AH 6809, with the microglia Ab 42 phagocytosis effect completely dependent on CD36 activity. Finally, our in vivo studies demonstrated that hippocampal CD36 mRNA levels were significantly reduced following ICV injection of theTLR3 activator PIC; this effect was reversed by an EP2 antagonist. Conclusions: Our results are consistent with those of other studies in supporting a key role for CD36 in Ab phagocytosis by microglia and highlight selective EP2 antagonists as an effective means to suppress this toxic reinforcing cycle. Our findings suggest that EP2 antagonists have potential as a therapeutic approach to suppressing key aspects of AD pathogenesis.

Myeloid-derived suppressor cell function is diminished in aspirin-triggered allergic airway hyperresponsiveness in mice

Myeloid-derived suppressor cells (MDSCs) have recently been implicated in the pathogenesis of asthma, but their regulation in patients with aspirin-intolerant asthma (AIA) remains unclear. We sought to characterize MDSC accumulation and pathogenic functions in allergic airway inflammation mediated by COX-1 deficiency or aspirin treatment in mice. Allergic airway inflammation was induced in mice by means of ovalbumin challenge. The distribution and function of MDSCs in mice were analyzed by using flow cytometry and pharmacologic/gene manipulation approaches. CD11b(+)Gr1(high)Ly6G(+)Ly6C(int) MDSCs (polymorphonuclear MDSCs [PMN-MDSCs]) recruited to the lungs are negatively correlated with airway inflammation in allergen-challenged mice. Aspirin-treated and COX-1 knockout (KO) mice showed significantly lower accumulation of PMN-MDSCs in the inflamed lung and immune organs accompanied by increased TH2 airway responses. The TH2-suppressive function of PMN-MDSCs was notably impaired by COX-1 deletion or inhibition, predominantly through downregulation of arginase-1. COX-1-derived prostaglandin E2 promoted PMN-MDSC generation in bone marrow through E prostanoid 2 and 4 receptors (EP2 and EP4), whereas the impaired arginase-1 expression in PMN-MDSCs in COX-1 KO mice was mediated by dysregulation of the prostaglandin E2/EP4/cyclic AMP/protein kinase A pathway. EP4 agonist administration alleviated allergy-induced airway hyperresponsiveness in COX-1 KO mice. Moreover, the immunosuppressive function of PMN-MDSCs from patients with AIA was dramatically decreased compared with that from patients with aspirin-tolerant asthma. The immunosuppressive activity of PMN-MDSCs was diminished in both allergen-challenged COX-1 KO mice and patients with AIA, probably through an EP4-mediated signaling pathway, indicating that activation of PMN-MDSCs might be a promising therapeutic strategy for asthma, particularly AIA.

Application of prostaglandin E2 improves ileal blood flow in NEC

PurposeIndomethacin, a nonselective prostaglandin inhibitor used to treat patent ductus arteriosus, is associated with intestinal perforation inducing an NEC-like illness. We sought to define the contribution of prostaglandin E2 (PG E2) and its receptor EP4 to intestinal blood flow regulation in premature neonates with NEC. MethodsNewborn Sprague-Dawley rats were randomized by litter to undergo experimental NEC induction or to serve as a CONTROL. At 48hours of age, intestinal laser Doppler blood flow was assessed at baseline and after intraperitoneal administration of indomethacin, PG E2, EP4 antagonist, or EP4 agonist. Data were analyzed using a 2-way ANOVA with post hoc Tukey-Kramer correction. ResultsAt baseline, NEC animals had lower intestinal blood flow than controls. Indomethacin, PG E2 and EP4 agonist all increased ileal blood flow, but PG E2 and EP4 agonist increased blood flow the most in NEC pups. EP4 antagonist decreased intestinal perfusion in both groups. ConclusionThe above evidence suggests the importance of PG E2 and EP4 in regulation of neonatal intestinal blood flow. Since indomethacin treatment of patent ductus arteriosus in the premature infant is associated with an increased risk of intestinal perforation owing to compromised blood flow, PG E2 supplementation might provide intestinal protection if administered simultaneously with indomethacin.

In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines.

Many chemical mediators regulate neutrophil recruitment to inflammatory sites. Although the actions of each chemical mediator have been demonstrated with neutrophils in vitro, how such chemical mediators act cooperatively or counteractively in vivo remains largely unknown. Here, by in vivo two-photon excitation microscopy with transgenic mice expressing biosensors based on Förster resonance energy transfer, we time-lapse-imaged the activities of extracellular signal-regulated kinase (ERK) and protein kinase A (PKA) in neutrophils in inflamed intestinal tissue. ERK activity in neutrophils rapidly increased during spreading on the endothelial cells and showed positive correlation with the migration velocity on endothelial cells or in interstitial tissue. Meanwhile, in the neutrophils migrating in the interstitial tissue, high PKA activity correlated negatively with migration velocity. In contradiction to previous in vitro studies that showed ERK activation by prostaglandin E2 (PGE2) engagement with prostaglandin receptor EP4, intravenous administration of EP4 agonist activated PKA, inhibited ERK, and suppressed migration of neutrophils. The opposite results were obtained using nonsteroidal antiinflammatory drugs (NSAIDs). Therefore, NSAID-induced enteritis may be caused at least partially by the inhibition of EP4 receptor signaling of neutrophils. Our results demonstrate that ERK positively regulates the neutrophil recruitment cascade by promoting adhesion and migration steps.

Prostaglandin E2 induces apoptosis in cultured rat microglia

Prostaglandin E2 (PGE2) plays a critical role in the modulation of microglial function including migration and phagocytosis through EP2, which increases intracellular cyclic adenosine monophosphate (AMP) concentration. In the present study, we found that PGE2 reduces cell viability in microglia. PGE2 decreased 3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) reduction and increased lactate dehydrogenase release, deoxyribonucleic acid fragmentation, and poly(ADP-ribose) polymerase cleavage after 24h incubation, suggesting that PGE2 induces apoptosis in these cells. An EP2 agonist, butaprost, and an EP4 agonist, PGE1 alcohol, also induced apoptosis, while an EP1 agonist, 17-phenyl trinor PGE2, or an EP3 agonist, sulprostone, at 10−6M did not. On the other hand, EP1–EP4 antagonists, SC-51322, AH6809, L-798106, or GW627368X, up to 10−5M did not affect the decrease in MTT reduction by PGE2. Intracellular cyclic AMP accumulation was induced by butaprost, but not 17-phenyl trinor PGE2, sulprostone, or PGE1 alcohol at 10−6M. Additionally, we previously reported that PGE2-induced intracellular cyclic AMP accumulation was reversed by AH6809. Besides EP receptors, one of other targets was thought to be prostaglandin transporter, but its inhibitors, bromocresol green or U-46619 up to 10−5M did not affect the decrease in MTT reduction by PGE2. These results suggest that PGE2 induces apoptosis in microglia independent of intracellular cyclic AMP concentration, and there are different mechanisms between PGE2-induced apoptosis and the modulation of microglial function.