The prostaglandin receptor EP2 determines prognosis in EP3-negative and galectin-3-high cervical cancer cases.

Recently our study identified EP3 receptor and galectin-3 as prognosticators of cervical cancer. The aim of the present study was the analysis of EP2 as a novel marker and its association to EP3, galectin-3, clinical pathological parameters and the overall survival rate of cervical cancer patients. Cervical cancer tissues (n = 250), as also used in our previous study, were stained with anti-EP2 antibodies employing a standardized immunohistochemistry protocol. Staining results were analyzed by the IRS scores and evaluated for its association with clinical-pathological parameters. H-test of EP2 percent-score showed significantly different expression in FIGO I-IV stages and tumor stages. Kaplan-Meier survival analyses indicated that EP3-negative/EP2-high staining patients (EP2 IRS score ≥2) had a significantly higher survival rate than the EP3-negative/EP2-low staining cases (p = 0.049). In the subgroup of high galectin-3 expressing patients, the group with high EP2 levels (IRS ≥2) had significantly better survival rates compared to EP2-low expressing group (IRS <2, p = 0.044). We demonstrated that the EP2 receptor is a prognostic factor for the overall survival in the subgroup of negative EP3 and high galectin-3 expressed cervical cancer patients. EP2 in combination with EP3 or galectin-3 might act as prognostic indicators of cervical cancer. EP2, EP3, and galectin-3 could be targeted for clinical diagnosis or endocrine treatment in cervical cancer patients, which demands future investigations.

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

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

E-Prostanoid 3 Receptor Mediates Sprouting Angiogenesis Through Suppression of the Protein Kinase A/ß-Catenin/Notch Pathway.

OBJECTIVE: Angiogenesis is a hallmark of embryonic development and various ischemic and inflammatory diseases. Prostaglandin E2 receptor subtype 3 (EP3) plays an important role in pathophysiologic angiogenesis; however, the precise mechanisms remain unknown. Here, we investigated the role of EP3 in zebra fish embryo and mouse retina angiogenesis and evaluated the underlying mechanisms. APPROACH AND RESULTS: The EP3 receptor was highly expressed in the vasculature in both zebra fish embryos and murine fetal retinas. Pharmacological inhibition or genetic deletion of EP3 significantly reduced vasculature formation in zebra fish embryos and mouse retinas. Further characterization revealed reduced filopodia extension of tip cells in embryonic retinas in EP3-deficient mice. EP3 deletion activated Notch activity by upregulation of delta-like ligand 4 expression in endothelial cells (ECs). Inhibition of Notch signaling rescued the angiogenic defects in EP3-deficient mouse retinas. Moreover, EP3 deficiency led to a significant increase in ß-catenin phosphorylation at Ser675 and nuclear accumulation of ß-catenin in ECs. Knockdown or inhibition of ß-catenin restored the impaired sprouting angiogenesis resulting from EP3 deficiency in ECs. The EP3 receptor depressed protein kinase A activity in ECs by coupling to Gαi. Inhibition of protein kinase A activity significantly reduced Ser675 phosphorylation and nuclear translocation of ß-catenin, abolished the increased delta-like ligand 4 expression, and subsequently restored the impaired angiogenic capacity of EP3-deficient ECs both in vitro and in vivo. CONCLUSIONS: Activation of the EP3 receptor facilitates sprouting angiogenesis through protein kinase A-dependent Notch signaling, suggesting that EP3 and its downstream pathways maybe potential therapeutic targets in the treatment of ischemic diseases.

Signaling of Prostaglandin E Receptors, EP3 and EP4 Facilitates Wound Healing and Lymphangiogenesis with Enhanced Recruitment of M2 Macrophages in Mice.

Lymphangiogenesis plays an important role in homeostasis, metabolism, and immunity, and also occurs during wound-healing. Here, we examined the roles of prostaglandin E2 (PGE2) receptor (EP) signaling in enhancement of lymphangiogenesis in wound healing processes. The hole-punch was made in the ears of male C57BL/6 mice using a metal ear punch. Healing process and lymphangiogenesis together with macrophage recruitment were analyzed in EP knockout mice. Lymphangiogenesis was up-regulated in the granulation tissues at the margins of punched-hole wounds in mouse ears, and this increase was accompanied by increased expression levels of COX-2 and microsomal prostaglandin E synthase-1. Administration of celecoxib, a COX-2 inhibitor, suppressed lymphangiogenesis in the granulation tissues and reduced the induction of the pro-lymphangiogenic factors, vascular endothelial growth factor (VEGF) -C and VEGF-D. Topical applications of selective EP receptor agonists enhanced the expressions of lymphatic vessel endothelial hyaluronan receptor-1 and VEGF receptor-3. The wound-healing processes and recruitment of CD11b-positive macrophages, which produced VEGF-C and VEGF-D, were suppressed under COX-2 inhibition. Mice lacking either EP3 or EP4 exhibited reduced wound-healing, lymphangiogenesis and recruitment of M2 macrophages, compared with wild type mice. Proliferation of cultured human lymphatic endothelial cells was not detected under PGE2 stimulation. Lymphangiogenesis and recruitment of M2 macrophages that produced VEGF-C/D were suppressed in mice treated with a COX-2 inhibitor or lacking either EP3 or EP4 during wound healing. COX-2 and EP3/EP4 signaling may be novel targets to control lymphangiogenesis in vivo.

Prostaglandin E2-EP3 signaling induces inflammatory swelling by mast cell activation.

PGE2 has long been known as a potentiator of acute inflammation, but its mechanisms of action still remain to be defined. In this study, we employed inflammatory swelling induced in mice by arachidonate and PGE2 as models and dissected the role and mechanisms of action of each EP receptor at the molecular level. Arachidonate- or PGE2-induced vascular permeability was significantly reduced in EP3-deficient mice. Intriguingly, the PGE2-induced response was suppressed by histamine H1 antagonist treatment, histidine decarboxylase deficiency, and mast cell deficiency. The impaired PGE2-induced response in mast cell-deficient mice was rescued upon reconstitution with wild-type mast cells but not with EP3-deficient mast cells. Although the number of mast cells, protease activity, and histamine contents in ear tissues in EP3-deficient mice were comparable to those in wild-type mice, the histamine contents in ear tissues were attenuated upon PGE2 treatment in wild-type but not in EP3-deficient mice. Consistently, PGE2-EP3 signaling elicited histamine release in mouse peritoneal and bone marrow-derived mast cells, and it exerted degranulation and IL-6 production in a manner sensitive to pertussis toxin and a PI3K inhibitor and dependent on extracellular Ca(2+) ions. These results demonstrate that PGE2 triggers mast cell activation via an EP3-Gi/o-Ca(2+) influx/PI3K pathway, and this mechanism underlies PGE2-induced vascular permeability and consequent edema formation.

Inflammatory prostaglandin E2 signaling in a mouse model of Alzheimer disease.

OBJECTIVE: 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 Aß peptide. METHODS: The function of PGE(2) signaling through its EP3 receptor was examined in vivo in a model of subacute neuroinflammation induced by administration of Aß(42) peptides. Our findings were then confirmed in young adult APPSwe-PS1ΔE9 transgenic mice. RESULTS: Deletion of the PGE(2) EP3 receptor in a model of Aß(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 Aß peptides were significantly decreased, as were ß-secretase and ß C-terminal fragment levels, suggesting that generation of Aß 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. INTERPRETATION: 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.

Inhibition of prostaglandin E2 EP3 receptors improves stroke injury via anti-inflammatory and anti-apoptotic mechanisms.

Although deletion of EP3 receptors is known to ameliorate stroke injury in experimental stroke models, the underlying mechanisms and the effects of EP3-specific antagonists remain poorly understood. Here we demonstrate the protective effect of postischemic treatment with an EP3 antagonist, ONO-AE3-240, through anti-inflammatory and anti-apoptotic effects. In transient focal ischemia models, peritoneal injection of an EP3 antagonist after occlusion-reperfusion reduced infarction, edema and neurological dysfunctions to almost the same levels of those in EP3 knockout (KO) mice. Furthermore, neuronal apoptosis in the ischemic cortex investigated by terminal dUTP nick-end labeling (TUNEL) and caspase-3 immunostaining were ameliorated in EP3 antagonist-treated mice or EP3 KO mice as compared with vehicle-treated mice or wild-type (WT) mice, respectively. There were no significant differences between ONO-AE3-240-injected or EP3 KO mice and vehicle-injected or WT mice, respectively, in mean arterial blood pressure, cerebral blood flow or body temperature. The double-immunostaining showed that EP3 receptor-positive cells were also positive for CD-11b and partially for Neu-N, the marker for microglia and neurons. Deletion of EP3 receptors also reduced damage of the blood-brain barrier, activation of microglia and infiltration of neutrophils into the ischemic cortex. These results suggest that EP3 receptors are involved in stroke injury through the enhancement of inflammatory and apoptotic reactions in the ischemic cortex. Thus, EP3 antagonists may be valuable for the treatment of human stroke.

Roles of prostaglandin E2-EP3/EP4 receptor signaling in the enhancement of lymphangiogenesis during fibroblast growth factor-2-induced granulation formation.

OBJECTIVE: One of the hallmarks of inflammation is lymphangiogesis that drains the interstitial fluids. During chronic inflammation, angiogenesis is induced by a variety of inflammatory mediators, such as prostaglandins (PGs). However, it remains unknown whether they enhance lymphangiogenesis. We examined the roles of cyclooxygenase-2 (COX-2) and PGE2 receptor signaling in enhancement of lymphangiogenesis during proliferative inflammation. METHODS AND RESULTS: Lymphangiogenesis estimated by podoplanin/vascular endothelial growth factor (VEGF) receptor-3/LYVE-1 expression was upregulated during proliferative inflammation seen around and into subcutaneous Matrigel plugs containing fibroblast growth factor-2 (125 ng/site). A COX-2 inhibitor (celecoxib) significantly reduced lymphangiogenesis in a dose-dependent manner, whereas topical PGE2 enhanced lymphangiogenesis. Topical injection of fluorescein isothiocyanate-dextran into the Matrigel revealed that lymphatic flow from the Matrigels was COX-2 dependent. Lymphangiogenesis was suppressed in the granulation tissues of mice lacking either EP3 or EP4, suggesting that these molecules are receptors in response to endogenous PGE2. An EP3-selective agonist (ONO-AE-248) increased the expression of VEGF-C and VEGF-D in cultured macrophages, whereas an EP4-selective agonist (ONO-AE1-329) increased VEGF-C expression in cultured macrophages and increased VEGF-D expression in cultured fibroblasts. CONCLUSIONS: Our findings suggest that COX-2 and EP3/EP4 signaling contributes to lymphangiogenesis in proliferative inflammation, possibly via induction of VEGF-C and VEGF-D, and may become a therapeutic target for controlling lymphangiogenesis.