EP3 Receptor Knockout Research Articles

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.

Knockout of the Prostaglandin E2 Receptor Subtype 3 Promotes Eccentric Cardiac Hypertrophy and Fibrosis in Mice.

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

Abstract TP100: PGE2 EP1-4 Receptor Expression Levels After Intracerebral Hemorrhage in Young, Aged, and EP1 Deficient Mice

Intracerebral hemorrhage (ICH) is the most severe form of stroke and is associated with high mortality and morbidity. Neuroinflammation significantly contributes to ICH-induced brain injury and upregulation of prostaglandin E 2 (PGE 2 ) has been implicated in modulating these deleterious neuroinflammatory pathways. PGE 2 acts on mainly the four G-protein-coupled E prostanoid (EP) receptors, EP1-4, which each have different downstream signaling pathways, tissue distributions, and expression profiles. We have previously demonstrated that EP1 receptor deletion promotes injury following ICH, whereas deletion of EP2 and EP3 is neuroprotective in an equivalent experimental approach. Here, we aimed to investigate the time course, brain sub-region expression profile, and relative level of EP1-4 mRNA expression in young (5-7mo) and aged (12-13mo) wildtype (WT) and EP1 receptor knockout (EP1 -/- ) mice. Following ICH or sham surgery, EP1-4 mRNA levels were assessed by RT-qPCR. Minimal EP1-4 expression changes are seen at 24h after ICH; although, EP2 (p=0.0.36) and EP4 (p=0.066) are 1.6X increased in the cortex of young WT mice. At 72h post-ICH, EP1 is 3.9x elevated in the cortex (p=0.0003) and 4.6x in the striatum (p=0.012). EP3 is also 1.6x elevated in the cortex (p=0.044). In the contralateral hemisphere, a mean 2.4x increase of EP1-4 (p<0.01) expression is seen. In contrast, at 72h after ICH, EP1 -/- mice have 0.53x reduced EP3 in the cortex (p=0.030) and 0.68x and 0.71x decreased EP3 (p=0.016) and EP4 (p=0.049), respectively, in the contralateral hemisphere. Aged EP1 -/- mice show significantly decreased expression levels of EP2-4 in nearly all areas (cortex, striatum, and contralateral hemisphere). Due to the contralateral differences, basal expression levels of EP2-4 were investigated in the EP1 -/- mice, where EP2 is 2.6x, 3.4x, and 3.8x increased in the cortex, hippocampus, and cerebellum; whereas, EP3 trended oppositely. These data indicate a cross-talk interaction between EP1 and the other EP receptors pre- and post-ICH and an association between age and EP receptor expression levels. A better understanding of EP receptor localization and dynamic expression levels after ICH will facilitate the development of effective pharmacological treatments.

Abstract P127: Angiotensin II Priming Enhances Prostaglandin E2 Vasoconstrictor Effects

Prostaglandins are key modulators of blood pressure and arterial tone. Prostaglandin E 2 (PGE 2 ), is a prostanoid that has vasodepressor effects; however, under certain circumstances PGE 2 can induce vasopressor responses. Recent reports demonstrated that sub-threshold concentrations of vasoconstrictors augment PGE 2 -mediated constriction in rat femoral arteries. However, whether angiotensin II (Ang II) could affect PGE 2 -mediated contraction is not known. Using a wire myograph, we demonstrated that PGE 2 had no significant effect on mouse femoral arterial rings at doses up to 1 μM. However, priming of arterial rings with 1 nM Ang II potentiated PGE 2 -evoked constriction in a concentration dependent manner (Area Under the Curve, AUC untreated 1.784 ± 0.353, AUC Ang II 23.27± 9.820, P<0.05). We tested femoral arteries from EP1, EP2, and EP3 receptor knockout mice. Only the EP3-/- arteries were unable to respond to PGE 2 after Ang II priming (figure below). Pretreatment of arterial rings with 1 μM losartan, an angiotensin receptor antagonist, blocked PGE 2 -induced constrictor effects primed with Ang II (% of KCl, Ang II 21.72 ± 5.296, Ang II + losartan 3.025 ± 1.046, n=3). We have determined that re-addition of extracellular Ca 2+ to a Ca 2+ -free artery restores PGE 2 -induced contractions (n=5) and that the Rho-kinase inhibitor Y-27632 blocks contraction (n=3). Taken together these data are consistent with angiotensin AT1 and prostaglandin EP3 receptors mediating a synergistic Rho-kinase-dependent contractile response. We are continuing to investigate the relationship between Ang II and PGE 2 to determine the physiological relevance this may have in modulating blood pressure.

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

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

Abstract W P260: Role of the Prostaglandin E2 EP1 Receptor in Traumatic Brain Injury

Introduction: Brain injuries promote upregulation of so-called proinflammatory prostaglandin E2 leading to overactivation of a class of its cognate G-protein coupled receptors, notably EP1, which is considered as a promising target for treatment of ischemic stroke and, possibly, other neurological disorders involving excitotoxicity. However, our recent data suggest that of EP1 receptor in intracerebral hemorrhage may play a protective role. The goal of this study was to investigate a translational potential of EP1 receptor for treatment of traumatic brain injury (TBI). Methods: The acute brain injury was induced using controlled cortical impact (CCI) in wildtype (WT) and genetic EP1 receptor knockout mice (EP1-/-). Neurological deficit scores (NDS) and anatomical brain pathology were accessed at 48h after injury. Results: CCI resulted in significant cortical lesions, localized hippocampal edema and neurological deficits compared to animals from sham group underwent craniotomy only. The NDS after CCI were significantly higher in older mice (7-11mo) compared to young adult animals (2-4mo) in both WT and EP1-/- groups. Treatment with a selective antagonist SC-51089 with repeated doses of 20-100μg/kg after CCI had no significant effects on cortical lesions, hippocampal edema and NDS in young adult mice of both WT and EP1-/- genotypes. Post-treatment with 17-pt-PGE2 (300μg/kg) had no significant effects on anatomical brain pathology in young adult mice, but improved NDS at 24h in WT but not in EP1-/- mice. Immunohistochemistry revealed significant increases in GFAP and Iba1 immunoreactivity in selected brain regions surrounding injury suggesting astrogliosis and microglia activation. EP1 receptor knockout had no effects on GFAP and Iba1 expression in young adult mice, whereas lead to a significant attenuation of GFAP immunoreactivity in older mice. Conclusions: This study provides, for the first time, a clarification on the role of EP1 receptor in a preclinical model of contusive TBI. The results suggest that EP1 receptor might be involved in complex pathways differentially associated with neurological deficits. In addition, this study provides further clarification on clinical use of EP1 receptor ligands for treatment of acute brain injuries.

Role of the prostaglandin E2 EP1 receptor in traumatic brain injury.

Brain injuries promote upregulation of so-called proinflammatory prostaglandins, notably prostaglandin E2 (PGE2), leading to overactivation of a class of its cognate G-protein-coupled receptors, including EP1, which is considered a promising target for treatment of ischemic stroke. However, the role of the EP1 receptor is complex and depends on the type of brain injury. This study is focused on the investigation of the role of the EP1 receptor in a controlled cortical impact (CCI) model, a preclinical model of traumatic brain injury (TBI). The therapeutic effects of post-treatments with a widely studied EP1 receptor antagonist, SC-51089, were examined in wildtype and EP1 receptor knockout C57BL/6 mice. Neurological deficit scores (NDS) were assessed 24 and 48 h following CCI or sham surgery, and brain immunohistochemical pathology was assessed 48 h after surgery. In wildtype mice, CCI resulted in an obvious cortical lesion and localized hippocampal edema with an associated significant increase in NDS compared to sham-operated animals. Post-treatments with the selective EP1 receptor antagonist SC-51089 or genetic knockout of EP1 receptor had no significant effects on cortical lesions and hippocampal swelling or on the NDS 24 and 48 h after CCI. Immunohistochemistry studies revealed CCI-induced gliosis and microglial activation in selected ipsilateral brain regions that were not affected by SC-51089 or in the EP1 receptor-deleted mice. This study provides further clarification on the respective contribution of the EP1 receptor in TBI and suggests that, under this experimental paradigm, the EP1 receptor would have limited effects in modulating acute neurological and anatomical pathologies following contusive brain trauma. Findings from this protocol, in combination with previous studies demonstrating differential roles of EP1 receptor in ischemic, neurotoxic, and hemorrhagic conditions, provide scientific background and further clarification of potential therapeutic application of prospective prostaglandin G-protein-coupled receptor drugs in the clinic for treatment of TBI and other acute brain injuries.

Role of PGE2 EP1 Receptor in Intracerebral Hemorrhage-Induced Brain Injury

Prostaglandin E₂ (PGE₂) has been described to exert beneficial and detrimental effects in various neurologic disorders. These conflicting roles of PGE₂ could be attributed to its diverse receptor subtypes, EP1-EP4. At present, the precise role of EP1 in intracerebral hemorrhage (ICH) is unknown. Therefore, to elucidate its possible role in ICH, intrastriatal injection of collagenase was given in randomized groups of adult male wildtype (WT) and EP1 receptor knockout (EP1⁻/⁻)C57BL/6 mice. Functional outcomes including neurologic deficits, rotarod performance, open field activity, and adhesive removal performance were evaluated at 24, 48, and 72 h post-ICH. Lesion volume, cell survival and death, were assessed using Cresyl Violet, and Fluoro-Jade staining, respectively. Microglial activation and phagocytosis were estimated using Iba1 immunoreactivity and fluorescently-labeled microspheres. Following 72 h post-ICH, EP1⁻/⁻ mice showed deteriorated outcomes compared to the WT control mice. These outcomes were demonstrated by elevated neurological deficits, exacerbated lesion volume, and significantly worsened sensorimotor functions. Fluoro-Jade staining showed significantly increased numbers of degenerating neurons and reduced neuronal survival in EP1⁻/⁻ compared to WT mice. To assess in vivo phagocytosis, the number of microspheres phagocytosed by Iba1-positive cells was 145.4 ± 15.4 % greater in WT compared to EP1⁻/⁻ mice. These data demonstrate that EP1 deletion exacerbates neuro-behavioral impairments following ICH, potentially by slowing down/impairing microglial phagocytosis. A better understanding of this EP1 mechanism could lead to improved intervention strategies for hemorrhagic stroke.

Editorial Comment from Dr Ishizuka to Effect of intrathecal administration of E‐series prostaglandin 1 receptor antagonist in a cyclophosphamide‐induced cystitis rat model

ganglia. J. Neurochem. 1991; 57: 167–74. 14 Hu VY, Malley S, Dattilio A et al. COX-2 and prostanoid expression in micturition pathways after cyclophosphamide-induced cystitis in the rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2003; 284: R574–85. 15 Schroder A, Newgreen D, Andersson KE. Detrusor responses to prostaglandin E2 and bladder outlet obstruction in wild-type and EP1 receptor knockout mice. J. Urol. 2004; 172: 1166–70. 16 Chuang YC, Yoshimura N, Huang CC et al. Expression of E-series prostaglandin (EP) receptors and urodynamic effects of an EP4 receptor antagonist on cyclophosphamide-induced overactive bladder in rats. BJU Int. 2010; 106: 1782–7. 17 Minami T, Nishihara I, Uda R et al. Characterization of EP-receptor subtype involved in allodynia and hyperalgesia induced by intrathecal administration of prostaglandin E2 to mice. Br. J. Pharmacol. 1994; 112: 735–40. 18 Uda R, Horiguchi S, Ito S et al. Nociceptive effects induced by intrathecal administration of prostaglandin D2, E2, or F2 alpha to conscious mice. Brain Res. 1990; 510: 26–32. 19 Malmberg AB, Rafferty MF, Yaksh TL. Antinociceptive effect of spinally delivered prostaglandin E receptor antagonist in the formalin test on the rat. Neurosci. Lett. 1994; 173: 193–6. 20 Miura A, Kawatani M, Maruyama T et al. Effect of prostaglandins on parasympathetic neurons in the rat lumbosacral spinal cord. Neuroreport 2002; 13: 1557–62.

Temporal expression of the PGE2 synthetic system in the kidney is associated with the time frame of renal developmental vulnerability to cyclooxygenase-2 inhibition

Pharmacological blockade of cyclooxygenase-2 (COX-2) causes impairment of kidney development. The present study was aimed at determining temporal expression pattern and activity of the PGE(2) synthetic pathway during postnatal nephrogenesis in mice and its association to the time window sensitive to COX-2 inhibition. During the first 10 days after birth, we observed transient induction of mRNA and protein for microsomal PGE synthase (mPGES)-1 between postnatal days 4 (P4) and P8, but not for mPGES-2 or cytosolic PGE synthase (cPGES). PGE(2) synthetic activity using arachidonic acid and PGH(2) as substrates and also urinary excretion of PGE(2) were enhanced during this time frame. In parallel to the PGE(2) system, COX-2 but not COX-1 expression was also transiently induced. Studying glomerulogenesis in EP receptor knockout mice revealed a reduction in glomerular size in EP1(-/-), EP2(-/-), and EP4(-/-) mice, supporting the developmental role of PGE(2). The most vulnerable time window to COX-2 inhibition by SC-236 was found closely related to the temporal expression of COX-2 and mPGES-1. The strongest effects of COX-2 inhibition were achieved following 8 days of drug administration. Similar developmental damage was caused by application of rofecoxib, but not by the COX-1-selective inhibitor SC-560. COX-2 inhibition starting after P10 has had no effect on the size of glomeruli or on the relative number of superficial glomeruli; however, growth of the renal cortex was significantly diminished, indicating the requirement of COX-2 activity after P10. Effects of COX-2 inhibition on renal cell differentiation and on renal fibrosis needed a prolonged time of exposition of at least 10 days. In conclusion, temporal expression of the PGE(2) synthetic system coincides with the most vulnerable age interval for the induction of irreversible renal abnormalities. We assume that mPGES-1 is coregulated with COX-2 for PGE(2) synthesis to orchestrate postnatal kidney development and growth.

Bidirectional regulation of adrenal catecholamine release by prostaglandin E2

Catecholamine secretion from adrenal chromaffin cells is elevated in response to adverse physiological conditions. Similarly, systemic immune challenge leads to intra‐adrenal inflammatory signaling and a resulting induction of synthetic enzymes for prostaglandin E2 (PGE2). In the present study we investigated the functional impact of PGE2 on calcium signaling and catecholamine release from mouse adrenal chromaffin cells. PGE2 potently (EC50 = 5.5 nM) inhibited Cav2 voltage‐gated calcium currents (ICa), while having no effect on resting intracellular [Ca2+] or the peak amplitude of nicotinic acetylcholine receptor currents. The inhibition of ICa is mediated by pertussis toxin‐sensitive G proteins, and reversed by a strongly depolarizing voltage step, characteristic of direct G protein βγ subunit binding to the channel. PGE2 acts primarily through a family of cognate GPCRs (EP1‐EP4). We detected mRNA for all four EP receptors in mouse adrenal gland, but using selective pharmacological tools and EP receptor knockout mice we show the inhibition of ICa is mediated by EP3 receptors. Monitoring changes in membrane capacitance in response to brief depolarizations, we show Ca2+‐ dependent exocytosis is reduced in parallel with ICa in wild‐type mice. Conversely, PGE2 potentiates exocytosis in chromaffin cells isolated from EP3 receptor knockout mice, revealing a signaling modality distinct from the inhibitory EP3 pathway. Using carbon fiber amperometry, we find the potentiation of exocytosis is also apparent in wild type cells during sustained depolarizations mimicking acute stress. Our data support a model in which distinct EP receptor subtypes mediate activity‐dependent, bidirectional modulation of catecholamine release by PGE2. This work is supported by the American Heart Association [11GRNT7890031] and the National Institutes of Health [R01 NS052446].

Regulation of Calcium Channels and Exocytosis in Mouse Adrenal Chromaffin Cells by Prostaglandin EP3 Receptors

Prostaglandin (PG) E(2) controls numerous physiological functions through a family of cognate G protein-coupled receptors (EP1-EP4). Targeting specific EP receptors might be therapeutically useful and reduce side effects associated with nonsteroidal anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors that block prostanoid synthesis. Systemic immune challenge and inflammatory cytokines have been shown to increase expression of the synthetic enzymes for PGE(2) in the adrenal gland. Catecholamines and other hormones, released from adrenal chromaffin cells in response to Ca(2+) influx through voltage-gated Ca(2+) channels, play central roles in homeostatic function and the coordinated stress response. However, long-term elevation of circulating catecholamines contributes to the pathogenesis of hypertension and heart failure. Here, we investigated the EP receptor(s) and cellular mechanisms by which PGE(2) might modulate chromaffin cell function. PGE(2) did not alter resting intracellular [Ca(2+)] or the peak amplitude of nicotinic acetylcholine receptor currents, but it did inhibit Ca(V)2 voltage-gated Ca(2+) channel currents (I(Ca)). This inhibition was voltage-dependent and mediated by pertussis toxin-sensitive G proteins, consistent with a direct Gβγ subunit-mediated mechanism common to other G(i/o)-coupled receptors. mRNA for all four EP receptors was detected, but using selective pharmacological tools and EP receptor knockout mice, we demonstrated that EP3 receptors mediate the inhibition of I(Ca). Finally, changes in membrane capacitance showed that Ca(2+)-dependent exocytosis was reduced in parallel with I(Ca). To our knowledge, this is the first study of EP receptor signaling in mouse chromaffin cells and identifies a molecular mechanism for paracrine regulation of neuroendocrine function by PGE(2).

Roles of a prostaglandin E‐type receptor, EP3, in upregulation of matrix metalloproteinase‐9 and vascular endothelial growth factor during enhancement of tumor metastasis

Cyclooxygenase (COX)-2 is known to correlate with poor cancer prognosis and to contribute to tumor metastasis. However, the precise mechanism of this phenomenon remains unknown. We have previously reported that host stromal prostaglandin E(2) (PGE(2))-prostaglandin E2 receptor (EP)3 signaling appears critical for tumor-associated angiogenesis and tumor growth. Here we tested whether the EP3 receptor has a critical role in tumor metastasis. Lewis lung carcinoma (LLC) cells were intravenously injected into WT mice and mice treated with the COX-2 inhibitor NS-398. The nonselective COX inhibitor aspirin reduced lung metastasis, but the COX-1 inhibitor SC560 did not. The expression of matrix metalloproteinases (MMP)-9 and vascular endothelial growth factor (VEGF)-A was suppressed in NS-398-treated mice compared with PBS-treated mice. Lungs containing LLC colonies were markedly reduced in EP3 receptor knockout (EP3(-/-)) mice compared with WT mice. The expression of MMP-9 and VEGF-A was downregulated in metastatic lungs of EP3(-/-) mice. An immunohistochemical study revealed that MMP-9-expressing endothelial cells were markedly reduced in EP3(-/-) mice compared with WT mice. When HUVEC were treated with agonists for EP1, EP2, EP3, or EP4, only the EP3 agonist enhanced MMP-9 expression. These results suggested that EP3 receptor signaling on endothelial cells is essential for the MMP-9 upregulation that enhances tumor metastasis and angiogenesis. An EP3 receptor antagonist may be useful to protect against tumor metastasis.

Host prostaglandin EP3 receptor signaling relevant to tumor-associated lymphangiogenesis

Prostaglandin E 2 (PGE 2) and prostaglandin E (EP) receptor signaling pathways have been implicated in the promotion of tumor growth and angiogenesis. However, little is known about their roles in lymphangiogenesis during tumor development. The present study evaluates whether endogenous PGE 2 exhibits a critical role in tumor-associated lymphangiogenesis. Treatment of male C57BL/6 mice with a cyclooxygenase-2 inhibitor, celecoxib, for seven days resulted in a 52.4% reduction in tumor size induced by subcutaneous injection of murine Lewis lung cells. Celecoxib treatment down-regulated the expression of vascular endothelial growth factor receptor (VEGFR)-3 in stromal tissues by 73.9%, and attenuated expression of podoplanin, a marker for lymphatic endothelial cells. To examine the role of host PGE receptor signaling, we tested four kinds of EP receptor knockout mice. At Day 7 after tumor cell implantation, EP3 receptor knockout mice, but not EP receptor knockout mice lacking EP1, EP2, or EP4, exhibited a 53.3% reduction in tumor weight, which was associated with a 74.5% reduction in VEGFR-3 mRNA expression in tumor stromal tissues. At Day 14, VEGFR-3 expression in EP3–/– mice remained significantly lower than that of their wild-type (WT) counterparts. The expression of VEGF-C in the tumor stromal tissues in EP3–/– mice were also reduced by 22.1% (Day 7) and 44.1% (Day 14), respectively. In addition, the level of immunoreactive podoplanin in the tumor tissues from EP3–/– mice was less than that of WT. These results suggest that host EP3 receptor signaling regulates tumor-associated lymphangiogenesis by up-regulating expression of VEGF-C and its receptor, VEGFR-3, in tumor stromal tissues. Host EP3 blockade together with COX-2 inhibition may be a novel therapeutic strategy to suppress tumor-associated lymphangiogenesis.

Prostanoids in health and disease

The prostanoids are a family of lipid mediators generated by the action of cyclooxygenase on a 20-carbon unsaturated fatty acid, arachidonic acid. Prostanoids are generated widely in response to diverse stimuli and, acting in a paracrine or autocrine manner, play important roles in normal physiology and disease. This review summarizes the current knowledge on prostanoid generation and the roles of individual mediators, their biosynthetic pathways, and their receptors in health and disease.

Enhanced bladder capacity and reduced prostaglandin E2-mediated bladder hyperactivity in EP3 receptor knockout mice

Nonsteroidal anti-inflammatory cyclooxygenase inhibitors that function to reduce prostaglandin E2 (PGE2) production have been widely reported as effective agents in models of urinary bladder overactivity. We therefore investigated a potential role for the PGE2 receptor, EP3, in urinary bladder function by performing conscious, freely moving cystometry on EP3 receptor knockout (KO) mice. EP3 KO mice demonstrated an enhanced bladder capacity compared with wild-type (WT) mice ( approximately 185% of WT) under control conditions, based on larger voided and infused bladder volumes. Infusion of the EP3 receptor agonist GR63799X into the bladder of WT mice reduced the bladder capacity. This was ineffective in EP3 KO mice that demonstrated a time-dependent increase in bladder capacity with GR63799X, an effect similar to that observed with vehicle in both genotypes. In addition, infusion of PGE2 into WT mice induced bladder overactivity, an effect that was significantly blunted in the EP3 KO mice. The data reported here provide the first evidence supporting a functional role for EP3 receptors in normal urinary bladder function and implicate EP3 as a contributor to bladder overactivity during pathological conditions of enhanced PGE2 production, as reported previously in overactive bladder patients.

Roles of Prostaglandins in Facilitation of Angiogenesis <I>in vivo</I>

Angiogenesis, the formation of new blood vessels from the pre-existent microvasculature, is an essential component of wound repair and tumor growth. Nonsteroidal anti-inflammatories (NSAIDs) are known to suppress the incidence and progression of malignancies including colorectal cancers, and also to delay the wound healing. However, the precise mechanisms are not fully elucidated. Recent results obtained from prostaglandin (PG) receptor knockout mice indicate that host stromal PGE type receptor signaling is crucial in tumor-associated angiogenesis. Implanted tumor growth and tumor-associated angiogenesis were markedly suppressed in EP3 receptor knockout mice (EP3-/-), in comparison with their wild-type counterparts (WT). Tumor-associated angiogenesis in WT depends on vascular endothelial growth factor (VEGF). Major VEGF-expressing cells in stroma were CD3/Mac-1 double-negative fibroblasts, and that stromal VEGF expression was markedly reduced in EP3-/-. An EP3 receptor antagonist inhibited tumor growth and angiogenesis in WT. The wound healing process was significantly delayed in EP3-/-. The bone marrow transplantation of EP3-/- bone marrow cells revealed that the recruitment of EP3-expressing bone marrow cells to the wound granulation tissues was critical to the healing of wounds. These demonstrate the significance of EP receptor signaling to the angiogenesis in vivo. Such signaling will be a good target for controlling angiogenesis in pathological conditions.

Prostaglandin E2 receptors in bone formation

Prostaglandins, PGE(2) in particular, have diverse actions on various organs, including inflammation, bone healing, bone formation, embryo implantation, induction of labour and vasodilatation, among others. However, systemic side effects have limited their clinical utility. The pharmacological activities of PGE(2) are mediated through four G protein-coupled receptor subtypes, EP1-EP4. Recent studies have shown that EP2 and EP4 receptors play important roles in regulating bone formation and resorption. EP2 and EP4 receptor-selective agonists have been shown to stimulate local or systemic bone formation, augment bone mass and accelerate the healing of fractures or bone defects in animal models upon local or systemic administration, thus, potentially offering new therapeutic options for enhancing bone formation and bone repair in humans. This review will focus on the studies related to bone formation and bone healing in the EP receptor knockout (KO) mice and the EP2 or EP4 receptor-selective agonist treated animal models.

Recruitment of a Prostaglandin E Receptor Subtype, EP3-Expressing Bone Marrow Cells Is Crucial in Wound-Induced Angiogenesis

E-type prostaglandins have been reported to be proangiogenic in vivo. Thus, we examined prostaglandin receptor signaling relevant to wound-induced angiogenesis. Full-thickness skin wounds were created on the backs of mice, and angiogenesis in wound granulation tissues was estimated. Wound closure and re-epithelization in EP3 receptor knockout mice (EP3-/-) were significantly delayed compared with their wild-type (WT) mice, whereas those in EP1-/-, EP2-/-, and EP4-/- were not delayed. Wound-induced angiogenesis estimated with CD31 immunohistochemistry in EP3-/- mice was significantly inhibited compared with that in WT mice. Immunoreactive vascular endothelial growth factor (VEGF) in wound granulation tissues in EP3-/- mice was markedly less than that in WT mice. Wound closure in WT mice was delayed significantly by VEGF neutralizing antibody compared with control IgG. Wound-induced angiogenesis and wound closure were significantly suppressed in EP3-/- bone marrow transplantation mice compared with those in WT bone marrow transplantation mice. These were accompanied with the reductions in accumulation of VEGF-expressing cells in wound granulation tissues and in mobilization of VEGF receptor 1-expressing leukocytes in peripheral circulation. These results indicate that the recruitment of EP3-expressing cells to wound granulation tissues is critical for surgical wound healing and angiogenesis via up-regulation of VEGF.

Prostaglandin EP1 Receptor Contributes to Excitotoxicity and Focal Ischemic Brain Damage

The clinical side effects associated with the inhibition of cyclooxygenase enzymes under pathologic conditions have recently raised concerns. A better understanding of neuroinflammatory mechanisms and neuronal survival requires knowledge of cyclooxygenase downstream pathways, especially PGE2 and its G-protein-coupled receptors. In this study, we postulate that EP1 receptor is one of the mechanisms that propagate neurotoxicity and could be a therapeutic target in brain injury. This hypothesis was tested by pretreating C57BL/6 wildtype mice with the EP1 receptor selective agonist ONO-DI-004 and the selective antagonist ONO-8713, followed by striatal unilateral NMDA injection. Results revealed that ONO-DI-004 increased NMDA-induced lesion volume up to 128.7 +/- 12.0%, while ONO-8713 significantly decreased lesion volume to 71.3 +/- 10.9%, as compared to the NMDA-control group. Neurotoxic EP1 receptor properties were also studied using C57BL/6 EP1 receptor knockout (EP1-/-) mice, which revealed a significant decrease to 74.5 +/- 8.2%, as compared to wildtype controls. The protective effect of the antagonist ONO-8713 was also tested in the EP1-/- mice, revealing no additional protection in these mice. Together, these results support the selectivity of ONO-8713 toward EP1 receptor and suggest the neurotoxic role of EP1 receptor. Furthermore, the EP1 receptor role in ischemic brain damage was investigated using a model of middle cerebral artery occlusion (MCAO) and reperfusion. The infarct volume was significantly reduced to 56.9 +/- 11.5% in EP1-/- mice, as compared to wildtype controls. This is the first study that demonstrates that EP1 receptor aggravates neurotoxicity and that modulation of this receptor can determine the outcomes in both excitotoxic and focal ischemic neuronal damage.