Excessive EP4 Signaling in Smooth Muscle Cells Induces Abdominal Aortic Aneurysm by Amplifying Inflammation.

OBJECTIVE: Excessive prostaglandin E2 production is a hallmark of abdominal aortic aneurysm (AAA). Enhanced expression of prostaglandin E2 receptor EP4 (prostaglandin E receptor 4) in vascular smooth muscle cells (VSMCs) has been demonstrated in human AAAs. Although moderate expression of EP4 contributes to vascular homeostasis, the roles of excessive EP4 in vascular pathology remain uncertain. We aimed to investigate whether EP4 overexpression in VSMCs exacerbates AAAs. Approach and Results: We constructed mice with EP4 overexpressed selectively in VSMCs under an SM22α promoter (EP4-Tg). Most EP4-Tg mice died within 2 weeks of Ang II (angiotensin II) infusion due to AAA, while nontransgenic mice given Ang II displayed no overt phenotype. EP4-Tg developed much larger AAAs than nontransgenic mice after periaortic CaCl2 application. In contrast, EP4fl/+;SM22-Cre;ApoE-/- and EP4fl/+;SM22-Cre mice, which are EP4 heterozygous knockout in VSMCs, rarely exhibited AAA after Ang II or CaCl2 treatment, respectively. In Ang II-infused EP4-Tg aorta, Ly6Chi inflammatory monocyte/macrophage infiltration and MMP-9 (matrix metalloprotease-9) activation were enhanced. An unbiased analysis revealed that EP4 stimulation positively regulated the genes binding cytokine receptors in VSMCs, in which IL (interleukin)-6 was the most strongly upregulated. In VSMCs of EP4-Tg and human AAAs, EP4 stimulation caused marked IL-6 production via TAK1 (transforming growth factor-ß-activated kinase 1), NF-κB (nuclear factor-kappa B), JNK (c-Jun N-terminal kinase), and p38. Inhibition of IL-6 prevented Ang II-induced AAA formation in EP4-Tg. In addition, EP4 stimulation decreased elastin/collagen cross-linking protein LOX (lysyl oxidase) in both human and mouse VSMCs. CONCLUSIONS: Dysregulated EP4 overexpression in VSMCs promotes inflammatory monocyte/macrophage infiltration and attenuates elastin/collagen fiber formation, leading to AAA exacerbation.

PGE2/EP4 receptor and TRPV1 channel are involved in repeated restraint stress-induced prolongation of sensitization pain evoked by subsequent PGE2 challenge.

Prevalence of prior stressful experience is linked to high incidence of chronic pain. Stress, particularly repeated stress, is known to induce maladaptive neuroplasticity along peripheral and central pain transmission pathways. These maladaptive neuroplastic events facilitate sensitization of nociceptive neurons and transition from acute to chronic pain. Pro-inflammatory and pain mediators are involved in inducing neuroplasticity. Pain mediators such as prostaglandin E2 (PGE2), EP4 receptor and transient receptor potential vanilloid-1 (TRPV1) contribute to the genesis of chronic pain. In this study, we examined the role of PGE2/EP4 signaling and TRPV1 signaling in repeated restraint stress-induced prolongation of sensitization pain, a model for transition from acute to chronic pain, in both in vivo and in vitro models. We found that pre-exposure to single restraint stress induced analgesia that masked sensitization pain evoked by subsequent PGE2 challenge. However, pre-exposure to 3d consecutive restraint stress not only prolonged sensitization pain, but also increased stress hormone corticosterone (CORT) in serum, COX2 levels in paw skin, and EP4 and TRPV1 levels in dorsal root ganglion (DRG) and paw skin. Pre-exposure to CORT for 3d, not 1d, also prolonged sensitization pain evoked by PGE2. Co-injection of glucocorticoid receptor (GR) antagonist RU486, COX2 inhibitor NS-398, EP4 receptor antagonist L161,982 or TRPV1 antagonist capsazepine prevented 3d restraint stress prolonged sensitization pain evoked by PGE2. In DRG cultures, CORT increased EP4 and TRPV1 protein levels through GR activation. These data suggest that PGE2/EP4 signaling and TRPV1 signaling in peripheral pain pathway contribute to repeated stress-predisposed transition from acute to chronic pain.

[Role of prostaglandin E2 receptor EP4 in the regulation of adipogenesis and adipose metabolism].

Adipose tissue is the energy storage organ of the body, and excess energy is stored in adipocytes in the form of lipid droplets. The homeostasis of adipose tissue is the basis for the body to maintain normal metabolic activity. Prostaglandin E2 (PGE2) is an important lipid mediator in the body. It is synthesized in almost all tissues and participates in the regulation of many physiological processes such as blood pressure, glucose and lipid metabolism, and inflammation. PGE2 is abundant in white adipose tissue, where it is involved in the regulation of fat metabolism. PGE2 plays its biological role through binding to four G protein coupled receptors (prostaglandin E2 receptors), including EP-1, -2, -3, and -4. The EP4 subtype has been proved to play an important role in adipogenesis and adipose metabolism: it could inhibit adipogenesis while it was activated, whereas its knockout could promote lipolysis. This review summarized the relationship between EP4 and adipose metabolism, hoping to identify new targets of drug development for metabolic disorders.

[Role of prostaglandin E2 receptor 4 in cardiovascular diseases].

Prostaglandin E2 (PGE2) is a cyclooxygenase metabolite of arachidonic acid. It acts as a bioactive lipid and plays an important role in regulating many biological processes. PGE2 binds to 4 different G protein-coupled receptors including prostaglandin E2 receptor subtypes EP1, EP2, EP3 and EP4. The EP4 receptor is widely expressed in most of human organs and tissues. Increasing evidence demonstrates that EP4 is essential for cardiovascular homeostasis and participates in the pathogenesis of many cardiovascular diseases. Here we summarize the role of EP4 in the regulation of cardiovascular function and discuss potential mechanisms by which EP4 is involved in the development of cardiovascular disorders with a focus on its effect on inflammation.

Reciprocal interaction of HOTAIR and SP1 together enhance the ability of Xiaoji decoction and gefitinib to inhibit EP4 expression.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese herbal prescription Xiaoji decoction (XJD) has long been used for cancer treatment. However, the molecular mechanisms underlying the effects of this medicine, particularly to enhance the efficiency of EGFR-TKI in the treatment of lung cancer have not been well elucidated. MATERIALS AND METHODS: Cell viability and cell cycle distribution were detected by MTT assay and flow cytometry, respectively. The phosphorylation of ERK1/2 and protein levels of SP1 and EP4 were determined by Western blot. The expression of the HOX transcript antisense RNA (HOTAIR) was measured by qRT-PCR. Transient transfection experiments were used to overexpress the HOTAIR, SP1 and EP4 genes. The interaction between HOTAIR and SP1 were further examined via RNA immunoprecipitation (RIP) assay. A tumor xenograft model was used to confirm the in vitro findings. RESULTS: We showed that XJD inhibited growth and induced cell arrest of human non-small cell lung cancer (NSCLC) cells. We also found that XJD increased the phosphorylation of ERK1/2 and inhibited levels of HOTAIR and SP1, EP4 proteins, which were blocked by inhibitor of MEK/ERK. There was reciprocal interaction between HOTAIR and SP1. Silencing of HOTAIR reduced EP4 protein levels and repressed the growth of NSCLC cells, while overexpression of HOTAIR and SP1 overcame XJD-reduced EP4 protein expression. Additionally, excessive expressed EP4 reversed the effect of XJD on cell growth. Importantly, there was synergy of XJD with another cancer treatment drug, EGFR-TKI gefitinib, in this process. We also found that XJD inhibited tumor growth in a xenograft nude mice model. CONCLUSIONS: Our results show that XJD inhibits NSCLC cell growth via ERK1/2-mediated reciprocal repression of HOTAIR and SP1 protein expression, followed by reduced EP4 gene expression. XJD and gefitinib exhibit synergy in this process. The in vitro and in vivo study provides a novel mechanism by which XJD enhances the growth inhibitory effect of gefitinib in gefitinib-resistant NSCLC cells.

Prostanoid EP4 Receptor-Mediated Augmentation of I h Currents in Aß Dorsal Root Ganglion Neurons Underlies Neuropathic Pain.

An injury of the somatosensory system causes neuropathic pain, which is usually refractory to conventional analgesics, thus warranting the development of novel drugs against this kind of pain. The mechanism of neuropathic pain in rats that had undergone left L5 spinal nerve transection was analyzed. Ten days after surgery, these rats acquired neuropathic pain. The patch-clamp technique was used on the isolated bilateral L5 dorsal root ganglion neurons. The current-clamped neurons on the ipsilateral side exhibited significantly higher excitability than those on the contralateral side. However, only neurons with diameters of 40-50 µm on the ipsilateral side exhibited significantly larger voltage sags in response to hyperpolarizing current pulses than those on the contralateral side. Under the voltage clamp, only these neurons on the ipsilateral side showed a significantly larger density of an inward current at < -80 mV [hyperpolarization-activated nonselective cation (I h) current] with a rightward-shifted activation curve than that on the contralateral side. Ivabradine-an I h current inhibitor-inhibited I h currents in these neurons on both sides in a similar concentration-dependent manner, with an IC50 value of ∼3 µM. Moreover, the oral administration of ivabradine significantly alleviated the neuropathic pain on the ipsilateral side. An inhibitor of adenylyl cyclase or an antagonist of prostanoid EP4 receptors (CJ-023423) inhibited ipsilateral, but not contralateral I h, currents in these neurons. Furthermore, the intrathecal administration of CJ-023423 significantly attenuated neuropathic pain on the ipsilateral side. Thus, ivabradine and/or CJ-023423 may be a lead compound for the development of novel therapeutics against neuropathic pain.

Regulation of the porcine corpus luteum during pregnancy.

The new corpora lutea (CLs) in pigs are formed from the preovulatory follicles after the luteinizing hormone (LH) surge. However, total autonomy and independence of CLs from LH up to Day 12 of cycle has recently been questioned. Transformation of estrous cycle CL to CL of pregnancy initiated by embryonic signals requires not only the cessation of prostaglandin F2 (PGF2α) supply to the luteal tissue but also needs the CL to overcome luteolytic acquisition and/or changing its sensitivity to PGF2α during Days 12-14 of pregnancy. The luteolytic cascade is prevented by inhibition of lymphocyte infiltration and leucocyte recruitment, limitation of cell apoptosis, upregulation of pregnancy-associated genes and an enhanced antiluteolytic role of PGE2 Our 'two-signal switch hypothesis' highlights the importance of post PGF2α and PGE2 receptor signaling pathways activation in CLs during luteolysis and rescue. The 'luteolytic switch' involves increased expression of many regression mediators and activation of the post PTGFR signaling pathway. The 'rescue switch' initiated by embryonic signals - estradiol 17ß and PGE2 - induces post PTGER2/4 pathway, turning the 'luteolytic switch' off and triggering activity of genes responsible for CL maintenance. In mid and late pregnancy, CLs are maintained by LH and the synergistic action of metabolic hormones. This paper provides an outline of recent views on CL regression, rescue and maintenance during pregnancy in pigs that conflict with previous paradigms and highlights new findings regarding the actions of prostaglandins, role of microRNAs (miRNA) and immune system and signaling pathways governing the life cycle of porcine CL.

PGE2 /EP4 receptor attenuated mucosal injury via ß-arrestin1/Src/EGFR-mediated proliferation in portal hypertensive gastropathy.

BACKGROUND AND PURPOSE: Portal hypertensive gastropathy (PHG) is a serious complication of liver cirrhosis and a potential cause of bleeding in patients with cirrhosis. Suppressed mucosal epithelial proliferation is a crucial pathological characteristic of PHG. Our studies demonstrated an important role for PGE2 and its EP4 receptor in the promotion of mucosal proliferation. However, whether ß-arrestin1 (ß-arr1), a well-established mediator of GPCRs, is involved in the PGE2 /EP4 receptor-mediated mucosal proliferation complex in PHG remains unclear. The aim of the study was to investigate whether ß-arr1 participated in PGE2 /EP4 receptor-mediated mucosal proliferation by recruiting the Src/EGF receptor (EGFR) complex to activate Akt/proliferating cell nuclear antigen (PCNA) signalling in PHG. EXPERIMENTAL APPROACH: Gastric mucosal proliferation was examined in patients with PHG and the PHG model of ß-arr1-knockout (ß-arr1-KO) and ß-arr1-wild type (ß-arr1-WT) mice. The induction of ß-arr1 and EP4 receptor expression and the Src/EGFR signalling elements was investigated, and the mechanisms underlying PGE2 -regulated gastric mucosal proliferation were analysed. KEY RESULTS: Portal hypertension suppressed COX-1 but not COX-2, which was accompanied by a down-regulation of PGE2 generation and EP4 receptor levels in the mucosa of patients with PHG. PGE2 administration markedly promoted mucosal proliferation in a mouse model of PHG. Targeted deletion of ß-arr1 abolished PGE2 /EP4 receptor-mediated gastric proliferation in PHG by repressing the Src/EGFR/Akt/PCNA signalling network. CONCLUSIONS AND IMPLICATIONS: These results indicate that ß-arr1 regulates PGE2 /EP4 receptor-mediated mucosal proliferation by promoting activation of the Src/EGFR/Akt/PCNA signalling pathway, and thus, this network is a potential therapeutic target for PHG.

Activation of calcitonin gene-related peptide signaling through the prostaglandin E2-EP1/EP2/EP4 receptor pathway in synovium of knee osteoarthritis patients.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is a 37-amino-acid vasodilatory neuropeptide that binds to receptor activity-modifying protein 1 (RAMP1) and the calcitonin receptor-like receptor (CLR). Clinical and preclinical evidence suggests that CGRP is associated with hip and knee joint pain; however, the regulation mechanisms of CGRP/CGRP receptor signaling in synovial tissue are not fully understood. METHODS: Synovial tissues were harvested from 43 participants with radiographic knee osteoarthritis (OA; unilateral Kellgren/Lawrence (K/L) grades 3-4) during total knee arthroplasty. Correlationships between the mRNA expression levels of CGRP and those of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, and cycloxygenase-2 (COX-2) were evaluated using real-time PCR analysis of total RNA extracted from the collected synovial tissues. To investigate the factors controlling the regulation of CGRP and CGRP receptor expression, cultured synovial cells were stimulated with TNF-α, IL-1ß, IL-6, and prostaglandin E2 (PGE2) and were also treated with PGE2 receptor (EP) agonist. RESULTS: CGRP and COX-2 localized in the synovial lining layer. Expression of COX-2 positively correlated with CGRP mRNA expression in the synovial tissue of OA patients. The gene expression of CGRP and RAMP1 increased significantly in synovial cells exogenously treated with PGE2 compared to untreated control cells. In cultured synovial cells, CGRP gene expression increased significantly following EP4 agonist treatment, whereas RAMP1 gene expression increased significantly in the presence of exogenously added EP1 and EP2 agonists. CONCLUSIONS: PGE2 appears to regulate CGRP/CGRP receptor signaling through the EP receptor in the synovium of knee OA patients.

Doxorubicin resistant cancer cells activate myeloid-derived suppressor cells by releasing PGE2.

Chemotherapies often induce drug-resistance in cancer cells and simultaneously stimulate proliferation and activation of Myeloid-Derived Suppressor Cells (MDSCs) to inhibit anti-tumor T cells, thus result in poor prognosis of patients with breast cancers. To date, the mechanism underlying the expansion of MDSCs in response to chemotherapies is poorly understood. In the present study, we used in vitro cell culture and in vivo animal studies to demonstrate that doxorubicin-resistant breast cancer cells secret significantly more prostaglandin E2 (PGE2) than their parental doxorubicin-sensitive cells. The secreted PGE2 can stimulate expansion and polymerization of MDSCs by directly target to its receptors, EP2/EP4, on the surface of MDSCs, which consequently triggers production of miR-10a through activating PKA signaling. More importantly, activated MDSCs can inhibit CD4(+)CD25(-) T cells as evidenced by reduced proliferation and IFN-γ release. In order to determine the molecular pathway that involves miR-10a mediated activation of MDSCs, biochemical and pharmacological studies were carried out. We found that miR-10a can activate AMPK signaling to promote expansion and activation of MDSCs. Thus, these results reveal, for the first time, a novel role of PGE2/miR-10a/AMPK signaling axis in chemotherapy-induced immune resistance, which might be targeted for treatment of chemotherapy resistant tumors.

The prostaglandin E2/EP4 receptor/cyclic AMP/T-type Ca(2+) channel pathway mediates neuritogenesis in sensory neuron-like ND7/23 cells.

We investigated mechanisms for the neuritogenesis caused by prostaglandin E2 (PGE2) or intracellular cyclic AMP (cAMP) in sensory neuron-like ND7/23 cells. PGE2 caused neuritogenesis, an effect abolished by an EP4 receptor antagonist or inhibitors of adenylyl cyclase (AC) or protein kinase A (PKA) and mimicked by the AC activator forskolin, dibutyryl cAMP (db-cAMP), and selective activators of PKA or Epac. ND7/23 cells expressed both Cav3.1 and Cav3.2 T-type Ca(2+) channels (T-channels). The neuritogenesis induced by db-cAMP or PGE2 was abolished by T-channel blockers. T-channels were functionally upregulated by db-cAMP. The PGE2/EP4/cAMP/T-channel pathway thus appears to mediate neuritogenesis in sensory neurons.

Antidiuretic Action of Collecting Duct (Pro)Renin Receptor Downstream of Vasopressin and PGE2 Receptor EP4.

Within the kidney, the (pro)renin receptor (PRR) is predominantly expressed in the collecting duct (CD), particularly in intercalated cells, and it is regulated by the PGE2 receptor EP4 Notably, EP4 also controls urinary concentration through regulation of aquaporin 2 (AQP2). Here, we tested the hypothesis that sequential activation of EP4 and PRR determines AQP2 expression in the CD, thus mediating the antidiuretic action of vasopressin (AVP). Water deprivation (WD) elevated renal PRR expression and urinary soluble PRR excretion in rats. Intrarenal infusion of a PRR decoy peptide, PRO20, or an EP4 antagonist partially prevented the decrease in urine volume and the increase in urine osmolality and AQP2 expression induced by 48-hour WD. In primary cultures of rat inner medullary CD cells, AQP2 expression induced by AVP treatment for 24 hours depended on sequential activation of the EP4 receptor and PRR. Additionally, mice lacking PRR in the CD exhibited increased urine volume and decreased urine osmolality under basal conditions and impaired urine concentrating capability accompanied by severe volume loss and a dangerous level of plasma hyperosmolality after WD. Together, these results suggest a previously undescribed linear AVP/PGE2/EP4/PRR pathway in the CD for regulation of AQP2 expression and urine concentrating capability.

Doxycycline Attenuates Endotoxin-Induced Uveitis by Prostaglandin E2-EP4 Signaling.

PURPOSE: We explored the anti-inflammatory effects of doxycycline in experimental uveitis and the underlying mechanisms. METHODS: Rats with endotoxin-induced uveitis (EIU) received doxycycline (1.5 mg/kg) or the control vehicle via intraperitoneal injection. Clinical scores were graded under a slit lamp. Rat peritoneal macrophages were used in vitro to further explore the anti-inflammatory mechanisms of doxycycline. The levels of nitric oxide (NO), TNF-α, IL-1ß, prostaglandin E2 (PGE2), cyclooxygenase (COX)-2, I kappa B-α (IκB-α), inducible nitric oxide synthase (iNOS), Akt, caspase-3, and nuclear factor-kappa B (NF-κB) were analyzed. RESULTS: Treatment with doxycycline dramatically reduced the clinical scores of EIU (P < 0.001), with significant decreases in inflammatory cell infiltration, protein concentrations, and the production of NO, TNF-α, and IL-1ß in the aqueous humor (AqH). In vitro, doxycycline significantly inhibited the production of NO, IL-1ß, and TNF-α in peritoneal macrophages by modulating the PI3K/Akt/IκB-α/NF-κB pathway. Importantly, we found that doxycycline significantly enhanced COX2 expression and PGE2 production both in vivo and in vitro. More importantly, blockade of the EP4 receptor of PGE2 significantly reversed the doxycycline-mediated inhibition of macrophages and the PI3K/Akt pathway in vitro. Furthermore, simultaneous injection of an EP4 antagonist and doxycycline significantly blocked the doxycycline-mediated attenuation of EIU. CONCLUSIONS: Doxycycline can ameliorate EIU, and PGE2-EP4 signaling is essential for the anti-inflammatory effects of doxycycline in vitro and in vivo.

The Prostaglandin E2 Receptor EP4 Regulates Obesity-Related Inflammation and Insulin Sensitivity.

With increasing body weight, macrophages accumulate in adipose tissue. There, activated macrophages secrete numerous proinflammatory cytokines and chemokines, giving rise to chronic inflammation and insulin resistance. Prostaglandin E2 suppresses macrophage activation via EP4; however, the role of EP4 signaling in insulin resistance and type 2 diabetes mellitus remains unknown. In this study, we treated db/db mice with an EP4-selective agonist, ONO-AE1-329, for 4 weeks to explore the role of EP4 signaling in obesity-related inflammation in vivo. Administration of the EP4 agonist did not affect body weight gain or food intake; however, in the EP4 agonist-treated group, glucose tolerance and insulin resistance were significantly improved over that of the vehicle-treated group. Additionally, administration of the EP4 agonist inhibited the accumulation of F4/80-positive macrophages and the formation of crown-like structures in white adipose tissue, and the adipocytes were significantly smaller. The treatment of the EP4 agonist increased the number of anti-inflammatory M2 macrophages, and in the stromal vascular fraction of white adipose tissue, which includes macrophages, it markedly decreased the levels of proinflammatory cytokines and chemokines. Further, EP4 activation increased the expression of adiponectin and peroxidase proliferator-activated receptors in white adipose tissue. Next, we examined in vitro M1/M2 polarization assay to investigate the impact of EP4 signaling on determining the functional phenotypes of macrophages. Treatment with EP4 agonist enhanced M2 polarization in wild-type peritoneal macrophages, whereas EP4-deficient macrophages were less susceptible to M2 polarization. Notably, antagonizing peroxidase proliferator-activated receptor δ activity suppressed EP4 signaling-mediated shift toward M2 macrophage polarization. Thus, our results demonstrate that EP4 signaling plays a critical role in obesity-related adipose tissue inflammation and insulin resistance by regulating macrophage recruitment and polarization. The activation of EP4 signaling holds promise for treating obesity and type 2 diabetes mellitus.

Importance of cyclooxygenase-1/prostacyclin in modulating gastric mucosal integrity under stress conditions.

BACKGROUND AND AIM: We investigated the roles of cyclooxygenase (COX) isozymes and prostaglandins (PGs) and their receptors in mucosal defense against cold-restraint stress (CRS)-induced gastric lesions. METHODS: Male C57BL/6 wild-type (WT) mice and those lacking COX-1 or COX-2 as well as those lacking EP1, EP3, or IP receptors were used after 18 h fasting. Animals were restrained in Bollman cages and kept in a cold room at 10°C for 90 min. RESULTS: CRS induced multiple hemorrhagic lesions in WT mouse stomachs. The severity of these lesions was significantly worsened by pretreatment with the nonselective COX inhibitors (indomethacin, loxoprofen) or selective COX-1 inhibitor (SC-560), while neither of the selective COX-2 inhibitors (rofecoxib and celecoxib) had any effect. These lesions were also aggravated in animals lacking COX-1, but not COX-2. The expression of COX-2 mRNA was not detected in the stomach after CRS, while COX-1 expression was observed under normal and stressed conditions. The gastric ulcerogenic response to CRS was similar between EP1 or EP3 knockout mice and WT mice, but was markedly worsened in animals lacking IP receptors. Pretreating WT mice with iloprost (the PGI2 analog) significantly prevented CRS-induced gastric lesions in the presence of indomethacin. PGE2 also reduced the severity of these lesions, and the effect was mimicked by the EP4 agonist, AE1-329. CONCLUSIONS: These results suggest that endogenous PGs derived from COX-1 play a crucial role in gastric mucosal defense during CRS, and this action is mainly mediated by PGI2 /IP receptors and partly by PGE2 /EP4 receptors.

EP2 and EP4 receptors mediate PGE2 induced relaxation in murine colonic circular muscle: pharmacological characterization.

BACKGROUND: Prostaglandin E2 (PGE2) is a regulator of gastrointestinal motility that might be involved in impaired motor function associated to gut inflammation. The aim of the present work is to pharmacologically characterize responses to exogenous and endogenous PGE2 in the mouse colon targeting EP2 and EP4 receptors. METHODS: Wild type (WT) and EP2 receptor knockout (EP2-KO) mice were used to characterize PGE2 and butaprost (EP2 receptor agonist) effects on smooth muscle resting membrane potential and myogenic contractility in circularly oriented colonic preparations. RESULTS: In WT animals, PGE2 and butaprost concentration-dependently inhibited spontaneous contractions and hyperpolarized smooth muscle cells. Combination of both EP2 (PF-04418948 0.1µM) and EP4 receptor antagonists (L-161,982 10µM) was needed to block both electrical and mechanical PGE2 responses. Butaprost inhibitory responses (both electrical and mechanical) were totally abolished by PF-04418948 0.1µM. In EP2-KO mice, PGE2 (but not butaprost) concentration-dependently inhibited spontaneous contractions and hyperpolarized smooth muscle cells. In EP2-KO mice, PGE2 inhibition of spontaneous contractility and hyperpolarization was fully antagonized by L-161,982 10µM. In WT animals, EP2 and EP4 receptor antagonists caused a smooth muscle depolarization and an increase in spontaneous mechanical activity. CONCLUSIONS: PGE2 responses in murine circular colonic layer are mediated by post-junctional EP2 and EP4 receptors. PF-04418948 and L-161,982 are selective EP2 and EP4 receptor antagonists that inhibit PGE2 responses. These antagonists might be useful pharmacological tools to limit prostaglandin effects associated to dismotility in gut inflammatory processes.

PGE2-induced colon cancer growth is mediated by mTORC1.

The inflammatory prostaglandin E2 (PGE2) cytokine plays a key role in the development of colon cancer. Several studies have shown that PGE2 directly induces the growth of colon cancer cells and furthermore promotes tumor angiogenesis by increasing the production of the vascular endothelial growth factor (VEGF). The signaling intermediaries implicated in these processes have however not been fully characterized. In this report, we show that the mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in PGE2-induced colon cancer cell responses. Indeed, stimulation of LS174T cells with PGE2 increased mTORC1 activity as observed by the augmentation of S6 ribosomal protein phosphorylation, a downstream effector of mTORC1. The PGE2 EP4 receptor was responsible for transducing the signal to mTORC1. Moreover, PGE2 increased colon cancer cell proliferation as well as the growth of colon cancer cell colonies grown in matrigel and blocking mTORC1 by rapamycin or ATP-competitive inhibitors of mTOR abrogated these effects. Similarly, the inhibition of mTORC1 by downregulation of its component raptor using RNA interference blocked PGE2-induced LS174T cell growth. Finally, stimulation of LS174T cells with PGE2 increased VEGF production which was also prevented by mTORC1 inhibition. Taken together, these results show that mTORC1 is an important signaling intermediary in PGE2 mediated colon cancer cell growth and VEGF production. They further support a role for mTORC1 in inflammation induced tumor growth.

Antagonism of the prostaglandin E2 EP1 receptor in MDCK cells increases growth through activation of Akt and the epidermal growth factor receptor.

The actions of prostaglandin E2 (PGE2) in the kidney are mediated by G protein-coupled E-prostanoid (EP) receptors, which affect renal growth and function. This report examines the role of EP receptors in mediating the effects of PGE2 on Madin-Darby canine kidney (MDCK) cell growth. The results indicate that activation of Gs-coupled EP2 and EP4 by PGE2 results in increased growth, while EP1 activation is growth inhibitory. Indeed, two EP1 antagonists (ONO-8711 and SC51089) stimulate, rather than inhibit, MDCK cell growth, an effect that is lost following an EP1 knockdown. Similar observations were made with M1 collecting duct and rabbit kidney proximal tubule cells. ONO-8711 even stimulates growth in the absence of exogenous PGE2, an effect that is prevented by ibuprofen (indicating a dependence upon endogenous PGE2). The involvement of Akt was indicated by the observation that 1) ONO-8711 and SC51089 increase Akt phosphorylation, and 2) MK2206, an Akt inhibitor, prevents the increased growth caused by ONO-8711. The involvement of the EGF receptor (EGFR) was indicated by 1) the increased phosphorylation of the EGFR caused by SC51089 and 2) the loss of the growth-stimulatory effect of ONO-8711 and SC51089 caused by the EGFR kinase inhibitor AG1478. The growth-stimulatory effect of ONO-8711 was lost following an EGFR knockdown, and transduction of MDCK cells with a dominant negative EGFR. These results support the hypothesis that 1) signaling via the EP1 receptor involves Akt as well as the EGFR, and 2), EP1 receptor pharmacology may be employed to prevent the aberrant growth associated with a number of renal diseases.

Prostaglandin I2 and prostaglandin E2 modulate human intrarenal artery contractility through prostaglandin E2-EP4, prostacyclin-IP, and thromboxane A2-TP receptors.

Cyclooxygenase inhibitors decrease renal blood flow in settings with decreased effective circulating volume. The present study examined the hypothesis that prostaglandins, prostaglandin E2 (PGE2) and prostacyclin (PGI2), induce relaxation of human intrarenal arteries through PGE2-EP and PGI2-IP receptors. Intrarenal arteries were microdissected from human nephrectomy samples (n=53, median diameter ≈362 µm, 88% viable, 76% relaxed in response to acetylcholine). Rings were suspended in myographs to record force development. In vessels with K(+)-induced tension (EC70: -log [mol/L]=1.36±0.03), PGE2 and PGI2 induced concentration-dependent relaxation (-log EC50: PGE2=7.1±0.3 and PGI2=7.7). The response to PGE2 displayed endothelium dependence and desensitization. Relaxation by PGE2 was mimicked by an EP4 receptor agonist (CAY10598, EC50=6.7±0.2). The relaxation after PGI2 was abolished by an IP receptor antagonist (BR5064, 10(-8) mol/L). Pretreatment of quiescent arteries with PGE2 for 5 minutes (10(-6) mol/L) led to a significant right shift of the concentration-response to norepinephrine (EC50 from 6.6±0.1-5.9±0.1). In intrarenal arteries with K(+)-induced tone, PGE2 and PGI2 at 10(-5) mol/L elicited increased tension. This was abolished by thromboxane receptor (TP) antagonist (S18886, 10(-6) mol/L). A TP agonist (U46619, n=6) evoked tension (EC50=8.1±0.2) that was inhibited by S18886. Polymerase chain reaction and immunoblotting showed EP4, IP, and TP receptors in intrarenal arteries. In conclusion, PGE2 and PGI2 may protect renal perfusion by activating cognate IP and EP4 receptors associated with smooth muscle cells and endothelium in human intrarenal arteries and contribute to increased renal vascular resistance at high pathological concentrations mediated by noncognate TP receptor.

Active smoking increases microsomal PGE2-synthase-1/PGE-receptor-4 axis in human abdominal aortic aneurysms.

BACKGROUND: The cyclooxygenase- (COX-) 2/microsomal PGE-synthase- (mPGES-) 1/PGE-receptor- (EP-) 4 axis could play a key role in the physiopathology of abdominal aortic aneurysm (AAA) in humans. In this study, we investigated the influence of cardiovascular risk factors on the expression of the PGE2 pathway in human AAA. METHODS: Aortic (n = 89) and plasma (n = 79) samples from patients who underwent AAA repair were collected. Patients were grouped according to risk factors. COX-isoenzymes, mPGES-1, EPs, α-actin, and CD45 and CD68 transcripts levels were quantified by QRT-PCR and plasma PGE2 metabolites by EIA. RESULTS: Current smoking (CS) patients compared to no-CS had significantly higher local levels of mPGES-1 (P = 0.009), EP-4 (P = 0.007), and PGE2 metabolites plasma levels (P = 0.008). In the multiple linear regression analysis, these parameters remained significantly enhanced in CS after adding confounding factors. Results from association studies with cell type markers suggested that the increased mPGES-1/EP-4 levels were mainly associated with microvascular endothelial cells. CONCLUSIONS: This study shows that elements of the PGE2 pathway, which play an important role in AAA development, are increased in CS. These results provide insight into the relevance of tobacco smoking in AAA development and reinforce the potential of mPGES-1 and EP-4 as targets for therapy in AAA patients.