EP3 prostanoid receptor isoforms utilize distinct mechanisms to regulate ERK 1/2 activation

Prostaglandin-E₂ (PGE₂) is a hormone derived from the metabolism of arachidonic acid whose functions include regulation of platelet aggregation, fever and smooth muscle contraction/relaxation. PGE₂ mediates its physiological and pathophysiological effects through its binding to four G-protein coupled receptor subtypes, named EP₁, EP₂, EP₃ and EP₄. The EP₃ prostanoid receptor is unique in that it has multiple isoforms generated by alternative mRNA splicing. These splice variants display differences in tissue expression, constitutive activity and regulation of signaling molecules. To date there are few reports identifying differential activities of EP₃ receptor isoforms and their effects on gene regulation. We generated HEK cell lines expressing the human EP_3-Ia, EP_3-II or EP_3-III isoforms. Using immunoblot analysis we found that nM concentrations of PGE₂ strongly stimulated the phosphorylation of ERK 1/2 by the EP_3-II and EP_3-III isoforms; whereas, ERK 1/2 phosphorylation by the EP_3-Ia isoform was minimal and only occurred at μM concentrations of PGE₂. Furthermore, the mechanisms of the PGE₂ mediated phosphorylation of ERK 1/2 by the EP_3-II and EP_3-III isoforms were different. Thus, PGE₂ stimulation of ERK 1/2 phosphorylation by the EP_3-III isoform involves activation of a Gα_i/PI3K/PKC/Src and EGFR-dependent pathway; while for the EP_3-II isoform it involves activation of a Gα_i/Src and EGFR-dependent pathway. These differences result in unique differences in the regulation of reporter plasmid activity for the downstream effectors ELK1 and AP-1 by the EP_3-II and EP_3-III prostanoid receptor isoforms.     

Prostaglandin EP1 receptor down-regulates expression of cyclooxygenase-2 by facilitating its proteasomal degradation

The enzyme cyclooxygenase-2 (COX-2) is rapidly and transiently up-regulated by a large variety of signals and implicated in pathologies such as inflammation and tumorigenesis. Although many signals cause COX-2 up-regulation, much less is known about mechanisms that actively down-regulate its expression. Here we show that the G protein-coupled receptor prostaglandin E(1) (EP(1)) reduces the expression of COX-2 in a concentration-dependent manner through a mechanism that does not require receptor activation. The reduction in COX-2 protein is not due to decreased protein synthesis and occurs because of enhancement of substrate-independent COX-2 proteolysis. Although EP(1) does not interfere with the entry of COX-2 into the endoplasmic reticulum-associated degradation cascade, it facilitates COX-2 ubiquitination through complex formation. Blockade of proteasomal activity results in degradation of the receptor and concomitant recovery in the expression of COX-2, suggesting that EP(1) may scaffold an unknown E3 ligase that ubiquitinates COX-2. These findings propose a new role for the EP(1) receptor in resolving inflammation through down-regulation of COX-2.     

Prostaglandin I2 as a potentiator of acute inflammation in rats

Prostaglandin I₂ potentiated the paw swelling induced by carrageenin in rats. Prostaglandin I₂ (0.1 μg) showed similar activity to PGE₁ (0.01 μg). This potentiating property disappeared in 60 minutes and was completely abolished by diphenhydramine (25 mg kg⁻¹, i.p.). In vascular permeability tests, PGI₂ itself (2.5 × 10⁻¹⁰ mol, 88 ng) caused no dye leakage reaction, but PGE₁ (2.5 × 10⁻¹⁰ mol, 88.5 ng) caused a significant dye leakage. This effect of PGE₁ was statistically significant compared with vehicle- or PGI₂-treated group (p<0.05). Prostaglandin I₂ potentiated the increased vascular permeability induced by 5-hydroxytriptamine (2.5 × 10⁻¹⁰ mol), bradykinin (5 × 10⁻¹⁰ mol) and histamine (2 × 10⁻¹⁰ to 2 × 10⁻⁸ mol). The potentiation was the most evidence in the case of histamine.     

Study on the heterogeneous degradation of chitosan with H2O2 catalyzed by a new supermolecular assembly crystal: [C6H8N2]6H3[PW12O40]·2H2O

A new supermolecular assembly crystal, [C₆H₈N₂]₆H₃[PW₁₂O₄₀]·2H₂O (DMB-PWA), was synthesized with phosphotungstic acid (PWA) and 1,2-diaminobenzene (DMB) under hydrothermal conditions and was characterized by Fourier-transform infrared spectra (FTIR) and single-crystal X-ray diffraction analysis. DMB-PWA could effectively catalyze oxidative degradation of chitosan with H₂O₂ in the heterogeneous phase. The optimum degradation conditions were determined by orthogonal tests as follows: amount of chitosan 1.00 g, 30% (wt %); H₂O₂, 3.0 mL; dosage of catalyst, 0.06 g; reaction temperature, 85 °C; and reaction time, 30 min. The water-soluble chitosan with a viscosity-average molecular weight (M_v) of 4900 was obtained under the optimum degradation conditions and was characterized by FTIR, ultraviolet-visible diffuse reflection spectra (UV-vis DRS), and X-ray powder diffraction analysis.     

Prostaglandin E2 down-regulation of cytochrome P-450 2B1 expression induced by phenobarbital is through EP2 receptor in rat hepatocytes

Cytochrome P-450 is an important bioactivation-detoxification system in vivo. Its expression is regulated by foreign chemicals and dietary factors, and lipids have been found to regulate its gene expression. We showed previously that prostaglandin E(2) (PGE(2)), a fatty acid metabolite, down-regulates cytochrome P-450 2B1 (CYP 2B1) expression induced by phenobarbital. The objective of the present study was to determine whether PGE(2) type 2 receptor (EP(2))-which is coupled to Gs-protein when bound by PGE(2), leading to cAMP production-is involved in this down-regulation. We also determined the possible roles of EP(2) downstream pathways in this down-regulation. We used a primary rat hepatocyte culture model in which EP(2) was shown to be present to study this question. The intracellular cAMP concentration in primary rat hepatocytes was significantly higher after treatment with 1microM PGE(2) than after treatment with 0, 0.01, or 0.1microM PGE(2). Butaprost, an EP(2) agonist, down-regulated CYP 2B1 expression in a dose-dependent manner. SQ22536, an adenylate cyclase inhibitor, reversed the down-regulation by PGE(2) as did H-89, a protein kinase A inhibitor. These results suggest that EP(2) and the downstream pathways of cAMP and protein kinase A are involved in the down-regulation of CYP 2B1 expression by PGE(2) in the presence of phenobarbital.     

EP2 receptor activation by prostaglandin E2 leads to induction of HO-1 via PKA and PI3K pathways in C6 cells

Recently we proposed that COX-2 induction precedes expression of HO-1 in ischemic preconditioned rat brain. In the current study, we investigated the molecular mechanism by which prostaglandin E₂, one of COX-2 metabolites, induces HO-1 in rat C6 brain cells. We demonstrated that concentration of PGE₂ increased HO-1 expression in C6 cells in vitro. The effects of PGE₂ were mimicked by PGE₂ receptor EP₂ agonists, 11-deoxy PGE₂, and cAMP analog, dibutyl-cAMP. HO-1 expression by PGE₂ was inhibited by LY294002, PI3K inhibitor and H89, PKA inhibitor. The EP₂-specific antagonist, AH8006 also inhibited PGE₂-mediated HO-1 expression in a concentration-dependent manner. Finally, PGE₂ inhibited GOX-induced apoptosis as assayed by FACS analysis or DNA strand breaks assay, and this cell death was reversed by ZnPPIX, HO-1 inhibitor. In addition to HO-1 induction, PGE₂ also increased phosphorylation of Bad by PKA- and PI3K-depednent manner. Taken together, we conclude that PGE₂ induces HO-1 protein expression through PKA and PI3K signaling pathways via EP₂ receptor in C6 cells. The induction of HO-1 along with increase of p-Bad by PGE₂ is responsible for anti-apoptosis against oxidant stress.     

Prostaglandin E1 or E2 inhibits an oxytocin-induced premature luteolysis in ewes when oxytocin is given early in the estrous cycle

The objective of this study was to determine whether PGE₁ or PGE₂ prevents a premature luteolysis when oxytocin is given on Days 1 to 6 of the ovine estrous cycle. Oxytocin given into the jugular vein every 8 hours on Days 1 to 6 postestrus in ewes decreased (P ≤ 0.05) luteal weights on Day 8 postestrus. Plasma progesterone differed (P ≤ 0.05) among the treatment groups; toward the end of the experimental period, concentrations of circulating progesterone in the oxytocin-only treatment group decreased (P ≤ 0.05) when compared with the other treatment groups. Plasma progesterone concentrations in ewes receiving PGE₁ or PGE₁ + oxytocin were greater (P ≤ 0.05) than in vehicle controls or in ewes receiving PGE₂ or PGE₂ + oxytocin and was greater (P ≤ 0.05) in all treatment groups receiving PGE₁ or PGE₂ than in ewes treated only with oxytocin. Chronic intrauterine treatment with PGE₁ or PGE₂ also prevented (P ≤ 0.05) oxytocin decreases in luteal unoccupied and occupied LH receptors on Day 8 postestrus. Oxytocin given alone on Days 1 to 6 postestrus in ewes advanced (P ≤ 0.05) increases in PGF_2α in inferior vena cava or uterine venous blood. PGE₁ or PGE₂ given alone did not affect (P ≥ 0.05) concentrations of PGF_2α in inferior vena cava and uterine venous blood when compared with vehicle controls or oxytocin-induced PGF_2α increases (P ≤ 0.05) in inferior vena cava or uterine venous blood. We concluded that PGE₁ or PGE₂ prevented oxytocin-induced premature luteolysis by preventing a loss of luteal unoccupied and occupied LH receptors.     

Prostaglandin E1 decreases circulating endothelial cells

Prostaglandin E₁ (PGE₁) has been claimed to have cytoprotective effects and also to decrease thrombogenicity. The effect of intraarterial (i.a.) and intravenous (i.v.) administration of PGE₁ on the number of circulating endothelial cells (CEC) was investigated in patients with peripheral vascular disease (PVD). Patients with hyperlipoproteinemia and also smokers exhibited higher numbers of CEC. PGE₁ significantly (p < 0.01) decreased CEC. In parallel, plate let survival was prolonged (r = −0.82). This effect lasted for more than a month after stopping PGE₁-therapy. The observed decrease in CEC reflects the decreased thrombogenicity and improved haemostasis achieved after PGE₁.     

Prostaglandin E2 produced by inducible COX-2 and mPGES-1 promoting cancer cell proliferation in vitro and in vivo

Aim

Many cancers originate and flourish in a prolonged inflammatory environment. Our aim is to understand the mechanisms of how the pathway of prostaglandin E₂ (PGE₂) biosynthesis and signaling can promote cancer growth in inflammatory environment at cellular and animal model levels.

Main methods

In this study, a chronic inflammation pathway was mimicked with a stable cell line that over-expressed a novel human enzyme consisting of cyclooxygenase isoform-2 (COX-2) linked to microsomal (PGE₂ synthase-1 (mPGES-1)) for the overproduction of pathogenic PGE₂. This PGE₂-producing cell line was co-cultured and co-implanted with three human cancer cell lines including prostate, lung, and colon cancers in vitro and in vivo, respectively.

Key findings

Increases in cell doubling rates for the three cancer cell types in the presence of the PGE₂-producing cell line were clearly observed. In addition, one of the four human PGE₂ subtype receptors, EP₁, was used as a model to identify PGE₂-signaling involved in promoting the cancer cell growth. This finding was further proven in vivo by co-implanting the PGE₂-producing cells line and the EP₁-positive cancer cells into the immune deficient mice, after that, it was observed that the PGE₂-producing cells promoted all three types of cancer formation in the mice.

Significance

This study clearly demonstrated that the human COX-2 linked to mPGES-1 is a pathway that, when mediated by the EP, is linked to promoting cancer growth in a chronic inflammatory environment. The identified pathway could be used as a novel target for developing and advancing anti-inflammation and anti-cancer interventions.     

Activation of cyclooxygenases by H2O2and t-butylhydroperoxide in aged rat lung

The arachidonate cascade is important for the generation of reactive species (RS), and cyclooxygenase (COX) is a key enzyme of this cascade. Tissues of 24-month-old rat lung showed a 2-fold increase in RS, malondialdehyde and thromboxane B₂ than those of 6-month-old rat. We found that the effects of 50 µM H₂O₂ and 200 µM t-butylhydroperoxide (t-BHP) specify on COX activity, and that their effects increased cytosolic COX activity in a concentration-dependent manner (1–50 µM) in 24-month-old rat. Our results suggested that COX activators such as t-BHP and H₂O₂, which are located in cytosol, are essential for the activation of COX in aged lung.     

Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents

Upregulation and activation of phospholipases A₂ (PLA₂) and cyclooxygenases (COX) leading to prostaglandin E₂(PGE₂) production have been implicated in a number of neurodegenerative diseases. In this study, we investigated PGE₂ production in primary rat astrocytes in response to agents that activate PLA₂ including pro-inflammatory cytokines (IL-1β, TNFα and IFNγ), the P2 nucleotide receptor agonist ATP, and oxidants (H₂O₂ and menadione). Exposure of astrocytes to cytokines resulted in a time-dependent increase in PGE₂ production that was marked by increased expression of secretory sPLA₂ and COX-2, but not COX-1 and cytosolic cPLA₂. Although astrocytes responded to ATP or phorbol ester (PMA) with increased cPLA₂ phosphorylation and arachidonic acid release, ATP or PMA only caused a small increase in levels of PGE₂. However, when astrocytes were first treated with cytokines, further exposure to ATP or PMA, but not H₂O₂ or menadione, markedly increased PGE₂ production. These results suggest that ATP release during neuronal excitation or injury can enhance the inflammatory effects of cytokines on PGE₂ production and may contribute to chronic inflammation seen in Alzheimer's disease.     

丁酸梭菌发酵培养基的优化及发酵产物对黄曲霉毒素B1的降解

【背景】丁酸梭菌是专性厌氧的新一代芽孢益生菌,耐热、耐酸、抗逆性强,极具应用价值和开发前景。【目的】优化丁酸梭菌发酵培养基并初步研究其发酵液对黄曲霉菌的抑制作用和降解黄曲霉毒素B₁ (aflatoxin B₁, AFB₁)的能力。【方法】利用响应面法对发酵培养基进行优化,采用牛津杯法对丁酸梭菌发酵液抑制黄曲霉菌生长进行研究,并通过酶联免疫法测定发酵液对AFB₁的降解能力。【结果】优化后的发酵培养基为：葡萄糖18.1g/L,大豆蛋白胨29.7g/L,磷酸氢二钾3.8 g/L,氯化钠2.0 g/L,乙酸钠4.0 g/L,结晶硫酸镁1.2 g/L,L-半胱氨酸盐酸盐0.3 g/L。优化后的丁酸梭菌生物量由8.99×10⁸个/mL提高至2.28×10⁹个/mL,是优化前的2.54倍。丁酸梭菌发酵液对致病真菌黄曲霉菌的抑菌效果十分显著,其上清液经浓缩后对AFB₁降解72h的降解率达到68.65%,初步分析表明上清液中对AFB₁     

Prostaglandin E2 receptor type 2-selective agonist prevents the degeneration of articular cartilage in rabbit knees with traumatic instability

Introduction

Osteoarthritis (OA) is a common cause of disability in older adults. We have previously reported that an agonist for subtypes EP2 of the prostaglandin E2 receptor (an EP2 agonist) promotes the regeneration of chondral and osteochondral defects. The purpose of the current study is to analyze the effect of this agonist on articular cartilage in a model of traumatic degeneration.

Methods

The model of traumatic degeneration was established through transection of the anterior cruciate ligament and partial resection of the medial meniscus of the rabbits. Rabbits were divided into 5 groups; G-S (sham operation), G-C (no further treatment), G-0, G-80, and G-400 (single intra-articular administration of gelatin hydrogel containing 0, 80, and 400 μg of the specific EP2 agonist, ONO-8815Ly, respectively). Degeneration of the articular cartilage was evaluated at 2 or 12 weeks after the operation.

Results

ONO-8815Ly prevented cartilage degeneration at 2 weeks, which was associated with the inhibition of matrix metalloproteinase-13 (MMP-13) expression. The effect of ONO-8815Ly failed to last, and no effects were observed at 12 weeks after the operation.

Conclusions

Stimulation of prostaglandin E2 (PGE2) via EP2 prevents degeneration of the articular cartilage during the early stages. With a system to deliver it long term, the EP2 agonist could be a new therapeutic tool for OA.     

Green tea epigallocatechin-3-gallate inhibits microsomal prostaglandin E2 synthase-1

Prostaglandin (PG)E₂ is a critical lipid mediator connecting chronic inflammation to cancer. The anti-carcinogenic epigallocatechin-3-gallate (EGCG) from green tea (Camellia sinensis) suppresses cellular PGE₂ biosynthesis, but the underlying molecular mechanisms are unclear. Here, we investigated the interference of EGCG with enzymes involved in PGE₂ biosynthesis, namely cytosolic phospholipase (cPL)A₂, cyclooxygenase (COX)-1 and -2, and microsomal prostaglandin E₂ synthase-1 (mPGES-1). EGCG failed to significantly inhibit isolated COX-2 and cPLA₂ up to 30 μM and moderately blocked isolated COX-1 (IC₅₀ > 30 μM). However, EGCG efficiently inhibited the transformation of PGH₂ to PGE₂ catalyzed by mPGES-1 (IC₅₀ = 1.8 μM). In lipopolysaccharide-stimulated human whole blood, EGCG significantly inhibited PGE₂ generation, whereas the concomitant synthesis of other prostanoids (i.e., 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-keto PGF_1α) was not suppressed. Conclusively, mPGES-1 is a molecular target of EGCG, and inhibition of mPGES-1 is seemingly the predominant mechanism underlying suppression of cellular PGE₂ biosynthesis by EGCG.     

c-Jun NH2-terminal kinase decreases ubiquitination and promotes stabilization of p21(WAF1/CIP1) in K562 cell

Proteasome-dependent degradation of regulatory proteins is a known mechanism of cell cycle control. p21(WAF1/CIP1) (p21), a negative regulator of the cell division cycle, exhibits proteasome-sensitive turnover and ubiquitination. In the present study, we analyzed the regulatory effects of JNK1 on p21 protein accumulation in p53 null K562 cells. We found that JNK1 (wild type, WT) mediated H(2)O(2)-induced p21 protein up-regulation. Over-expression of JNK1 (WT) could elevate endogenous p21 protein level but did not affect p21 mRNA level and also prolong the p21 half-life as well as inhibited the p21 ubiquitination. These findings indicated that JNK1 could regulate cellular p21 level via inhibiting ubiquitination of p21, which provided a new insight for analyzing the regulatory effect of JNK after stress.     

Prostaglandin E2 increases the calcium concentration in rat brown adipocytes and their consumption of oxygen

Effects of prostaglandin E₂ (PGE₁) were examined on the oxygen consumption and intracellular calcium concentration of rat brown adipose tissue (BAT). PGE₂ 0.1 nM-1 μM increased oxygen consumption of the tissue blocks of BAT, with a maximum 2–13 min after PGE2 administration. PGE₂ was most effective at 1 and 10 nM, and the oxygen consumption was elevated for over 40 min. Pretreatment of BAT with indomethacin, a prostaglandin synthesis inhibitor, did not affect the increase in oxygen consumption induced by noradrenaline. PGE₂ at 1–10 nM gradually increased the intracellular calcium concentration of freshly dispersed single brown adipocytes by 3–4 times in 30 min. PGE₂ also increased the intracellular calcium concentration of brown adipocytes in calcium-free medium. These results raise the possibility that PGE₂ and noradrenaline affect heat genesis and metabolism of BAT independently.     

Intracellular- and extracellular-derived Ca influence phospholipase A2-mediated fatty acid release from brain phospholipids

Docosahexaenoic acid (DHA) and arachidonic acid (AA) are found in high concentrations in brain cell membranes and are important for brain function and structure. Studies suggest that AA and DHA are hydrolyzed selectively from the sn-2 position of synaptic membrane phospholipids by Ca²⁺-dependent cytosolic phospholipase A₂ (cPLA₂) and Ca²⁺-independent phospholipase A₂ (iPLA₂), respectively, resulting in increased levels of the unesterified fatty acids and lysophospholipids. Cell studies also suggest that AA and DHA release depend on increased concentrations of Ca²⁺, even though iPLA₂ has been thought to be Ca²⁺-independent. The source of Ca²⁺ for activation of cPLA₂ is largely extracellular, whereas Ca²⁺ released from the endoplasmic reticulum can activate iPLA₂ by a number of mechanisms. This review focuses on the role of Ca²⁺ in modulating cPLA₂ and iPLA₂ activities in different conditions. Furthermore, a model is suggested in which neurotransmitters regulate the activity of these enzymes and thus the balanced and localized release of AA and DHA from phospholipid in the brain, depending on the primary source of the Ca²⁺ signal.     

Sphingosine-1-phosphate receptor S1P1 is regulated by direct interactions with P-Rex1, a Rac guanine nucleotide exchange factor

Sphingosine-1-phosphate (S1P) receptors S1P₁ are emerging molecular targets for the treatment of cancer, vascular and immune diseases, due to their pivotal role in cell migration and survival of immune and endothelial cells. A therapeutic strategy to control S1P₁ function is based on agonists that promote changes on S1P₁ expression at the plasma membrane. Here, we explored the hypothesis that cell surface expression and function of S1P₁ are influenced by direct interactions with P-Rex1, a guanine nucleotide exchange factor for Rac. We demonstrate that P-Rex1-PDZ domains interact with S1P₁-carboxyl terminal tail and full length receptor monomers and dimers. Endothelial cells transfected with P-Rex1-PDZ domains show an increased migratory response to S1P. S1P₁ trafficking to intracellular compartments is diminished by coexpression of P-Rex1. We conclude that S1P₁ signaling linked to cell migration is facilitated by a functional interaction with P-Rex1 via a mechanism that involves the maintenance of S1P₁ receptors at the cell membrane.     

Discovery of brain penetrant, soluble, pyrazole amide EP1 receptor antagonists

We describe the discovery of a series of pyrazole amide EP₁ receptor antagonists with good aqueous solubility and CNS penetration. In order to achieve solubility we investigated the incorporation of a basic group in the region of the molecule previously occupied by a carboxylic acid, which was known to be a key element of the pharmacophore. This study led to the identification of compounds such as 4h, 4j and 10b which demonstrated brain-to-blood ratios of 0.8:1–2.0:1 in addition to good solubility and metabolic stability.     

The identification of substituted benzothiophene derivatives as PGE(2) subtype 4 receptor antagonists: From acid to non-acid

We disclose herein our preliminary SAR study on the identification of substituted benzothiophene derivatives as PGE₂ subtype 4 receptor antagonists. A potent EP₄ antagonist 6a (K_i = 1.4 nM with 10% HSA) was identified. Furthermore, we found that an acidic group was not essential for the EP₄ antagonizing activity in the series and neutral replacements were identified. This opens a new direction for future EP₄ antagonist design.