Effect of early pregnancy on the expression of prostaglandin synthases in the ovine thymus.

Thymus is a primary lymphoid organ, must adapt to the presence of fetal alloantigens. Prostaglandins (PGs) have diverse effects to activate or inhibit the immune response, but effects of early pregnancy on the expression of PG synthases in ovine maternal thymus are unclear. In this study, ovine thymic samples were obtained at day 16 of the estrous cycle, and days 13, 16 and 25 of pregnancy. The expression of PG synthases, including cyclooxygenase 1 (COX-1), COX-2, PGE2 synthase (PTGES), and a prostaglandin F2α synthase (Aldo-keto reductase family 1, member B1, AKR1B1), was evaluated using quantitative real-time PCR, Western blot and immunohistochemistry analysis. In addition, the thymus/body ratio was also calculated. Our results showed that the expression of COX-2 mRNA and protein, AKR1B1 mRNA and dimer were up-regulated on day 25 of pregnancy (P < 0.05), and expression of COX-1, PTGES mRNA and protein, AKR1B1 monomer and thymus/body ratio were similar at different stages of pregnancy and the estrous cycle. The immunohistochemistry results showed that the COX-2 and AKR1B1 proteins were located in the stromal cells, capillaries and thymic corpuscles. This is the first study to report that expression of COX-2 and AKR1B1 dimer is up-regulated in the maternal thymus during early pregnancy, suggesting that early pregnancy exerts its effects on maternal thymus, which is involved in immunomodulation during early pregnancy in sheep.

Characterization of a novel glycosylated glutathione transferase of Onchocerca ochengi, closest relative of the human river blindness parasite.

Filarial nematodes possess glutathione transferases (GSTs), ubiquitous enzymes with the potential to detoxify xenobiotic and endogenous substrates, and modulate the host immune system, which may aid worm infection establishment, maintenance and survival in the host. Here we have identified and characterized a σ class glycosylated GST (OoGST1), from the cattle-infective filarial nematode Onchocerca ochengi, which is homologous (99% amino acid identity) with an immunodominant GST and potential vaccine candidate from the human parasite, O. volvulus, (OvGST1b). Onchocerca ochengi native GSTs were purified using a two-step affinity chromatography approach, resolved by 2D and 1D SDS-PAGE and subjected to enzymic deglycosylation revealing the existence of at least four glycoforms. A combination of lectin-blotting and mass spectrometry (MS) analyses of the released N-glycans indicated that OoGST1 contained mainly oligomannose Man5GlcNAc2 structure, but also hybrid- and larger oligommanose-type glycans in a lower proportion. Furthermore, purified OoGST1 showed prostaglandin synthase activity as confirmed by Liquid Chromatography (LC)/MS following a coupled-enzyme assay. This is only the second reported and characterized glycosylated GST and our study highlights its potential role in host-parasite interactions and use in the study of human onchocerciasis.

Expression profiles of interferon-stimulated gene 15 and prostaglandin synthases in the ovine lymph nodes during early pregnancy.

Lymph nodes are distributed all over the body and are part of the lymphatic system. The interferon-stimulated gene 15 kDa protein (ISG15) and prostaglandins (PGs) are involved in the establishment of pregnancy and are expressed in the uterus during early pregnancy in sheep. In this study, the ovine lymph nodes were obtained on Day 16 of the estrous cycle, and Days 13, 16, and 25 of pregnancy, and the expression of ISG15 and PG synthases, including cyclooxygenase 1 (COX-1), COX-2, prostaglandin E (PGE) synthase (PTGES), and a PGF synthase (aldo-keto reductase family 1, member B1, AKR1B1) were detected by quantitative real-time polymerase chain reaction, western blot analysis, and immunohistochemistry analysis. Our results showed that there were peaks in the expression of mRNAs and the proteins of ISG15, COX-1, COX-2, PTGES, and AKR1B1 in the lymph nodes during early pregnancy and that the COX-2 and AKR1B1 proteins were limited to the subcapsular sinus and lymph sinuses. In conclusion, the ISG15, COX-1, COX-2, PTGES, and AKR1B1 were upregulated in the maternal lymph nodes, which may be beneficial for the development of conceptus, maternal systemic immunoregulation, and anti-luteolysis during early pregnancy in sheep.

Expression of interferon-stimulated gene 15-kDa protein, cyclooxygenase (COX) 1, COX-2, aldo-keto reductase family 1, member B1, and prostaglandin E synthase in the spleen during early pregnancy in sheep.

The spleen is a unique lymphoid organ that plays a key role in immune regulation. Interferon-tau induces upregulation of interferon-stimulated gene 15-kDa protein (ISG15) in the uterus during early pregnancy in sheep. Prostaglandins (PGs) have important effects in both the activation and the inhibition of immune response through an autocrine and paracrine manner. In this study, splenic samples were obtained at Day 16 of the estrous cycle, and Days 13, 16, and 25 of pregnancy from ewes, and the expression of ISG15 and PG synthases, including cyclooxygenase 1 (COX-1), COX-2, PGE synthase (PTGES), and PGF synthase (aldo-keto reductase family 1, member B1, AKR1B1), was detected through quantitative real-time PCR, western blot, and immunohistochemistry analysis. Our results showed that there was upregulation of COX-2 mRNA and protein at Day 25 of pregnancy, and ISG15 mRNA and conjugated proteins, and AKR1B1 mRNA and dimer at Days 16 and 25 of pregnancy. COX-2 and AKR1B1 proteins were limited to the capsule, trabeculae, and splenic cords. However, the expression of COX-1 and PTGES was not affected by early pregnancy. In conclusion, ISG15-conjugated proteins, COX-2 and AKR1B1 upregulated in maternal spleen during early pregnancy, which may be beneficial for the placentation in sheep.

Genetic and Histological Alterations Reveal Key Role of Prostaglandin Synthase and Cyclooxygenase 1 and 2 in Traumatic Brain Injury-Induced Neuroinflammation in the Cerebral Cortex of Rats Exposed to Moderate Fluid Percussion Injury.

After the initial insult in traumatic brain injury (TBI), secondary neurodegeneration occurs that is intimately associated with neuroinflammation. Prostaglandin (PG) synthases and cyclooxygenase (COX) 1 and 2 may contribute to inflammation in the brain. Temporal and spatial expression features of PG and COX1 and 2 following trauma may guide the development of antineuroinflammation strategies. Here, we examined PG synthase signaling and COX1 and 2 gene expression levels and COX-1- and 2-positive cell types and their temporal localization in TBI-induced brain in an effort to reveal their participation in the disease's evolving neuroinflammation. Using brain samples from the cerebral cortex of rats subjected to TBI model of lateral moderate fluid percussion injury (FPI), we sought to characterize the temporal (subacute TBI) and spatial (lateral cortical lesion) brain alterations accompanying the disease progression. Temporal gene expression changes of PG synthase signaling were compared between sham-operated and TBI-treated rats using microarray pathway analysis. Moreover, we examined COX1 and 2 expression patterns and their intracellular distribution in sham-operated and TBI-treated rats by immunohistochemistry. After FPI, COX1 and 2 gene expression levels, and PGE2 synthase increased while PGD2 synthase decreased, suggesting that PGE2 and PGD2 afforded contraindicative effects of inflammation and anti-inflammation, respectively. Immunohistochemical analyses showed that both COX1 and COX2 increased in a time-dependent manner in the brain, specifically in degenerating neurons of the cortex. Interestingly, the expression of COX cell type was cell-specific, in that COX1 was particularly increased in degenerating neurons while COX2 was expressed in macrophages. In view of the dynamic temporal and spatial expression of PG, COX1 and 2 gene expression and localization in the injured brain regulating PG synthase and COX1 and 2 activity will require a careful disease-specific tailoring of treatments to abrogate the neuroinflammation-plagued secondary cell death due to TBI.

Computational models for the classification of mPGES-1 inhibitors with fingerprint descriptors.

Human microsomal prostaglandin [Formula: see text] synthase (mPGES)-1 is a promising drug target for inflammation and other diseases with inflammatory symptoms. In this work, we built classification models which were able to classify mPGES-1 inhibitors into two groups: highly active inhibitors and weakly active inhibitors. A dataset of 1910 mPGES-1 inhibitors was separated into a training set and a test set by two methods, by a Kohonen's self-organizing map or by random selection. The molecules were represented by different types of fingerprint descriptors including MACCS keys (MACCS), CDK fingerprints, Estate fingerprints, PubChem fingerprints, substructure fingerprints and 2D atom pairs fingerprint. First, we used a support vector machine (SVM) to build twelve models with six types of fingerprints and found that MACCS had some advantage over the other fingerprints in modeling. Next, we used naïve Bayes (NB), random forest (RF) and multilayer perceptron (MLP) methods to build six models with MACCS only and found that models using RF and MLP methods were better than NB. Finally, all the models with MACCS keys were used to make predictions on an external test set of 41 compounds. In summary, the models built with MACCS keys and using SVM, RF and MLP methods show good prediction performance on the test sets and the external test set. Furthermore, we made a structure-activity relationship analysis between mPGES-1 and its inhibitors based on the information gain of fingerprints and could pinpoint some key functional groups for mPGES-1 activity. It was found that highly active inhibitors usually contained an amide group, an aromatic ring or a nitrogen heterocyclic ring, and several heteroatoms substituents such as fluorine and chlorine. The carboxyl group and sulfur atom groups mainly appeared in weakly active inhibitors.

Prosurvival effect of cumulus prostaglandin G/H synthase 2/prostaglandin2 signaling on bovine blastocyst: impact on in vivo posthatching development.

Apoptotic activity is a common physiological process which culminates at the blastocyst stage in the preimplantation embryo of many mammals. The degree of embryonic cell death can be influenced by the oocyte microenvironment. However, the prognostic significance of the incidence of apoptosis remains undefined. Prostaglandin E2 (PGE2) derived from prostaglandin G/H synthase-2 (PTGS2) activity is a well-known prosurvival factor that is mainly studied in oncology. PGE2 is the predominant PTGS2-derived prostaglandin present in the oocyte microenvironment during the periconceptional period. Using an in vitro model of bovine embryo production followed by transfer and collection procedures, we investigated the impact of periconceptional PGE2 on the occurrence of spontaneous apoptosis in embryos and on subsequent in vivo posthatching development. Different periconceptional PGE2 environments were obtained using NS-398, a specific inhibitor of PTGS2 activity, and exogenous PGE2. We assessed the level of embryonic cell death in blastocysts at day 8 postfertilization by counting total cell numbers, by the immunohistochemical staining of active caspase-3, and by quantifying terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling signals and apoptosis regulator (BCL-2/BAX) mRNA expression. Morphometric parameters were used to estimate the developmental stage of the embryonic disk and the extent of trophoblast elongation on day 15 conceptuses. Our findings indicate that periconceptional PGE2 signaling durably impacts oocytes, conferring increased resistance to spontaneous apoptosis in blastocysts and promoting embryonic disk development and the elongation process during preimplantation development.

Acetylsalicylic acid interferes with embryonic kidney growth and development by a prostaglandin-independent mechanism.

AIM: To evaluate the effects of the non-selective, non-steroidal anti-inflammatory drug (NSAID) acetylsalicylic acid (ASA), on ex vivo embryonic kidney growth and development. METHODS: Pairs of fetal mouse kidneys at embryonic day 12.5 were cultured ex vivo in increasing concentrations of ASA (0.04-0.4 mg/mL) for up to 7 d. One organ from each pair was grown in control media and was used as the internal control for the experimental contralateral organ. In some experiments, organs were treated with ASA for 48 h and then transferred either to control media alone or control media containing 10 µmol/L prostaglandin E2 (PGE2) for a further 5 d. Fetal kidneys were additionally obtained from prostaglandin synthase 2 homozygous null or heterozygous (PTGS2-/- and PTGS2-/+) embryos and grown in culture. Kidney cross-sectional area was used to determine treatment effects on kidney growth. Whole-mount labelling to fluorescently detect laminin enabled crude determination of epithelial branching using confocal microscopy. RESULTS: Increasing ASA concentration (0.1, 0.2 and 0.4 mg/mL) significantly inhibited metanephric growth (P < 0.05). After 7 d of culture, exposure to 0.2 mg/mL and 0.4 mg/mL reduced organ size to 53% and 23% of control organ size respectively (P < 0.01). Addition of 10 µmol/L PGE2 to culture media after exposure to 0.2 mg/mL ASA for 48 h resulted in a return of growth area to control levels. Application of control media alone after cessation of ASA exposure showed no benefit on kidney growth. Despite the apparent recovery of growth area with 10 µmol/L PGE2, no obvious renal tubular structures were formed. The number of epithelial tips generated after 48 h exposure to ASA was reduced by 40% (0.2 mg/mL; P < 0.05) and 47% (0.4 mg/mL; P < 0.01). Finally, growth of PTGS2-/- and PTGS2+/- kidneys in organ culture showed no differences, indicating that PTGS2 derived PGE2 may at best have a minor role. CONCLUSION: ASA reduces early renal growth and development but the role of prostaglandins in this may be minor.

[Gene regulation of prostaglandin synthase and prostate diseases].

Prostaglandin synthase (PGS) can catalyze the production of various types of prostaglandins and regulate the expression levels of related substances. The regulation mechanisms of the PGS gene are closely related with the occurrence and development of prostate diseases. However, few studies are reported on the regulation mechanisms of PGS in prostatic diseases, such as benign prostatic hyperplasia (BPH) and prostate cancer (PCa), or on the relationship between PGS gene regulation and prostate diseases. This review aims to analyze their correlation and provide some ideas for the prevention and control of BPH and PCa by intervention of the prostaglandin synthase regulatory pathway.

Gene regulation of prostaglandin synthase and prostate diseases / 中华男科学杂志

Prostaglandin synthase (PGS) can catalyze the production of various types of prostaglandins and regulate the expression levels of related substances. The regulation mechanisms of the PGS gene are closely related with the occurrence and development of prostate diseases. However, few studies are reported on the regulation mechanisms of PGS in prostatic diseases, such as benign prostatic hyperplasia (BPH) and prostate cancer (PCa), or on the relationship between PGS gene regulation and prostate diseases. This review aims to analyze their correlation and provide some ideas for the prevention and control of BPH and PCa by intervention of the prostaglandin synthase regulatory pathway.

mRNA expression pattern of selected candidate genes differs in bovine oviductal epithelial cells in vitro compared with the in vivo state and during cell culture passages.

BACKGROUND: The mammalian oviduct provides the optimal environment for gamete maturation including sperm capacitation, fertilization, and development of the early embryo. Various cell culture models for primary bovine oviductal epithelial cells (BOEC) were established to reveal such physiological events. The aim of this study was to evaluate 17 candidate mRNA expression patterns in oviductal epithelial cells (1) in transition from in vivo cells to in vitro cells; (2) during three consecutive cell culture passages; (3) affected by the impact of LOW or HIGH glucose content media; and (4) influenced by different phases of the estrous cycle in vivo and in vitro. In addition, the release of a metabolite and proteins from BOEC at two distinct cell culture passage numbers was estimated to monitor the functionality. METHODS: BOEC from 8 animals were isolated and cultured for three consecutive passages. Total RNA was extracted from in vivo and in vitro samples and subjected to reverse transcription quantitative polymerase chain reaction to reveal mRNA expression of selected candidate genes. The release of prostaglandin E2 (PGE2), oviduct-specific glycoprotein 1 (OVGP1) and interleukin 8 (IL8) by BOEC was measured by EIA or ELISA after 24 h. RESULTS: Almost all candidate genes (prostaglandin synthases, enzymes of cellular metabolism and mucins) mRNA expression pattern differed compared in vivo with in vitro state. In addition, transcription of most candidate genes was influenced by the number of cell culture passages. Different glucose medium content did not affect mRNA expression of most candidate genes. The phase of the estrous cycle altered some candidate mRNA expression in BOEC in vitro at later passages. The release of PGE2 and OVGP1 between passages did not differ. However, BOEC in passage 3 released significantly higher amount of IL8 compared with cells in passage 0. CONCLUSION: This study supports the hypothesis that candidate mRNA expression in BOEC was influenced by transition from the in vivo situation to the new in vitro environment and during consecutive passages. The consequence of cell culture passaging on BOEC ability to release bioactive compounds should be considered.

Identification of prostamides, fatty acyl ethanolamines, and their biosynthetic precursors in rabbit cornea.

Arachidonoyl ethanolamine (anandamide) and pros-taglandin ethanolamines (prostamides) are biologically active derivatives of arachidonic acid. Although available through different precursor phospholipids, there is considerable overlap between the biosynthetic pathways of arachidonic acid-derived eicosanoids and anandamide-derived prostamides. Prostamides exhibit physiological actions and are involved in ocular hypotension, smooth muscle contraction, and inflammatory pain. Although topical application of bimatoprost, a structural analog of prostaglandin F2α ethanolamide (PGF2α-EA), is currently a first-line treatment for ocular hypertension, the endogenous production of prostamides and their biochemical precursors in corneal tissue has not yet been reported. In this study, we report the presence of anandamide, palmitoyl-, stearoyl-, α-linolenoyl docosahexaenoyl-, linoleoyl-, and oleoyl-ethanolamines in rabbit cornea, and following treatment with anandamide, the formation of PGF2α-EA, PGE2-EA, PGD2-EA by corneal extracts (all analyzed by LC/ESI-MS/MS). A number of N-acyl phosphatidylethanolamines, precursors of anandamide and other fatty acyl ethanolamines, were also identified in corneal lipid extracts using ESI-MS/MS. These findings suggest that the prostamide and fatty acid ethanolamine pathways are operational in the cornea and may provide valuable insight into corneal physiology and their potential influence on adjacent tissues and the aqueous humor.

Expression of AKR1B1, AKR1C3 and other genes of prostaglandin F2α biosynthesis and action in ovarian endometriosis tissue and in model cell lines.

Endometriosis is a frequent benign gynecological disease characterized by endometrial tissue outside the uterine cavity. The estimated prevalence in the general population is 6-10%, but this reaches 30-50% in women with infertility and/or pain. As ectopic tissue within the pelvic cavity provokes inflammation, endometriosis is also considered a chronic inflammatory disease, and is characterized by increased peritoneal fluid levels of prostaglandin (PG)E2 and PGF2α. The AKR1B1 and AKR1C3 enzymes act as PG synthases and catalyze reduction of PGH2 to PGF2α, and PGD2 to 9α,11ß-PGF2α, respectively. AKR1B1 and AKR1C3 may thus be associated with increased PGF2α production in endometriosis patients, as supported by our previous report of increased AKR1C1-AKR1C3 mRNA levels in endometriotic tissue, compared to control endometrium. Here, we initially evaluated PGF2α concentrations in peritoneal fluid from endometriosis patients and healthy women. We also examined expression of AKR1B1, AKR1C3 and other genes involved in PGF2α biosynthesis, metabolism, and action in ovarian endometriosis tissue versus healthy endometrium, and in peritoneal endometriosis and control endometrium model cell lines. Compared to controls, increased PGF2α concentrations in peritoneal fluid of patients were supported by endometriotic tissue showing increased AKR1B1 mRNA and protein levels, but unchanged AKR1C3 protein levels. Among genes involved in PGF2α biosynthesis, metabolism and action PLA2G2A, PTGS2/COX-2, ABCC4 and PTGFR were up-regulated, mRNA levels of SLCO2A, PTGDS and HPGDS were unchanged, and genes PLA2G4A and HPGD were down-regulated in diseased tissue. All of these PGF2α-associated genes were also expressed in control endometrial HIEEC epithelial and HIESC stromal cell lines, and in peritoneal endometriosis 12-Z epithelial and 22-B stromal cell lines. Higher expression of PLA2G2A, PTGS2, AKR1B1, AKR1C3 and ABCC4 was seen in 22-B endometriosis cells compared to HIESC control cells. These cell models characterized in this study will enable further investigations into the role of PGF2α in the pathophysiology of endometriosis and the involvement of AKR1B1 and AKR1C3.

Bacterial and algal orthologs of prostaglandin H2synthase: novel insights into the evolution of an integral membrane protein.

Prostaglandin H2synthase (PGHS; EC 1.14.99.1), a bi-functional heme enzyme that contains cyclooxygenase and peroxidase activities, plays a central role in the inflammatory response, pain, and blood clotting in higher eukaryotes. In this review, we discuss the progenitors of the mammalian enzyme by using modern bioinformatics and homology modeling to draw comparisons between this well-studied system and its orthologs from algae and bacterial sources. A clade of bacterial and algal orthologs is described that have salient structural features distinct from eukaryotic counterparts, including the lack of a dimerization and EGF-like domains, the absence of gene duplicates, and minimal membrane-binding domains. The functional implications of shared and variant features are discussed.

COX-2-derived endocannabinoid metabolites as novel inflammatory mediators.

Cyclooxygenase-2 (COX-2) is an enzyme that plays a key role in inflammatory processes. Classically, this enzyme is upregulated in inflammatory situations and is responsible for the generation of prostaglandins (PGs) from arachidonic acid (AA). One lesser-known property of COX-2 is its ability to metabolize the endocannabinoids, N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG). Endocannabinoid metabolism by COX-2 is not merely a means to terminate their actions. On the contrary, it generates PG analogs, namely PG-glycerol esters (PG-G) for 2-AG and PG-ethanolamides (PG-EA or prostamides) for AEA. Although the formation of these COX-2-derived metabolites of the endocannabinoids has been known for a while, their biological effects remain to be fully elucidated. Recently, several studies have focused on the role of these PG-G or PG-EA in vivo. In this review we take a closer look at the literature concerning these novel bioactive lipids and their role in inflammation.

Prospects for lentiviral vector mediated prostaglandin F synthase gene delivery in monkey eyes in vivo.

Currently, the most effective outflow drugs approved for clinical use are prostaglandin F2α analogues, but these require daily topical self-dosing and have various intraocular, ocular surface and extraocular side effects. Lentiviral vector-mediated delivery of the prostaglandin F synthase (PGFS) gene, resulting in long-term reduction of intraocular pressure (IOP), may eliminate off-target tissue effects and the need for daily topical PGF2α self-administration. Lentiviral vector-mediated delivery of the PGFS gene to the anterior segment has been achieved in cats and non-human primates. Although these results are encouraging, our studies have identified a number of challenges that need to be overcome for prostaglandin gene therapy to be translated into the clinic. Using examples from our work in non-human primates, where we were able to achieve a significant reduction in IOP (2 mm Hg) for 5 months after delivery of the cDNA for bovine PGF synthase, we identify and discuss these issues and consider several possible solutions.

Alteration of energy metabolism gene expression in cumulus cells affects oocyte maturation via MOS-mitogen-activated protein kinase pathway in dairy cows with an unfavorable "Fertil-" haplotype of one female fertility quantitative trait locus.

Prim'Holstein heifers selected for the "Fertil-" homozygous haplotype of QTL-Female-Fert ility-BTA3 showed a greater rate of early pregnancy failure and slower embryo development after IVM suggesting lower oocyte quality than those selected for "Fertile+". We aimed to ascertain intrafollicular factors related to lower oocyte quality in "Fertil-" cows. Analysis of individual oocytes showed meiotic progression delay in "Fertil-" compared with "Fertil+" dairy cows after in vivo maturation and IVM (P < 0.05). Expression of several genes localized to QTL-F-Fert-BTA3 or related to meiosis and mitogen-activated protein kinase pathway was analyzed in individual metaphase-II oocytes using reverse transcription- real-time polymerase chain reaction. Energy metabolism, apoptosis, extracellular matrix, and QTL-F-Fert-BTA3 genes were analyzed in surrounding cumulus cells (CC). In vivo, a significant decrease in prostaglandin synthase PTGES1 and PTGS2 expression coupled with lower PTGS2 protein abundance in CC and reduced expression of MOS in enclosed metaphase-II oocytes from "Fertil-" cows was observed. IVM strongly deregulated gene expression in CC and in oocytes compared with in vivo; nevertheless, differential expression of several genes including PEX19, NAMPT and MOS was observed between the two haplotypes. During IVM, PTGS2 activity inhibitor NS398 (50 µM) led to lower expression of fatty acid synthase (FASN) in CC and of MOS in treated metaphase-II oocytes. Using immunofluorescence, MOS protein was localized to a midbody-like contractile ring separating the polar body from the ooplasm, suggesting a role in the terminal stage of oocyte maturation. Our results suggest that factors involved in prostaglandin synthesis and lipid metabolism in CC could impair oocyte maturation, and might be involved in the reduced fertility of "Fertil-" cows.

New insights into the catalytic mechanism of Bombyx mori prostaglandin E synthase gained from structure-function analysis.

Prostaglandin E synthase (PGES) catalyzes the isomerization of PGH2 to PGE2. We previously reported the identification and structural characterization of Bombyx mori PGES (bmPGES), which belongs to Sigma-class glutathione transferase. Here, we extend these studies by determining the structure of bmPGES in complex with glutathione sulfonic acid (GTS) at a resolution of 1.37 Å using X-ray crystallography. GTS localized to the glutathione-binding site. We found that electron-sharing network of bmPGES includes Asn95, Asp96, and Arg98. Site-directed mutagenesis of these residues to create mutant forms of bmPGES mutants indicate that they contribute to catalytic activity. These results are, to our knowledge, the first to reveal the presence of an electron-sharing network in bmPGES.

Implication of the anti-inflammatory bioactive lipid prostaglandin D2-glycerol ester in the control of macrophage activation and inflammation by ABHD6.

Proinflammatory macrophages are key mediators in several pathologies; thus, controlling their activation is necessary. The endocannabinoid system is implicated in various inflammatory processes. Here we show that in macrophages, the newly characterized enzyme α/ß-hydrolase domain 6 (ABHD6) controls 2-arachidonoylglycerol (2-AG) levels and thus its pharmacological effects. Furthermore, we characterize a unique pathway mediating the effects of 2-AG through its oxygenation by cyclooxygenase-2 to give rise to the anti-inflammatory prostaglandin D2-glycerol ester (PGD2-G). Pharmacological blockade of cyclooxygenase-2 or of prostaglandin D synthase prevented the effects of increasing 2-AG levels by ABHD6 inhibition in vitro, as well as the 2-AG-induced increase in PGD2-G levels. Together, our data demonstrate the physiological relevance of the interaction between the endocannabinoid and prostanoid systems. Moreover, we show that ABHD6 inhibition in vivo allows for fine-tuning of 2-AG levels in mice, therefore reducing lipopolysaccharide-induced inflammation, without the characteristic central side effects of strong increases in 2-AG levels obtained following monoacylglycerol lipase inhibition. In addition, administration of PGD2-G reduces lipopolysaccharide-induced inflammation in mice, thus confirming the biological relevance of this 2-AG metabolite. This points to ABHD6 as an interesting therapeutic target that should be relevant in treating inflammation-related conditions, and proposes PGD2-G as a bioactive lipid with potential anti-inflammatory properties in vivo.