Membrane-bound prostaglandin E synthase-1-mediated prostaglandin E2 production by osteoblast plays a critical role in lipopolysaccharide-induced bone loss associated with inflammation

PGE(2) acts as a potent stimulator of bone resorption in several disorders including osteoarthritis and periodontitis. Three PGE synthases (PGES) were isolated for PGE(2) production, but which PGES has the major role in inflammatory bone resorption is still unclear. In this study, we examined the role of PGE(2) in LPS-induced bone resorption using membrane-bound PGES (mPGES)-1-deficient mice (mPges1(-/-)). In osteoblasts from wild-type mice, PGE(2) production was greatly stimulated by LPS following the expression of cyclooxygenase 2 and mPGES-1 mRNA, whereas no PGE(2) production was found in osteoblasts from mPges1(-/-). LPS administration reduced the bone volume in wild-type femur that was associated with an increased number of osteoclasts. In mPges1(-/-), however, LPS-induced bone loss was reduced. We next examined whether mPGES-1 deficiency could alter the alveolar bone loss in LPS-induced experimental periodontitis. LPS was injected into the lower gingiva and bone mineral density of alveolar bone was measured. LPS induced the loss of alveolar bone in wild-type, but not in mPges1(-/-) mice, suggesting an mPGES-1 deficiency resistant to LPS-induced periodontal bone resorption. To understand the pathway of LPS-induced PGE(2) production in osteoblast, we used C3H/HeJ mice with mutated tlr4. Osteoblasts from C3H/HeJ mice did not respond to LPS, and PGE(2) production was not altered at all. LPS-induced bone loss in the femur was also impaired in C3H/HeJ mice. Thus, LPS binds to TLR4 on osteoblasts that directly induce mPGES-1 expression for PGE(2) synthesis, leading to subsequent bone resorption. Therefore, mPGES-1 may provide a new target for the treatment of inflammatory bone disease.     

Microglia-specific expression of microsomal prostaglandin E2 synthase-1 contributes to lipopolysaccharide-induced prostaglandin E2 production

Microsomal prostaglandin E2 synthase (mPGES)-1 is an inducible protein recently shown to be an important enzyme in inflammatory prostaglandin E2 (PGE2) production in some peripheral inflammatory lesions. However, in inflammatory sites in the brain, the induction of mPGES-1 is poorly understood. In this study, we demonstrated the expression of mPGES-1 in the brain parenchyma in a lipopolysaccharide (LPS)-induced inflammation model. A local injection of LPS into the rat substantia nigra led to the induction of mPGES-1 in activated microglia. In neuron-glial mixed cultures, mPGES-1 was co-induced with cyclooxygenase-2 (COX-2) specifically in microglia, but not in astrocytes, oligodendrocytes or neurons. In microglia-enriched cultures, the induction of mPGES-1, the activity of PGES and the production of PGE2 were preceded by the induction of mPGES-1 mRNA and almost completely inhibited by the synthetic glucocorticoid dexamethasone. The induction of mPGES-1 and production of PGE2 were also either attenuated or absent in microglia treated with mPGES-1 antisense oligonucleotide or microglia from mPGES-1 knockout (KO) mice, respectively, suggesting the necessity of mPGES-1 for microglial PGE2 production. These results suggest that the activation of microglia contributes to PGE2 production through the concerted de novo synthesis of mPGES-1 and COX-2 at sites of inflammation of the brain parenchyma.     

Genetic deletion of mPGES-1 abolishes PGE2 production in murine dendritic cells and alters the cytokine profile, but does not affect maturation or migration

We undertook this study to determine the role of Microsomal PGE Synthase-1 (mPGES-1), and mPGES-1-generated Prostaglandin (PG) E2 on Dendritic Cell (DC) phenotype and function. Using mPGES-1 KnockOut (KO) mice, we generated bone marrow derived DCs and determined their eicosanoid production profile, cell surface marker expression, and cytokine production. We also assessed DC migratory and functional capacity in vivo. Compared to wild-type, mPGES-1 deficient DCs exhibited a markedly attenuated increase in PGE2 production upon LPS stimulation, and displayed preferential shunting towards PGD2 production. mPGES-1 KO DCs did not display deficiencies in maturation, migration or ability to sensitize T cells. However, mPGES-1 deficient DCs generated reduced amounts of the Th1 cytokine IL-12, which may in part be due to increased PGD2 rather than decreased PGE2. These findings provide useful information on the effects of inducible PGE2 on the innate immune system, and have important implications regarding potential consequences of pharmacologic mPGES-1 inhibition.     

Liver X receptor ligands inhibit the lipopolysaccharide-induced expression of microsomal prostaglandin E synthase-1 and diminish prostaglandin E2 production in murine peritoneal macrophages

Microsomal prostaglandin E synthase (mPGES)-1, which is dramatically induced in macrophages by inflammatory stimuli such as lipopolysaccharide (LPS), catalyzes the conversion of cyclooxygenase-2 (COX-2) reaction product prostaglandin H(2) (PGH(2)) into prostaglandin E(2) (PGE(2)). The mPGES-1-derived PGE(2) is thought to help regulate inflammatory responses. On the other hand, excess PGE(2) derived from mPGES-1 contributes to the development of inflammatory diseases such as arthritis and inflammatory pain. Here, we examined the effects of liver X receptor (LXR) ligands on LPS-induced mPGES-1 expression in murine peritoneal macrophages. The LXR ligands 22(R)-hydroxycholesterol (22R-HC) and T0901317 reduced LPS-induced expression of mPGES-1 mRNA and mPGES-1 protein as well as that of COX-2 protein. However, LXR ligands did not influence the expression of microsomal PGES-2 (mPGES-2) or cytosolic PGES (cPGES) protein. Consequently, LXR ligands suppressed the production of PGE(2) in macrophages. These results suggest that LXR ligands diminish PGE(2) production by inhibiting the LPS-induced gene expression of the COX-2-mPGES-1 axis in LPS-activated macrophages.     

mPGES-1 deletion potentiates urine concentrating capability after water deprivation

PGE(2) plays an important role in the regulation of fluid metabolism chiefly via antagonizing vasopressin-induced osmotic permeability in the distal nephron, but its enzymatic sources remain uncertain. The present study was undertaken to investigate the potential role of microsomal PGE synthase (mPGES)-1 in the regulation of urine concentrating ability after water deprivation (WD). Following 24-h WD, wild-type (WT) mice exhibited a significant reduction in urine volume, accompanied by a significant elevation in urine osmolality compared with control groups. In contrast, in response to WD, mPGES-1 knockout (KO) mice had much less urine volume and higher urine osmolality. Analysis of plasma volume by measurement of hematocrit and by using a nanoparticle-based method consistently demonstrated that dehydrated WT mice were volume depleted, which was significantly improved in the KO mice. WD induced a twofold increase in urinary PGE(2) output in WT mice, which was completely blocked by mPGES-1 deletion. At baseline, the KO mice had a 20% increase in V(2) receptor mRNA expression in the renal medulla but not the cortex compared with WT controls; the expression was unaffected by WD irrespective of the genotype. In response to WD, renal medullary aquaporin-2 (AQP2) mRNA exhibited a 60% increase in WT mice, and this increase was greater in the KO mice. Immunoblotting demonstrated increased renal medullary AQP2 protein abundance in both genotypes following WD, with a greater increase in the KO mice. Similar results were obtained by using immunohistochemistry. Paradoxically, plasma AVP response to WD seen in WT mice was absent in the KO mice. Taken together, these results suggest that mPGES-1-derived PGE(2) reduces urine concentrating ability through suppression of renal medullary expression of V(2) receptors and AQP2 but may enhance it by mediating the central AVP response.     

Induced microsomal PGE synthase-1 is involved in cyclooxygenase-2-dependent PGE2 production in gastric fibroblasts

We have previously shown that the cyclooxygenase (COX)-2/PGE2 pathway plays a key role in VEGF production in gastric fibroblasts. Recent studies have identified three PGE synthase (PGES) isozymes: cytosolic PGES (cPGES) and microsomal PGES (mPGES)-1 and -2, but little is known regarding the expression and roles of these enzymes in gastric fibroblasts. Thus we examined IL-1beta-stimulated mPGES-1 and cPGES mRNA and protein expression in gastric fibroblasts by quantitative PCR and Western blot analysis, respectively, and studied both their relationship to COX-1 and -2 and their roles in PGE2 and VEGF production in vitro. IL-1beta stimulated increases in both COX-2 and mPGES-1 mRNA and protein expression levels. However, COX-2 mRNA and protein expression were more rapidly induced than mPGES-1 mRNA and protein expression. Furthermore, MK-886, a nonselective mPGES-1 inhibitor, failed to inhibit IL-1beta-induced PGE2 release at the 8-h time point, while totally inhibiting PGE2 at the later stage. However, MK-886 did inhibit IL-1beta-stimulated PGES activity in vitro by 86.8%. N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS-398), a selective COX-2 inhibitor, totally inhibited PGE2 production at both the 8-h and 24-h time points, suggesting that COX-2-dependent PGE2 generation does not depend on mPGES-1 activity at the early stage. In contrast, NS-398 did not inhibit VEGF production at 8 h, and only partially at 24 h, whereas MK-886 totally inhibited VEGF production at each time point. These results suggest that IL-1beta-induced mPGES-1 protein expression preferentially coupled with COX-2 protein at late stages of PGE2 production and that IL-1beta-stimulated VEGF production was totally dependent on membrane-associated proteins involved in eicosanoid and glutathione metabolism (MAPEG) superfamily proteins, which includes mPGES-1, but was partially dependent on the COX-2/PGE2 pathway.     

Microsomal prostaglandin E synthase-1 is a major terminal synthase that is selectively up-regulated during cyclooxygenase-2-dependent prostaglandin E2 production in the rat adjuvant-induced arthritis model

To better define the role of the various prostanoid synthases in the adjuvant-induced arthritis (AIA) model, we have determined the temporal expression of the inducible PGE synthase (mPGES-1), mPGES-2, the cytosolic PGES (cPGES/p23), and prostacyclin synthase, and compared with that of cyclooxygenase-1 (COX-1) and COX-2. The profile of induction of mPGES-1 (50- to 80-fold) in the primary paw was similar to that of COX-2 by both RNA and protein analysis. Quantitative PCR analysis indicated that induction of mPGES-1 at day 15 was within 2-fold that of COX-2. Increased PGES activity was measurable in membrane preparations of inflamed paws, and the activity was inhibitable by MK-886 to >or=90% with a potency similar to that of recombinant rat mPGES-1 (IC(50) = 2.4 microM). The RNA of the newly described mPGES-2 decreased by 2- to 3-fold in primary paws between days 1 and 15 postadjuvant. The cPGES/p23 and COX-1 were induced during AIA, but at much lower levels (2- to 6-fold) than mPGES-1, with the peak of cPGES/p23 expression occurring later than that of COX-2 and PGE(2) production. Prostacyclin (measured as 6-keto-PGF(1alpha)) was transiently elevated on day 1, and prostacyclin synthase was down-regulated at the RNA level after day 3, suggesting a diminished role of prostacyclin during the maintenance of chronic inflammation in the rat AIA. These results show that mPGES-1 is up-regulated throughout the development of AIA and suggest that it plays a major role in the elevated production of PGE(2) in this model.     

Cellular prostaglandin E2 production by membrane-bound prostaglandin E synthase-2 via both cyclooxygenases-1 and -2

Current evidence suggests that two forms of prostaglandin (PG) E synthase (PGES), cytosolic PGES and membrane-bound PGES (mPGES) -1, preferentially lie downstream of cyclooxygenase (COX) -1 and -2, respectively, in the PGE2 biosynthetic pathway. In this study, we examined the expression and functional aspects of the third PGES enzyme, mPGES-2, in mammalian cells and tissues. mPGES-2 was synthesized as a Golgi membrane-associated protein, and spontaneous cleavage of the N-terminal hydrophobic domain led to the formation of a truncated mature protein that was distributed in the cytosol with a trend to be enriched in the perinuclear region. In several cell lines, mPGES-2 promoted PGE2 production via both COX-1 and COX-2 in the immediate and delayed responses with modest COX-2 preference. In contrast to the marked inducibility of mPGES-1, mPGES-2 was constitutively expressed in various cells and tissues and was not increased appreciably during tissue inflammation or damage. Interestingly, a considerable elevation of mPGES-2 expression was observed in human colorectal cancer. Collectively, mPGES-2 is a unique PGES that can be coupled with both COXs and may play a role in the production of the PGE2 involved in both tissue homeostasis and disease.     

Deletion of microsomal prostaglandin E2 (PGE2) synthase-1 reduces inducible and basal PGE2 production and alters the gastric prostanoid profile

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible protein recently shown to be an important source of inflammatory PGE2. Here we have used mPGES-1 wild type, heterozygote, and null mice to assess the impact of reduction or absence mPGES-1 protein on the production of PGE2 and other prostaglandins in lipopolysaccharide (LPS)-treated macrophages and mice. Thioglycollate-elicited peritoneal macrophages with mPGES-1 deficiency were found to lose their ability to produce PGE2 upon LPS stimulation. Resident mPGES-1(-/-) peritoneal macrophages exhibited severely impaired PGE2-releasing activity but retained some LPS-inducible PGE2 production capacity. Both macrophage types showed a 50% decrease in PGE2 production with removal of one copy of the mPGES-1 gene. In vivo, mPGES-1 deletion abolished the LPS-stimulated production of PGE2 in spleen, kidney, and brain. Surprisingly, lack of mPGES-1 activity resulted in an 80-90% decrease in basal, cyclooxygenase-1 (COX-1)-dependent PGE2 production in stomach and spleen, and a 50% reduction in brain and kidney. Other prostaglandins (thromboxane B2, PGD2, PGF(2alpha), and 6-keto-PGF(1alpha)) were significantly elevated in stomachs of mPGES-1-null mice but not in other tissues. Examination of mRNA for several terminal prostaglandin synthases did not reveal changes in expression levels associated with mPGES-1 deficiency, indicating that gastric prostaglandin changes may be due to shunting of cyclooxygenase products to other terminal synthases. These data demonstrate for the first time a dual role for mPGES-1 in both inflammatory and COX-1-mediated PGE2 production and suggest an interdependence of prostanoid production with tissue-specific alterations of prostaglandin levels in the absence of mPGES-1.     

COX-2 but Not mPGES-1 Contributes to Renal PGE2 Induction and Diabetic Proteinuria in Mice with Type-1 Diabetes

Prostaglandin E2 (PGE2) has been implicated to play a pathogenic role in diabetic nephropathy (DN) but its source remains unlcear. To elucidate whether mPGES-1, the best characterized PGE2 synthase, was involved in the development of DN, we examined the renal phenotype of mPGES-1 KO mice subjected to STZ-induced type-1 diabetes. After STZ treatment, mPGES-1 WT and KO mice presented the similar onset of diabetes as shown by similar elevation of blood glucose. Meanwhile, both genotypes of mice exhibited similar increases of urinary and renal PGE2 production. In parallel with this comparable diabetic status, the kidney injury indices including the urinary albumin excretion, kidney weight and the kidney histology (PAS staining) did not show any difference between the two genotypes. By Western-blotting and quantitative qRT-PCR, mPGES-1, mPGES-2, cPGES and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) remain unaltered following six weeks of diabetes. Finally, a selective COX-2 inhibitor celecoxib (50 mg/kg/day) was applied to the STZ-treated KO mice, which resulted in significant reduction of urinary albumin excretion (KO/STZ: 141.5±38.4 vs. KO/STZ + Celebrex: 48.7±20.8 ug/24 h, p<0.05) and the blockade of renal PGE2 induction (kidney: KO/STZ: 588.7±89.2 vs. KO/STZ + Celebrex: 340.8±58.7 ug/24 h, p<0.05; urine: KO/STZ 1667.6±421.4 vs. KO/STZ + Celebrex 813.6±199.9 pg/24 h, p<0.05), without affecting the blood glucose levels and urine volume. Taken together, our data suggests that an as yet unidentified prostaglanind E synthase but not mPGES-1 may couple with COX-2 to mediate increased renal PGE2 sythsesis in DN.     

Molecular cloning and tissue distribution of microsomal-1 and cytosolic prostaglandin E synthases in macaque

Prostaglandins derived from arachidonic acid are involved in a wide variety of physiological and pathological processes. The primary enzymes involved in the production of PGE2 from arachidonic acid are cyclooxygenases and prostaglandin E synthases. These enzymes have been identified in human, but only partially in the monkey where microsomal PGES-1 and cytosolic PGES have not been characterized. The present study was undertaken to clone these enzymes and to study their tissue distribution, along with mPGES-2. The coding sequence of Macaque mPGES-1 is 98% homologous to human mPGES-1 at the nucleic acid level and the deduced amino acid sequence has 98% homology with the human protein. The Macaque cPGES cDNA is more than 99% homologous to the human and the deduced amino acids sequence is identical to that of the human cPGES. By Northern blot analysis, we found that mPGES-2 and cPGES mRNA were expressed in the endometrium, myometrium, ovary and oviduct, albeit at different levels, while mPGES-1 mRNA was detected at a weak level, mainly in the oviduct. Western Blot analysis revealed that mPGES-2, mPGES-1 and cPGES proteins were present in all tissues tested. These results suggest that production of PGE2 in Macaque may involve more than one PGES and that further studies will be needed to fully understand the conditions under which each PGES contributes to PGE2 production.     

Cyclooxygenases and prostaglandin E synthases in preimplantation mouse embryos

Prostaglandin E2 (PGE2) is shown to be essential for female reproduction. Cyclooxygenase (COX) is a rate-limiting enzyme in prostaglandin synthesis from arachidonic acid and exists in two isoforms: COX-1 and COX-2. Prostaglandin E synthase (PGES) is a terminal prostanoid synthase and can catalyse the isomerization of the COX product PGH2 to PGE2, including microsomal PGES-1 (mPGES-1), cytosolic PGES (cPGES) and mPGES-2. This study examined the protein expression of COX-1, COX-2, mPGES-1, cPGES and mPGES-2 in preimplantation mouse embryos by immunohistochemistry. Embryos at different stages collected from oviducts or uteri were transferred into a flushed oviduct of non-pregnant mice. The oviducts containing embryos were paraffin-embedded and processed for immunostaining. COX-1 immunostaining was at a basal level in zygotes and a low level at the 2-cell stage, reaching a high level from the 4-cell to blastocyst stage. COX-2 immunostaining was at a low level at the zygote stage and was maintained at a high level from the 2-cell to blastocyst stages. A low level of mPGES-1 immunostaining was observed from the zygote to 8-cell stages. The signal for mPGES-1 immunostaining became stronger at the morula stage and was strongly seen at the blastocyst stage. cPGES immunostaining was strongly observed in zygotes, 2-cell and 8-cell embryos. There was a slight decrease in cPGES immunostaining at the 4-cell, morula and blastocyst stages. mPGES-2 immunostaining was at a low level from the zygote to morula stages and at a high level at the blastocyst stage. We found that the COX-1, COX-2, mPGES-1, cPGES and mPGES-2 protein signals were all at a high level at the blastocyst stage. PGE2 produced during the preimplantation development may play roles during embryo transport and implantation.     

Mechanical stress and prostaglandin E2 synthesis in cartilage

Knee osteoarthritis (OA) results, at least in part, from overloading and inflammation leading to cartilage degradation. Prostaglandin E2 (PGE2) is one of the main catabolic factors involved in OA in which metalloproteinase (MMP) is crucial for cartilage degradation. Its synthesis is the result of cyclooxygenase (COX) and prostaglandin E synthase (PGES) activities whereas NAD+-dependent 15 hydroxy-prostaglandin dehydrogenase (15-PGDH) is the key enzyme implicated in the catabolism of PGE2. Among the isoforms described, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. We investigated the regulation of the COX, PGES and 15-PGDH and MMP-2, MMP-9 and MMP-13 genes by mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) from 2 to 24 h. After determination of the PGE2 release in the media, mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blot respectively. Mechanical compression of cartilage explants significantly increased PGE2 production in a time dependent manner. This was not due to the synthesis of IL-1, since pretreatment with IL1-Ra did not alter the PGE2 synthesis. Interestingly, COX-2 and mPGES-1 mRNA expression significantly increased after 2 hours, in parallel with protein expression. Moreover, we observed a delayed overexpression of 15-PGDH just before the decline of PGE2 synthesis after 18 hours suggesting that PGE2 synthesis could be altered by the induction of 15-PGDH expression. MAPK are involved in signaling, since specific inhibitors partially inhibited COX-2 and mPGES-1 expressions. Lastly, compression induced MMP-2, -9, -13 mRNA expressions in cartilage. We conclude that dynamic compression induces pro-inflammatroy mediators release and matrix degradating enzymes synthesis. Notably, compression increases mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.     

15-deoxy-Delta12,14-prostaglandin J2 inhibits the expression of microsomal prostaglandin E synthase type 2 in colon cancer cells

Prostaglandin (PG) E(2) (PGE(2)) plays a predominant role in promoting colorectal carcinogenesis. The biosynthesis of PGE(2) is accomplished by conversion of the cyclooxygenase (COX) product PGH(2) by several terminal prostaglandin E synthases (PGES). Among the known PGES isoforms, microsomal PGES type 1 (mPGES-1) and type 2 (mPGES-2) were found to be overexpressed in colorectal cancer (CRC); however, the role and regulation of these enzymes in this malignancy are not yet fully understood. Here, we report that the cyclopentenone prostaglandins (CyPGs) 15-deoxy-Delta(12,14)-PGJ(2) and PGA(2) downregulate mPGES-2 expression in the colorectal carcinoma cell lines Caco-2 and HCT 116 without affecting the expression of any other PGES or COX. Inhibition of mPGES-2 was subsequently followed by decreased microsomal PGES activity. These effects were mediated via modulation of the cellular thiol-disulfide redox status but did not involve activation of the peroxisome proliferator-activated receptor gamma or PGD(2) receptors. CyPGs had antiproliferative properties in vitro; however, this biological activity could not be directly attributed to decreased PGES activity because it could not be reversed by adding PGE(2). Our data suggest that there is a feedback mechanism between PGE(2) and CyPGs that implicates mPGES-2 as a new potential target for pharmacological intervention in CRC.     

Deletion of microsomal prostaglandin E synthase-1 protects neuronal cells from cytotoxic effects of β-amyloid peptide fragment 31-35

Epidemiological studies have suggested that the long-term use of nonsteroidal anti-inflammatory drugs that inhibit cyclooxygenase (COX) activity moderates the onset or progression of Alzheimer's disease (AD). Thus it has been suggested that prostaglandin E(2) (PGE(2)), a major end-product of COX, may play a pathogenic role in AD, but the involvement of PGE synthase (PGES), a terminal enzyme downstream from COX, has not been fully elucidated. To examine the involvement in AD pathology of microsomal PGES-1 (mPGES-1), a PGES enzyme, we here prepared primary cerebral neuronal cells from the cerebri of wild-type and mPGES-1-deficient mice and then treated them with β-amyloid (Aβ) fragment 31-35 (Aβ(31-35)), which represents the shortest sequence of native Aβ peptide required for neurotoxicity. Treatment of wild-type neuronal cells with Aβ(31-35) induced mPGES-1 gene expression and PGE(2) production, followed by significant apoptotic cell death, but apoptosis was not induced in mPGES-1-deficient cells. Furthermore, the combined treatment of Aβ(31-35) and PGE(2) induced apoptosis in mPGES-1-deficient neuronal cells. These results indicated that mPGES-1 is induced during Aβ-mediated neuronal cell death and is involved in Aβ-induced neurotoxicity associated with AD pathology.     

Evidence that PAR2-triggered prostaglandin E2 (PGE2) formation involves the ERK-cytosolic phospholipase A2-COX-1-microsomal PGE synthase-1 cascade in human lung epithelial cells

We investigated possible involvement of three isozymes of prostaglandin E synthase (PGES), microsomal PGES-1 (mPGES-1), mPGES-2 and cytosolic PGES (cPGES) in COX-2-dependent prostaglandin E(2) (PGE(2)) formation following proteinase-activated receptor-2 (PAR2) stimulation in human lung epithelial cells. PAR2 stimulation up-regulated mPGES-1 as well as COX-2, but not mPGES-2 or cPGES, leading to PGE(2) formation. The PAR2-triggered up-regulation of mPGES-1 was suppressed by inhibitors of COX-1, cytosolic phospholipase A(2) (cPLA(2)) and MEK, but not COX-2. Finally, a selective inhibitor of mPGES-1 strongly suppressed the PAR2-evoked PGE(2) formation. PAR2 thus appears to trigger specific up-regulation of mPGES-1 that is dependent on prostanoids formed via the MEK/ERK/cPLA(2)/COX-1 pathway, being critical for PGE(2) formation.     

Anti-inflammatory role of microsomal prostaglandin E synthase-1 in a model of neuroinflammation

A major immunological response during neuroinflammation is the activation of microglia, which subsequently release proinflammatory mediators such as prostaglandin E(2) (PGE(2)). Besides its proinflammatory properties, cyclooxygenase-2 (COX-2)-derived PGE(2) has been shown to exhibit anti-inflammatory effects on innate immune responses. Here, we investigated the role of microsomal PGE(2) synthase-1 (mPGES-1), which is functionally coupled to COX-2, in immune responses using a model of lipopolysaccharide (LPS)-induced spinal neuroinflammation. Interestingly, we found that activation of E-prostanoid (EP)2 and EP4 receptors, but not EP1, EP3, PGI(2) receptor (IP), thromboxane A(2) receptor (TP), PGD(2) receptor (DP), and PGF(2) receptor (FP), efficiently blocked LPS-induced tumor necrosis factor α (TNFα) synthesis and COX-2 and mPGES-1 induction as well as prostaglandin synthesis in spinal cultures. In vivo, spinal EP2 receptors were up-regulated in microglia in response to intrathecally injected LPS. Accordingly, LPS priming reduced spinal synthesis of TNFα, interleukin 1β (IL-1β), and prostaglandins in response to a second intrathecal LPS injection. Importantly, this reduction was only seen in wild-type but not in mPGES-1-deficient mice. Furthermore, intrathecal application of EP2 and EP4 agonists as well as genetic deletion of EP2 significantly reduced spinal TNFα and IL-1β synthesis in mPGES-1 knock-out mice after LPS priming. These data suggest that initial inflammation prepares the spinal cord for a negative feedback regulation by mPGES-1-derived PGE(2) followed by EP2 activation, which limits the synthesis of inflammatory mediators during chronic inflammation. Thus, our data suggest a role of mPGES-1-derived PGE(2) in resolution of neuroinflammation.     

Inhibition of matrix metalloproteinase-3 and -13 synthesis induced by IL-1beta in chondrocytes from mice lacking microsomal prostaglandin E synthase-1

Joint destruction in arthritis is in part due to the induction of matrix metalloproteinase (MMP) expression and their inhibitors, especially MMP-13 and -3, which directly degrade the cartilage matrix. Although IL-1β is considered as the main catabolic factor involved in MMP-13 and -3 expression, the role of PGE(2) remains controversial. The goal of this study was to determine the role of PGE(2) on MMP synthesis in articular chondrocytes using mice lacking microsomal PGE synthase-1 (mPGES-1), which catalyses the rate-limiting step of PGE(2) synthesis. MMP-3 and MMP-13 mRNA and protein expressions were assessed by real-time RT-PCR, immunoblotting, and ELISA in primary cultures of articular chondrocytes from mice with genetic deletion of mPGES-1. IL-1β-induced PGE(2) synthesis was dramatically reduced in mPGES-1(-/-) and mPGES-1(+/-) compared with mPGES-1(+/+) chondrocytes. A total of 10 ng/ml IL-1β increased MMP-3 and MMP-13 mRNA, protein expression, and release in mPGES-1(+/+) chondrocytes in a time-dependent manner. IL-1β-induced MMP-3 and MMP-13 mRNA expression, protein expression, and release decreased in mPGES-1(-/-) and mPGES-1(+/-) chondrocytes compared with mPGES-1(+/+) chondrocytes from 8 up to 24 h. Otherwise, MMP inhibition was partially reversed by addition of 10 ng/ml PGE(2) in mPGES-1(-/-) chondrocytes. Finally, in mPGES-1(-/-) chondrocytes treated by forskolin, MMP-3 protein expression was significantly decreased compared with wild-type, suggesting that PGE(2) regulates MMP-3 expression via a signaling pathway dependent on cAMP. These results demonstrate that PGE(2) plays a key role in the induction of MMP-3 and MMP-13 in an inflammatory context. Therefore, mPGES-1 could be considered as a critical target to counteract cartilage degradation in arthritis.