Signal pathways involved in the regulation of prostaglandin E synthase-1 in human gingival fibroblasts

Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme regulating the synthesis of prostaglandin E2 (PGE2) in inflammatory conditions. In this study we investigated the regulation of mPGES-1 in gingival fibroblasts stimulated with the inflammatory mediators interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNFalpha). The results showed that IL-1beta and TNFalpha induce the expression of mPGES-1 without inducing the expression of early growth response factor-1 (Egr-1). Treatment of the cells with the PLA2 inhibitor 4-bromophenacyl bromide (BPB) decreased the cytokine-induced mPGES-1 expression accompanied by decreased PGE2 production whereas the addition of arachidonic acid (AA) upregulated mPGES-1 expression and PGE2 production. The protein kinase C (PKC) activator PMA did not upregulate the expression of mPGES-1 in contrast to COX-2 expression and PGE2 production. In addition, inhibitors of PKC, tyrosine and p38 MAP kinase markedly decreased the cytokine-induced PGE2 production but not mPGES-1 expression. Moreover, the prostaglandin metabolites PGE2 and PGF2alpha induced mPGES-1 expression as well as upregulated the cytokine-induced mPGES-1 expression indicating positive feedback regulation of mPGES-1 by prostaglandin metabolites. The peroxisome proliferator-activated receptor-gamma (PPARgamma) ligand, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), decreased mPGES-1 expression but not COX-2 expression or PGE2 production. The results indicate that the inflammatory-induced mPGES-1 expression is regulated by PLA2 and 15d-PGJ2 but not by PKC, tyrosine kinase or p38 MAP kinase providing new insights into the regulation of mPGES-1.     

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.     

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.     

Regulation of prostaglandin E synthases: effects of siRNA-mediated inhibition of microsomal prostaglandin E synthase-1

Prostaglandin E2 (PGE2) is a key mediator involved in several inflammatory conditions. In this study, we investigated the expression and regulation of the terminal PGE2 synthesizing enzyme prostaglandin E synthases (mPGES-1, mPGES-2 and cPGES) in gingival fibroblasts stimulated with pro-inflammatory cytokines. We used siRNA knockdown of mPGES-1 to elucidate the impact of mPGES-1 inhibition on mPGES-2 and cPGES expression, as well as on PGE2 production. The cytokines TNFalpha and IL-1beta increased protein expression and activity of mPGES-1, accompanied by increased COX-2 expression and PGE2 production. The isoenzymes mPGES-2 and cPGES, constitutively expressed at mRNA and protein levels, were unaffected by the pro-inflammatory cytokines. We show for the first time that treatment with mPGES-1 siRNA down-regulated the cytokine-induced mPGES-1 protein expression and activity. Interestingly, mPGES-1 siRNA did not affect the cytokine-stimulated PGE2 production, whereas PGF(2alpha) levels were enhanced. Neither mPGES-2 nor cPGES expression was affected by siRNA silencing of mPGES-1. Dexamethasone and MK-886 both inhibited the cytokine-induced mPGES-1 expression while mPGES-2 and cPGES expression remained unaffected. In conclusion, mPGES-1 siRNA down-regulates mPGES-1 expression, and neither mPGES-2 nor cPGES substituted for mPGES-1 in a knockdown setting in gingival fibroblasts. Moreover, mPGES-1 siRNA did not affect PGE2 levels, whereas PGF(2alpha) increased, suggesting a compensatory pathway of PGE2 synthesis when mPGES-1 is knocked down.     

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.     

Diverse effects of ethanol on the pathway of inducible prostaglandin E2 production in macrophages.

The effects of ethanol on inducible prostaglandin production in RAW macrophages were investigated. Indomethacin (1 microM) or cycloheximide (1 microM) abolished prostaglandin E2 (PGE2) production induced by lipopolysaccharide (LPS, 1 microg/ml). Ethanol at concentrations from 100 mM to 600 mM concentration-dependently inhibited inducible PGE2 production, while ethanol only at higher concentrations (400 mM or more) showed cytotoxity to the cells. Cyclooxygenase-2 (COX-2) activity, estimated by transformation of exogenous arachidonic acid into PGE2, was not affected by ethanol (100-400 mM). LPS-induced expression of COX-2 mRNA was inhibited by ethanol (50-400 mM). On the other hand, protein expression of COX-2 by LPS was significantly increased by ethanol (100-400 mM). Ethanol alone at concentrations up to 600 mM did not induce expression of COX-2 protein. In a medium containing arachidonic acid (1 microM), ethanol at a low concentration (100 mM) did not significantly affect LPS-induced PGE2 production. These results suggest that ethanol shows diverse effects on the pathway of inducible PGE2 production in macrophages. Finally, ethanol may suppress utilization of arachidonic acid, resulting in reduction of inducible PGE2 production. Further study is needed to elucidate the mechanism of dissociation of ethanol effects on protein and mRNA expression.     

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.     

Potential role of microsomal prostaglandin E synthase-1 in tumorigenesis

Microsomal prostaglandin E2 synthase-1 (mPGES-1) is a stimulus-inducible enzyme that functions downstream of cyclooxygenase (COX)-2 in the PGE2-biosynthetic pathway. Given the accumulating evidence that COX-2-derived PGE2 participates in the development of various tumors, including colorectal cancer, we herein examined the potential involvement of mPGES-1 in tumorigenesis. Immunohistochemical analyses demonstrated the expression of both COX-2 and mPGES-1 in human colon cancer tissues. HCA-7, a human colorectal adenocarcinoma cell line that displays COX-2- and PGE2-dependent proliferation, expressed both COX-2 and mPGES-1 constitutively. Treatment of HCA-7 cells with an mPGES-1 inhibitor or antisense oligonucleotide attenuated, whereas overexpression of mPGES-1 accelerated, PGE2 production and cell proliferation. Moreover, cotransfection of COX-2 and mPGES-1 into HEK293 cells resulted in cellular transformation manifested by colony formation in soft agar culture and tumor formation when implanted subcutaneously into nude mice. cDNA array analyses revealed that this mPGES-1-directed cellular transformation was accompanied by changes in the expression of a variety of genes related to proliferation, morphology, adhesion, and the cell cycle. These results collectively suggest that aberrant expression of mPGES-1 in combination with COX-2 can contribute to tumorigenesis.     

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.     

Prostaglandin E2 production in ovarian cancer cell lines is regulated by cyclooxygenase-1, not cyclooxygenase-2

The significance of cyclooxygenase-2 (COX-2) expression in ovarian cancer has been discussed. In this study, we found increased expression of COX-1 mRNA and protein in three out of 10 ovarian cancer cell lines. Prostaglandin E 2 (PGE2) production was elevated in these three cell lines, but not in other seven cell lines. COX-2 protein was not detected in any of the cell lines. Cytosolic prostaglandin E synthase (cPGES) mRNA and protein were detected in all 10 cell lines. Membrane-associated PGES-1 (mPGES-1) was detected in some of the ovarian cell lines, but its presence did not correspond with PGE2 production. In contrast, mPGES-2 mRNA and protein were detected in all 10 cell lines. A nonselective COX inhibitor (indometacin) and a selective COX-1 inhibitor (SC-560) strongly inhibited PGE2 production by the three cell lines, while selective COX-2 inhibitors (NS-398 and rofecoxib) did not inhibit PGE2 production. In addition, increased expression of COX-1, not COX-2 protein was observed in the mass of ovarian cancer tissues from 22 patients when compared with that in normal tissue. These findings suggest that COX-1 might be a major enzyme regulating PGE2 production in ovarian cancer cells.     

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.     

Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)gamma agonists on membrane-associated prostaglandin E2 synthase-1 in IL-1beta-stimulated rat chondrocytes: evidence for PPARgamma-independent inhibition by 15-deoxy-Delta12,14prostaglandin J2

Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF1alpha and PGE2 in rat chondrocytes stimulated with 10 ng/ml IL-1beta, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)gamma agonists. Real-time PCR analysis showed that IL-1beta induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF1alpha and PGE2 peaked 24 hours after stimulation with IL-1beta; the induction of PGE2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Delta12,14prostaglandin J2 (15d-PGJ2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 microM), with more potency on PGE2 level than on 6-keto-PGF1alpha level (-90% versus -66% at 10 microM). A high dose of 15d-PGJ2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 microM. Inhibitory effects of 10 microM 15d-PGJ2 were neither reduced by PPARgamma blockade with GW-9662 nor enhanced by PPARgamma overexpression, supporting a PPARgamma-independent mechanism. EMSA and TransAM analyses demonstrated that mutated IkappaBalpha almost completely suppressed the stimulating effect of IL-1beta on mPGES-1 expression and PGE2 production, whereas 15d-PGJ2 inhibited NF-kappaB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE2 synthesis; second, activation of the prostaglandin cascade requires NF-kappaB activation; third, 15d-PGJ2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ2 occurs independently of PPARgamma through inhibition of the NF-kappaB pathway; fifth, mPGES-1 is the main target of 15d-PGJ2.     

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.     

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.