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.     

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.     

Carrageenan-induced paw edema in rat elicits a predominant prostaglandin E2 (PGE2) response in the central nervous system associated with the induction of microsomal PGE2 synthase-1

Peripheral inflammation involves an increase in cyclooxygenase-2 (COX-2)-mediated prostaglandin (PG) synthesis in the central nervous system (CNS), which contributes to allodynia and hyperalgesia. In the present study we have determined the changes in prostanoid tissue levels and in expression of terminal prostanoid synthases in both the CNS and inflamed peripheral tissue during carrageenan-induced paw inflammation in the rat. Prostanoid levels were measured by liquid chromatography-mass spectrometry and enzyme expression at the RNA level by quantitative PCR analysis during both the early (1-6 h) and late (12 and 24 h) phases of the inflammatory response. In the paw, the early phase was associated with increases in PGE(2) and thromboxane (TX)B(2) levels and with a peak of COX-2 expression that preceded that of microsomal prostaglandin-E(2) synthase-1 (mPGES-1). COX-2 and mPGES-1 remained elevated during the late phase, and PGE(2) continued to further increase through 24 h. The cytosolic PGE(2) synthase (cPGES) showed a small transient increase during the early phase, whereas mPGES-2 expression was not affected by inflammation. In the cerebrospinal fluid, elevated levels of PGE(2), 6-keto-PGF(1alpha), PGD(2), and TXB(2) were detected during the early phase. PGE(2) levels also increased in the spinal cord and, to a lesser extent, in the brain and remained elevated in both the cerebrospinal fluid and the spinal cord during the late phase. The expression of mPGES-1 was strongly up-regulated in the brain and spinal cord during inflammation, whereas no change was detected for the expression of cPGES, mPGES-2, COX-1, and terminal PGD, TX, or PGI synthases. The results show that the carrageenan-induced edema in the paw elicits an early phase of COX-2 induction in the CNS leading to an increase synthesis in PGD(2), 6-keto-PGF(1alpha), and TXB(2) in addition to the major PGE(2) response. The data also indicate that the up-regulation of mPGES-1 contributes to COX-2-mediated PGE(2) production in the CNS during peripheral inflammation.     

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.     

COX-1 is coupled with mPGES-1 and ABCC4 in human cervix cancer cells

Cyclooxygenases are key enzymes in the arachidonic acid metabolism. Their unstable intermediate, prostaglandin H₂, is further metabolized to bioactive lipids by various downstream enzymes. In this study, utilizing short hairpin RNAs, we prepared a cell line of human cervix carcinoma with stable down-regulated cyclooxygenase-1 (COX-1) to assess the impact of COX-1 reduction on the downstream enzymes. We found a significant microsomal prostaglandin E synthase-1 (mPGES-1) suppression. In addition, mRNA expression of multidrug resistance protein 4 (MRP4, ABCC4), supposed to take part in antiviral and anticancer drug transport from cells, was up-regulated after COX-1 down-regulation. Our findings indicate that mPGES-1, believed to be coexpressed preferentially with cyclooxygenase-2, may be coupled to COX-1. ABCC4 up-regulation further supports the assumption of its involvement in prostanoid transport.     

Conditional expression of RET/PTC induces a weak oncogenic drive in thyroid PCCL3 cells and inhibits thyrotropin action at multiple levels

Chromosomal rearrangements linking the promoter(s) and N-terminal domain of unrelated gene(s) to the C terminus of RET result in constitutively activated chimeric forms of the receptor in thyroid cells (RET/PTC). RET/PTC rearrangements are thought to be tumor-initiating events; however, the early biological consequences of RET/PTC activation are unknown. To explore this, we generated clonal lines derived from well-differentiated rat thyroid PCCL3 cells with doxycycline-inducible expression of either RET/PTC1 or RET/PTC3. As previously shown in other cell types, RET/PTC1 and RET/PTC3 oligomerized and displayed constitutive tyrosine kinase activity. Neither RET/PTC1 nor RET/PTC3 conferred cells with the ability to grow in the absence of TSH, likely because of concomitant stimulation of both DNA synthesis and apoptosis, resulting in no net growth in the cell population. Effects of RET/PTC on DNA synthesis and apoptosis did not require direct interaction of the oncoprotein with either Shc or phospholipase Cgamma. Acute expression of the oncoprotein decreased TSH-mediated growth stimulation due to interference of TSH signaling by RET/PTC at multiple levels. Taken together, these data indicate that RET/PTC is a weak tumor-initiating event and that TSH action is disrupted by this oncoprotein at several points, and also predict that secondary genetic or epigenetic changes are required for clonal expansion.     

Cyclooxygenase-1 and -2 enzymes differentially regulate the brain upstream NF-kappa B pathway and downstream enzymes involved in prostaglandin biosynthesis

We have recently reported that cyclooxygenase (COX)-2-deficiency affects brain upstream and downstream enzymes in the arachidonic acid (AA) metabolic pathway to prostaglandin E2 (PGE2), as well as enzyme activity, protein and mRNA levels of the reciprocal isozyme, COX-1. To gain a better insight into the specific roles of COX isoforms and characterize the interactions between upstream and downstream enzymes in brain AA cascade, we examined the expression and activity of COX-2 and phospholipase A2 enzymes (cPLA2 and sPLA2), as well as the expression of terminal prostaglandin E synthases (cPGES, mPGES-1, and - 2) in wild type and COX-1(-/-) mice. We found that brain PGE2 concentration was significantly increased, whereas thromboxane B2 (TXB2) concentration was decreased in COX-1(-/-) mice. There was a compensatory up-regulation of COX-2, accompanied by the activation of the NF-kappaB pathway, and also an increase in the upstream cPLA2 and sPLA2 enzymes. The mechanism of NF-kappaB activation in the COX-1(-/-) mice involved the up-regulation of protein expression of the p50 and p65 subunits of NF-kappaB, as well as the increased protein levels of phosphorylated IkappaBalpha and of phosphorylated IKKalpha/beta. Overall, our data suggest that COX-1 and COX-2 play a distinct role in brain PG biosynthesis, with basal PGE2 production being metabolically coupled with COX-2 and TXB2 production being preferentially linked to COX-1. Additionally, COX-1 deficiency can affect the expression of reciprocal and coupled enzymes, COX-2, Ca2+ -dependent PLA2, and terminal mPGES-2, to overcome defects in brain AA cascade.     

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.     

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.     

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.     

Legionella pneumophila-induced PKCalpha-, MAPK-, and NF-kappaB-dependent COX-2 expression in human lung epithelium

Legionella pneumophila causes community- and hospital-acquired pneumonia. Lung airway and alveolar epithelial cells comprise an important barrier against airborne pathogens. Cyclooxygenase (COX) and microsomal PGE(2) synthase-1 (mPGES-1)-derived prostaglandins like prostaglandin E(2) (PGE(2)) are considered as important regulators of lung function. Herein we tested the hypothesis that L. pneumophila induced COX-2 and mPGES-1-dependent PGE(2) production in pulmonary epithelial cells. Legionella induced the release of PGE(2) in primary human small airway epithelial cells and A549 cells. This was accompanied by an increased expression of COX-2 and mPGES-1 as well as an increased PLA(2) activity in infected cells. Deletion of the type IV secretion system Dot/Icm did not impair Legionella-related COX-2 expression or PGE(2) release in A549 cells. L. pneumophila induced the degradation of IkappaBalpha and activated NF-kappaB. Inhibition of IKK blocked L. pneumophila-induced PGE(2) release and COX-2 expression. We noted activation of p38 and p42/44 MAP kinase in Legionella-infected A549 cells. Moreover, membrane translocation and activation of PKCalpha was observed in infected cells. PKCalpha and p38 and p42/44 MAP kinase inhibitors reduced PGE(2) release and COX-2 expression. In summary, PKCalpha and p38 and p42/44 MAP kinase controlled COX-2 expression and subsequent PGE(2) release by Legionella-infected lung epithelial cells. These pathways may significantly contribute to the host response in Legionnaires' disease.     

Prostaglandin E synthase, a terminal enzyme for prostaglandin E2 biosynthesis

Biosynthesis of prostanoids is regulated by three sequential enzymatic steps, namely phospholipase A2 enzymes, cyclooxygenase (COX) enzymes, and various lineagespecific terminal prostanoid synthases. Prostaglandin E synthase (PGES), which isomerizes COX-derived PGH2 specifically to PGE2, occurs in multiple forms with distinct enzymatic properties, expressions, localizations and functions. Two of them are membrane-bound enzymes and have been designated as mPGES-1 and mPGES-2. mPGES-1 is a perinuclear protein that is markedly induced by proinflammatory stimuli, is down-regulated by antiinflammatory glucocorticoids, and is functionally coupled with COX-2 in marked preference to COX-1. Recent gene targeting studies of mPGES-1 have revealed that this enzyme represents a novel target for anti-inflammatory and anti-cancer drugs. mPGES-2 is synthesized as a Golgi membrane-associated protein, and the proteolytic removal of the N-terminal hydrophobic domain leads to the formation of a mature cytosolic enzyme. This enzyme is rather constitutively expressed in various cells and tissues and is functionally coupled with both COX-1 and COX-2. Cytosolic PGES (cPGES) is constitutively expressed in a wide variety of cells and is functionally linked to COX-1 to promote immediate PGE2 production. This review highlights the latest understanding of the expression, regulation and functions of these three PGES enzymes.     

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.     

RET/PTC (rearranged in transformation/papillary thyroid carcinomas) tyrosine kinase phosphorylates and activates phosphoinositide-dependent kinase 1 (PDK1): an alternative phosphatidylinositol 3-kinase-independent pathway to activate PDK1

Thyroid cancers are a leading cause of death due to endocrine malignancies. RET/PTC (rearranged in transformation/papillary thyroid carcinomas) gene rearrangements are the most frequent genetic alterations identified in papillary thyroid carcinoma. Although the oncogenic potential of RET/PTC is related to intrinsic tyrosine kinase activity, the substrates for this enzyme are yet to be identified. In this report, we show that phosphoinositide-dependent kinase 1 (PDK1), a pivotal serine/threonine kinase in growth factor-signaling pathways, is a target of RET/PTC. RET/PTC and PDK1 colocalize in the cytoplasm. RET/PTC phosphorylates a specific tyrosine (Y9) residue located in the N-terminal region of PDK1. Y9 phosphorylation of PDK1 by RET/PTC requires an intact catalytic kinase domain. The short (iso 9) and long forms (iso 51) of the RET/PTC kinases (RET/PTC1 and RET/PTC3) induce Y9 phosphorylation of PDK1. Moreover, Y9 phosphorylation of PDK1 by RET/PTC does not require phosphatidylinositol 3-kinase or Src activity. RET/PTC-induced phosphorylation of the Y9 residue results in increased PDK1 activity, decrease of cellular p53 levels, and repression of p53-dependent transactivation. In conclusion, RET/PTC-induced tyrosine phosphorylation of PDK1 may be one of the mechanisms by which it acts as an oncogenic tyrosine kinase in thyroid carcinogenesis.     

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.