Prostaglandin E synthases: Understanding their pathophysiological roles through mouse genetic models

Prostaglandin E synthase (PGES), which converts cyclooxygenase (COX)-derived prostaglandin H₂ (PGH₂) to PGE₂, is known to comprise a group of at least three structurally and biologically distinct enzymes. Two of them are membrane-bound and have been designated as mPGES-1 and mPGES-2. mPGES-1 is a perinuclear protein that is markedly induced by proinflammatory stimuli and downregulated by anti-inflammatory glucocorticoids as in the case of COX-2. It is functionally coupled with COX-2 in marked preference to COX-1. 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. Recently, mice have been engineered with specific deletions in each of these three PGES enzymes. In this review, we summarize the current understanding of the in vivo roles of PGES enzymes by knockout mouse studies and provide an overview of their biochemical properties.     

Prostaglandin E2 synthesis in cartilage explants under compression: mPGES-1 is a mechanosensitive gene

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. 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. For both COX and PGES, three isoforms have been described: in cartilage, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. COX-3 (a variant of COX-1) and mPGES-2 have been recently cloned but little is known about their expression and regulation in cartilage, as is also the case for 15-PGDH. We investigated the regulation of the genes encoding COX and PGES isoforms during mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) for 2 to 24 hours. After determination of the amount of PGE₂ released in the media (enzyme immunoassay), mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blotting 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 interleukin 1 receptor antagonist (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, whereas COX-3 and mPGES-2 mRNA expression was not modified. 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. We conclude that, along with COX-2, dynamic compression induces mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.     

Up-regulation of microsomal prostaglandin E synthase-1 in COX-1 and COX-2 knock-out mouse fibroblast cell lines

In this paper we investigated the possible involvement of prostaglandin E synthases (PGESs) in compensatory mechanism. Our findings showed that microsomal (m)PGES-1 expression was significantly up-regulated in COX knock-out (K/O) cells whereas the expression of cytosolic PGES was not changed indicating that the induction of mPGES-1 may, at least in part, contribute to the substantial increase of PGE₂ production in COX K/O cell lines. The selective up-regulation of mPGES-1 in COX-2 K/O cells suggests that mPGES-1 may be metabolically coupled with COX-1 for PGE₂ formation. Addition of arachidonic acid caused significant induction of mPGES-1 and COX-2 in WT cells, whereas COX-1 and cPGES were not affected. Our earlier and the current studies demonstrate the coregulation of cPLA₂, COX, and mPGES-1, in PGE₂ synthesis pathway, and that these enzymes contribute to the elevation of PGE₂ level when one COX isoform is absent.     

Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)γ agonists on membrane-associated prostaglandin E2 synthase-1 in IL-1β-stimulated rat chondrocytes: evidence for PPARγ-independent inhibition by 15-deoxy-Δ12,14prostaglandin 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-PGF_1α and PGE₂ in rat chondrocytes stimulated with 10 ng/ml IL-1β, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)γ agonists. Real-time PCR analysis showed that IL-1β induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF_1α and PGE₂ peaked 24 hours after stimulation with IL-1β; the induction of PGE₂ was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Δ^12,14prostaglandin J₂ (15d-PGJ₂) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 μM), with more potency on PGE₂ level than on 6-keto-PGF_1α level (-90% versus -66% at 10 μM). 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 μM. Inhibitory effects of 10 μM 15d-PGJ₂ were neither reduced by PPARγ blockade with GW-9662 nor enhanced by PPARγ overexpression, supporting a PPARγ-independent mechanism. EMSA and TransAM^? analyses demonstrated that mutated IκBα almost completely suppressed the stimulating effect of IL-1β on mPGES-1 expression and PGE₂ production, whereas 15d-PGJ₂ inhibited NF-κB 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-κB activation; third, 15d-PGJ₂ strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ₂ occurs independently of PPARγ through inhibition of the NF-κB pathway; fifth, mPGES-1 is the main target of 15d-PGJ₂.     

Genetic deletion of mPGES-1 accelerates intestinal tumorigenesis in APC(Min/+) mice

The induced synthesis of bioactive prostanoids downstream of cyclooxygenase-2 (COX-2) and prostaglandin H₂ (PGH₂) exerts a critical event in colorectal carcinogenesis. Here we demonstrate that APC^Min/+ mice with genetic deletion of microsomal prostaglandin E synthase-1 (mPGES-1), which catalyses the terminal conversion of PGH₂ into PGE₂, surprisingly develop more and generally larger intestinal tumors than do mPGES-1 wild type littermates (mean number of tumors/intestine 80 vs. 38, p < 0.0005, mean tumor diameter 1.64 vs. 1.12 mm, p < 0.0005). No deviation regarding the expression of other PGE₂ related enzymes (COX-1, COX-2, mPGES-2, cPGES, and 15-PGDH) or receptors (EP1-4) was obvious among the mPGES-1 deficient mice. PGE₂ levels were suppressed in tumors of mPGES-1 deficient animals, but the concentrations of other PGH₂ derived prostanoids were generally enhanced, being most prominent for TxA₂ and PGD₂. Thus, we hypothesise that a redirected synthesis towards other lipid mediators might (over)compensate for loss of mPGES-1/PGE₂ during intestinal tumorigenesis. Nevertheless, our results question the suitability for mPGES-1 targeting therapy in the treatment or prevention of colorectal cancer.     

Expression of prostaglandin E synthases in the bovine oviduct

The oviduct is a specialized organ responsible for the storage and the transport of male and female gametes. It also provides an optimal environment for final gamete maturation, fertilization, and early embryo development. Prostaglandin (PG) E₂ is involved in many female reproductive functions, including ovulation, fertilization, implantation, and parturition. However, the control of its synthesis in the oviduct is not fully understood. Cyclooxygenases (COXs) are involved in the first step of the transformation of arachidonic acid to PGH_2. The prostaglandin E synthases (PGESs) constitute a family of enzymes that catalyze the conversion of PGH₂ to PGE₂, the terminal step in the formation of this bioactive prostaglandin. Quantitative real-time PCR was used to determine the expression of COX-1, COX-2, microsomal prostaglandin E synthase-1 (mPGES-1), microsomal prostaglandin E synthase-2 (mPGES-2), and cytosolic prostaglandin E synthase (cPGES) mRNA in various sections of the oviduct, both ipsilateral and contralateral (to the ovary on which ovulation occurred) at various stages of the estrous cycle. Furthermore, protein expression and localization of cPGES, mPGES-1, and mPGES-2 were determined by Western blot and immunohistochemistry. All three PGESs were detected at both mRNA and protein levels in the oviduct. These PGESs were mostly concentrated in the oviductal epithelial layer and primarily expressed in the ampulla section of the oviduct and to a lesser extent in the isthmus and the isthmic-ampullary junction. The mPGES-1 protein was highly expressed in the contralateral oviduct, which contrasted with mPGES-2 mostly expressed in the ipsilateral oviduct. This is apparently the first report documenting that the three PGESs involved in PGE₂ production were present in the Bos taurus oviduct.     

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.     

Prostaglandin PGE2 at very low concentrations suppresses collagen cleavage in cultured human osteoarthritic articular cartilage: this involves a decrease in expression of proinflammatory genes, collagenases and COL10A1, a gene linked to chondrocyte hypertrophy

Suppression of type II collagen (COL2A1) cleavage by transforming growth factor (TGF)-β2 in cultured human osteoarthritic cartilage has been shown to be associated with decreased expression of collagenases, cytokines, genes associated with chondrocyte hypertrophy, and upregulation of prostaglandin (PG)E₂ production. This results in a normalization of chondrocyte phenotypic expression. Here we tested the hypothesis that PGE₂ is associated with the suppressive effects of TGF-β2 in osteoarthritic (OA) cartilage and is itself capable of downregulating collagen cleavage and hypertrophy in human OA articular cartilage. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with a wide range of concentrations of exogenous PGE₂ (1 pg/ml to 10 ng/ml). COL2A1 cleavage was measured by ELISA. Proteoglycan content was determined by a colorimetric assay. Gene expression studies were performed with real-time PCR. In explants from patients with OA, collagenase-mediated COL2A1 cleavage was frequently downregulated at 10 pg/ml (in the range 1 pg/ml to 10 ng/ml) by PGE₂ as well as by 5 ng/ml TGF-β2. In control OA cultures (no additions) there was an inverse relationship between PGE₂ concentration (range 0 to 70 pg/ml) and collagen cleavage. None of these concentrations of added PGE₂ inhibited the degradation of proteoglycan (aggrecan). Real-time PCR analysis of articular cartilage from five patients with OA revealed that PGE₂ at 10 pg/ml suppressed the expression of matrix metalloproteinase (MMP)-13 and to a smaller extent MMP-1, as well as the proinflammatory cytokines IL-1β and TNF-α and type X collagen (COL10A1), the last of these being a marker of chondrocyte hypertrophy. These studies show that PGE₂ at concentrations much lower than those generated in inflammation is often chondroprotective in that it is frequently capable of selectively suppressing the excessive collagenase-mediated COL2A1 cleavage found in OA cartilage. The results also show that chondrocyte hypertrophy in OA articular cartilage is functionally linked to this increased cleavage and is often suppressed by these low concentrations of added PGE₂. Together these initial observations reveal the importance of very low concentrations of PGE₂ in maintaining a more normal chondrocyte phenotype.     

The shunt from the cyclooxygenase to lipoxygenase pathway in human osteoarthritic subchondral osteoblasts is linked with a variable expression of the 5-lipoxygenase-activating protein

Osteoarthritis (OA) is characterized by articular cartilage degradation and hypertrophic bone changes with osteophyte formation and abnormal bone remodeling. Two groups of OA patients were identified via the production of variable and opposite levels of prostaglandin E₂ (PGE₂) or leukotriene B₄ (LTB₄) by subchondral osteoblasts, PGE₂ levels discriminating between low and high subgroups. We studied whether the expression of 5-lipoxygenase (5-LO) or 5-LO-activating protein (FLAP) is responsible for the shunt from prostaglandins to leukotrienes. FLAP mRNA levels varied in low and high OA groups compared with normal, whereas mRNA levels of 5-LO were similar in all osteoblasts. Selective inhibition of cyclooxygenase-2 (COX-2) with NS-398-stimulated FLAP expression in the high OA osteoblasts subgroup, whereas it was without effect in the low OA osteoblasts subgroup. The addition of PGE₂ to the low OA osteoblasts subgroup decreased FLAP expression but failed to affect it in the high OA osteoblasts subgroup. LTB₄ levels in OA osteoblasts were stimulated about twofold by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) plus transforming growth factor-β (TGF-β), a situation corresponding to their effect on FLAP mRNA levels. Treatments with 1,25(OH)₂D₃ and TGF-β also modulated PGE₂ production. TGF-β stimulated PGE₂ production in both OA osteoblast groups, whereas 1,25(OH)₂D₃ alone had a limited effect but decreased the effect of TGF-β in the low OA osteoblasts subgroup. This modulation of PGE₂ production was mirrored by the synthesis of COX-2. IL-18 levels were only slightly increased in a subgroup of OA osteoblasts compared with normal; however, no relationship was observed overall between IL-18 and PGE₂ levels in normal and OA osteoblasts. These results suggest that the shunt from the production of PGE₂ to LTB₄ is through regulation of the expression of FLAP, not 5-LO, in OA osteoblasts. The expression of FLAP in OA osteoblasts is also modulated differently by 1,25(OH)₂D₃ and TGF-β depending on their endogenous low and high PGE₂ levels.     

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.     

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.     

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.     

Intracellular-specific colocalization of prostaglandin E2 synthases and cyclooxygenases in the brain

Prostaglandin E2 (PGE2) is the major primary prostaglandin generated by brain cells. However, the coordination and intracellular localization of the cyclooxygenases (COXs) and prostaglandin E synthases (PGESs) that convert arachidonic acid to PGE2 in brain tissue are not known. We aimed to determine whether microsomal and cytosolic PGES (mPGES-1 and cPGES) colocalize and coordinate activity with either COX-1 or COX-2 in brain tissue, particularly during development. Importantly, we found that cytosolic PGES also associates with microsomes (cPGES-m) from the cerebrum and cerebral vasculature of the pig and rat as well as microsomes from various cell lines; this seemed dependent on the carboxyl terminal 35-amino acid domain and a cysteine residue (C58) of cPGES. In microsomal membranes from the postnatal brain and cerebral microvessels of mature animals, cPGES-m colocalized with both COX-1 and COX-2, whereas mPGES-1 was undetectable in these microsomes. Accordingly, in this cell compartment, cPGES could coordinate its activity with COX-2 and COX-1 (partly inhibited by NS398); albeit in microsomes of the brain microvasculature from newborns, mPGES-1 was also present. In contrast, in nuclei of brain parenchymal and endothelial cells, mPGES-1 and cPGES colocalized exclusively with COX-2 (determined by immunoblotting and immunohistochemistry); these PGESs contributed to conversion of PGH2 into PGE2. Hence, contrary to a previously proposed model of exclusive COX-2/mPGES-1 coordination, COX-2 can coordinate with mPGES-1 and/or cPGES in the brain, depending on the cell compartment and the age group.