Elevated PGE2 is a hallmark of most inflammatory lesions. This lipid mediator can induce the cardinal signs of inflammation, and the beneficial actions of non-steroidal anti-inflammatory drugs are attributed to inhibition of cyclooxygenase COX-1 and COX-2, enzymes essential in the biosynthesis of PGE2 from arachidonic acid. However, both clinical studies and rodent models suggest that, in the asthmatic lung, PGE2 acts to restrain the immune response and limit physiological change secondary to inflammation. To directly address the role of PGE2 in the lung, we examined the development of disease in mice lacking microsomal prostaglandin E synthase 1 (mPGES1), which converts COX-1/COX-2 derived PGH2 to PGE2. We show that mPGES1 determines PGE2 levels in the naïve lung and is required for increases in PGE2 after ovalbumin (OVA) induced allergy. While loss of either COX-1 or COX-2 increases the disease severity, surprisingly mPGES1 −/− mice show reduced inflammation. However, an increase in serum IgE is still observed in the mPGES1 −/− mice, suggesting that loss of PGE2 does not impair induction of a TH2 response. Furthermore, mPGES1 −/− mice expressing a transgenic OVA-specific T cell receptor are also protected, indicating that PGE2 acts primarily after challenge with inhaled antigen. PGE2 produced by the lung plays the critical role in this response, as loss of lung mPGES1 is sufficient to protect against disease. Together this supports a model in which mPGES1-dependent PGE2 produced by populations of cells native to the lung contributes to the effector phase of some allergic responses.
Microsomal prostaglandin E2 synthase-1 (mPGES-1) is an inducible enzyme that acts downstream of cyclooxygenase (COX) to specifically catalyze the conversion of prostaglandin (PG) H2 to PGE2. mPGES-1 plays a key role in inflammation, pain and arthritis; however, the role of mPGES-1 in fibrogenesis is largely unknown. Herein, we examine the role of mPGES-1 in a mouse model of skin scleroderma using mice deficient in mPGES-1.
Wild type (WT) and mPGES-1 null mice were subjected to the bleomycin model of cutaneous skin scleroderma. mPGES-1 expressions in scleroderma fibroblasts and in fibroblasts derived from bleomycin-exposed mice were assessed by Western blot analysis. Degree of fibrosis, dermal thickness, inflammation, collagen content and the number of α-smooth muscle actin (α-SMA)-positive cells were determined by histological analyses. The quantity of the collagen-specific amino acid hydroxyproline was also measured.
Compared to normal skin fibroblasts, mPGES-1 protein expression was elevated in systemic sclerosis (SSc) fibroblasts and in bleomycin-exposed mice. Compared to WT mice, mPGES-1-null mice were resistant to bleomycin-induced inflammation, cutaneous thickening, collagen production and myofibroblast formation.
mPGES-1 expression is required for bleomycin-induced skin fibrogenesis. Inhibition of mPGES-1 may be a viable method to alleviate the development of cutaneous sclerosis and is a potential therapeutic target to control the onset of fibrogenesis.
Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that acts downstream of cyclooxygenase and specifically catalyzes the conversion of PGH2 to PGE2. The present study demonstrates the effect of genetic deletion of mPGES-1 on the developing immunologic responses and its impact on the clinical model of bovine collagen-induced arthritis. mPGES-1 null and heterozygous mice exhibited decreased incidence and severity of arthritis compared with wild-type mice in a gene dose-dependent manner. Histopathological examination revealed significant reduction in lining hyperplasia and tissue destruction in mPGES-1 null mice compared with their wild-type littermates. mPGES-1 deficient mice also exhibited attenuation of mechanical nociception in a gene dose-dependent manner. In addition, mPGES-1 null and heterozygous mice showed a marked reduction of serum IgG against type II collagen (CII), including subclasses IgG1, IgG2a, IgG2b, IgG2c, and IgG3, compared with wild-type mice, which correlated with the reduction in observed inflammatory features. These results demonstrate for the first time that deficiency of mPGES-1 inhibits the development of collagen-induced arthritis, at least in part, by blocking the development of a humoral immune response against type II collagen. Pharmacologic inhibition of mPGES-1 may therefore impact both the inflammation and the autoimmunity associated with human diseases such as rheumatoid arthritis.
Background & Aims
Microsomal prostaglandin E synthase-1 (mPGES-1) is a rate-limiting enzyme that is coupled with cyclooxygenase-2 (COX-2) in the synthesis of prostaglandin E2 (PGE2). Although COX-2 is involved in development and progression of various human cancers, the role of mPGES-1 in carcinogenesis has not been determined. We investigated the role of mPGES-1 in human cholangiocarcinoma growth.
We used immunohistochemical analyses to examine the expression of mPGES-1 in formalin-fixed, paraffin-embedded human cholangiocarcinoma tissues. The effects of mPGES-1 on human cholangiocarcinoma cells were determined in vitro and in SCID mice. Immunoblotting and immunoprecipitation assays were performed to determine the levels of PTEN and related signaling molecules in human cholangiocarcinoma cells with overexpression or knockdown of mPGES-1.
mPGES-1 is overexpressed in human cholangiocarcinoma tissues. Overexpression of mPGES-1 in human cholangiocarcinoma cells increased tumor cell proliferation, migration, invasion, and colony formation; in contrast, RNAi knockdown of mPGES-1 inhibited tumor growth parameters. In SCID mice with tumor xenografts, mPGES-1 overexpression accelerated tumor formation and increased tumor weight (P<0.01), whereas mPGES-1 knockdown delayed tumor formation and reduced tumor weight (P<0.01). mPGES-1 inhibited the expression of PTEN, leading to activation of the EGFR–PI3K–AKT–mTOR signaling pathways in cholangiocarcinoma cells. mPGES-1–mediated inhibition of PTEN is regulated through blocking of EGR-1 sumoylation and binding to the 5′-UTR of the PTEN gene.
mPGES-1 promotes experimental cholangiocarcinogenesis and tumor progression by inhibiting PTEN.
cancer cell signaling; biliary tract cancer; bile duct; liver
mPGES-1 (microsomal prostaglandin E synthase-1) is a stimulus-inducible enzyme that functions downstream of COX (cyclo-oxygenase)-2 in the PGE2 (prostaglandin E2)-biosynthesis pathway. Although COX-2-derived PGE2 is known to play a role in the development of various tumours, the involvement of mPGES-1 in carcinogenesis has not yet been fully understood. In the present study, we used LLC (Lewis lung carcinoma) cells with mPGES-1 knockdown or overexpression, as well as mPGES-1-deficient mice to examine the roles of cancer cell- and host-associated mPGES-1 in the processes of tumorigenesis in vitro and in vivo. We found that siRNA (small interfering RNA) silencing of mPGES-1 in LLC cells decreased PGE2 synthesis markedly, accompanied by reduced cell proliferation, attenuated Matrigel™ invasiveness and increased extracellular matrix adhesion. Conversely, mPGES-1-overexpressing LLC cells showed increased proliferating and invasive capacities. When implanted subcutaneously into wild-type mice, mPGES-1-silenced cells formed smaller xenograft tumours than did control cells. Furthermore, LLC tumours grafted subcutaneously into mPGES-1-knockout mice grew more slowly than did those grafted into littermate wild-type mice, with concomitant decreases in the density of microvascular networks, the expression of pro-angiogenic vascular endothelial growth factor, and the activity of matrix metalloproteinase-2. Lung metastasis of intravenously injected LLC cells was also significantly less obvious in mPGES-1-null mice than in wild-type mice. Thus our present approaches provide unequivocal evidence for critical roles of the mPGES-1-dependent PGE2 biosynthetic pathway in both cancer cells and host microenvironments in tumour growth and metastasis.
knockout mouse; metastasis; microsomal prostaglandin E synthase-1; prostaglandin E2; tumorigenesis; COX, cyclo-oxygenase; cPGES, cytosolic prostaglandin E synthase; DMEM, Dulbecco's modified Eagle's medium; dmPGE2, 16,16-dimethyl prostaglandin E2; ECM, extracellular matrix; EP, prostaglandin E receptor; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human embryonic kidney; KD, knockdown; KO, knockout; LLC, Lewis lung carcinoma; MMP, matrix metalloproteinase; mPGES, microsomal prostaglandin E synthase; NSAID, non-steroidal anti-inflammatory drug; PG, prostaglandin; PGES, PGE synthase; RT, reverse transcriptase; siRNA, small interfering RNA; TBS, Tris-buffered saline; TBS-Tween, TBS containing 0.05% Tween 20; VEGF, vascular endothelial growth factor; WT, wild-type
Prostaglandin (PG) E2 has multiple actions that may affect blood pressure. It is synthesized from arachidonic acid by the sequential actions of phospholipases, cyclooxygenases, and PGE synthases. While microsomal PGE synthase 1 (mPGES1) is the only genetically-verified PGE synthase, results of previous studies examining the consequences of mPGES1-deficiency on blood pressure (BP) are conflicting. To determine whether genetic background modifies the impact of mPGES1 on BP, we generated mPGES1−/− mice on two distinct inbred backgrounds, DBA/1lacJ and 129/SvEv. On the DBA/1 background, baseline BP was similar between wild-type (WT) and mPGES1−/− mice. By contrast, on the 129 background, baseline BPs were significantly higher in mPGES1−/− animals than WT controls. During angiotensin II infusion, the DBA/1 mPGES1−/− and WT mice developed mild hypertension of similar magnitude, while 129-mPGES1−/− mice developed more severe hypertension than WT controls. DBA/1 animals developed only minimal albuminuria in response to angiotensin II infusion. By contrast, WT 129 mice had significantly higher levels of albumin excretion than WT DBA/1 and the extent of albuminuria was further augmented in 129 mPGES1−/− animals. In WT mice of both strains, the increase in urinary excretion of PGE2 with angiotensin II was attenuated in mPGES1−/− animals. Urinary excretion of thromboxane was unaffected by angiotensin II in the DBA/1 lines but increased more than 4-fold in 129 mPGES1−/− mice. These data indicate that genetic background significantly modifies the BP response to mPGES1 deficiency. Exaggerated production of thromboxane may contribute to the robust hypertension and albuminuria in 129 mPGES1-deficient mice.
prostanoids; PGE synthase; blood pressure; strain; hypertension
Although cyclooxygenases (COX) and prostaglandin E synthases (PGES) have been implicated in ischemic stroke injury, little is known about their role in intracerebral hemorrhage (ICH)-induced brain damage. This study examines the expression and cellular localization of COX-1, COX-2, microsomal PGES-1 (mPGES-1), mPGES-2, and cytosolic PGES (cPGES) in mice that have undergone hemorrhagic brain injury.
ICH was induced in C57BL/6 mice by intrastriatal injection of collagenase. Expression and cellular localization of COX-1, COX-2, mPGES-1, mPGES-2, and cPGES were examined by immunofluorescence staining.
In the hemorrhagic brain, COX-1, mPGES-2, and cPGES were expressed constitutively in neurons; COX-1 was also constitutively expressed in microglia. The immunoreactivity of COX-2 was increased in neurons and astrocytes surrounding blood vessels at 5 h and then tended to decrease in neurons and increase in astrocytes at 1 day. At 3 days after ICH, COX-2 was observed primarily in astrocytes but was absent in neurons. Interestingly, the immunoreactivity of mPGES-1 was increased in neurons in the ipsilateral cortex and astrocytes in the ipsilateral striatum at 1 day post-ICH; the immunoreactivity of astrocytic mPGES-1 further increased at 3 days.
Our data suggest that microglial COX-1, neuronal COX-2, and astrocytic COX-2 and mPGES-1 may work sequentially to affect ICH outcomes. These findings have implications for efforts to develop anti-inflammatory strategies that target COX/PGES pathways to reduce ICH-induced secondary brain damage.
The potent inflammatory mediator prostaglandin E2 (PGE2) is implicated in the pathogenesis of several chronic inflammatory conditions, including periodontitis. The inducible enzyme microsomal prostaglandin E synthase-1 (mPGES-1), catalyzing the terminal step of PGE2 biosynthesis, is an attractive target for selective PGE2 inhibition. To identify mPGES-1 inhibitors, we investigated the effect of aminothiazoles on inflammation-induced PGE2 synthesis in vitro, using human gingival fibroblasts stimulated with the cytokine IL-1β and a cell-free mPGES-1 activity assay, as well as on inflammation-induced bone resorption in vivo, using ligature-induced experimental periodontitis in Sprague-Dawley rats. Aminothiazoles 4-([4-(2-naphthyl)-1,3-thiazol-2-yl]amino)phenol (TH-848) and 4-(3-fluoro-4-methoxyphenyl)-N-(4-phenoxyphenyl)-1,3-thiazol-2-amine (TH-644) reduced IL-1β-induced PGE2 production in fibroblasts (IC50 1.1 and 1.5 μM, respectively) as well as recombinant mPGES-1 activity, without affecting activity or expression of the upstream enzyme cyclooxygenase-2. In ligature-induced experimental periodontitis, alveolar bone loss, assessed by X-ray imaging, was reduced by 46% by local treatment with TH-848, compared to vehicle, without any systemic effects on PGE2, 6-keto PGF1α, LTB4 or cytokine levels. In summary, these results demonstrate that the aminothiazoles represent novel mPGES-1 inhibitors for inhibition of PGE2 production and reduction of bone resorption in experimental periodontitis, and may be used as potential anti-inflammatory drugs for treatment of chronic inflammatory diseases, including periodontitis.—Kats, A., Båge, T., Georgsson, P., Jönsson, J., Quezada, H. C., Gustafsson, A., Jansson, L., Lindberg, C., Näsström, K., Yucel-Lindberg, T. Inhibition of microsomal prostaglandin E synthase-1 by aminothiazoles decreases prostaglandin E2 synthesis in vitro and ameliorates experimental periodontitis in vivo.
cyclooxygenase-2; gingival fibroblasts; interleukin-1β; PGE2; mPGES-1 inhibitor; anti-inflammatory
Cytokine-induced prostaglandin (PG) E2 synthesis requires increased expression of cyclooxygenase-2 (COX-2) in human WISH epithelial cells. Recently, an inducible downstream PGE synthase (microsomal PGE synthase-1, mPGES-1) has been implicated in this inflammatory pathway. We evaluated cooperation between COX-2 and mPGES-1 as a potential mechanism for induced PGE2 production in WISH cells. Cytokine stimulation led to increased expression of both enzymes. Selective pharmacological inhibition of these enzymes demonstrated that induced PGE2 release occurred through a dominant COX-2/mPGES-1 pathway. Unexpectedly, immunofluorescent microscopy revealed that the expression of these enzymes was not tightly coordinated among cells following cytokine challenge. Within cells expressing high levels of both mPGES-1 and COX-2, immunolabeling of high-resolution semithin cryosections revealed that COX-2 and mPGES-1 were largely segregated to distinct regions within continuous intracellular membranes. Using biochemical means, it was further revealed that the majority of mPGES-1 resided within detergent-insoluble membrane fractions, whereas COX-2 was found only in detergent-soluble fractions. We conclude that although mPGES-1 and COX-2 show transcriptional and functional coordination in cytokine-induced PGE2 synthesis, complementary morphological and biochemical data suggest that a majority of intracellular mPGES-1 and COX-2 are segregated to discrete lipid microdomains in WISH epithelial cells.
Inflammation; cytokines; prostaglandin E2; cyclooxygenase-2; microsomal prostaglandin E synthase-1; lipid microdomains; epithelial cells
Chronic inflammation is associated with 25% of all cancers. In the inflammation-cancer axis, prostaglandin E2 (PGE2) is one of the major players. PGE2 synthases (PGES) are the enzymes downstream of the cyclooxygenases (COXs) in the PGE2 biosynthesis pathway. Microsomal prostaglandin E2 synthase 1 (mPGES-1) is inducible by pro-inflammatory stimuli and constitutively expressed in a variety of cancers. The potential role for this enzyme in tumorigenesis has been reported and mPGES-1 represents a novel therapeutic target for cancers. In order to identify novel small molecule inhibitors of mPGES-1, we screened the ChemBridge library and identified 13 compounds as potential hits. These compounds were tested for their ability to bind directly to the enzyme using surface plasmon resonance spectroscopy and to decrease cytokine-stimulated PGE2 production in various cancer cell lines. We demonstrate that the compound PGE0001 (ChemBridge ID number 5654455) binds to human mPGES-1 recombinant protein with good affinity (KD = 21.3 ± 7.8 μM). PGE0001 reduces IL-1β-induced PGE2 release in human HCA-7 colon and A549 lung cancer cell lines with EC50 in the submicromolar range. Although PGE0001 may have alternative targets based on the results from in vitro assays, it shows promising effects in vivo. PGE0001 exhibits significant anti-tumor activity in SW837 rectum and A549 lung cancer xenografts in SCID mice. Single injection i.p. of PGE0001 at 100 mg/kg decreases serum PGE2 levels in mice within 5 h. In summary, our data suggest that the identified compound PGE0001 exerts anti-tumor activity via the inhibition of the PGE2 synthesis pathway.
prostaglandin E2; drug design; inflammation; cancer; anti-tumor
Background and Purpose
Cyclooxygenase-2 (COX-2) and Microsomal Prostaglandin E2 Synthase-1 (mPGES-1) catalyze isomerization of the cyclooxygenase product PGH2 into PGE2. Deletion of COX-2/mPGES-1 suppresses carotid artery atherogenesis, angiotensin II-induced aortic aneurysms formation, and attenuates neointimal hyperplasia after vascular injury in mice. The upregulation of COX-2/mPGES-1 in the wall of ruptured human cerebral aneurysms is not known.
Ten patients with intracranial aneurysms (five ruptured and five non-ruptured) underwent microsurgical clipping. During the procedure, a segment of the aneurysm dome was resected and immunostained with monoclonal antibodies for COX-1, COX2 and mPGES-1. A segment of the superficial temporal artery (STA) was also removed and immunostained with monoclonal antibodies for COX-1, COX2 and mPGES-1.
All ten aneurysm tissues stained positive for mPGES-1 monoclonal antibody. Expression of mPGES-1 was more abundant in ruptured aneurysm tissue than non-ruptured aneurysms, based on a semiquantitative grading. None of the STA specimens expressed mPGES-1. COX-2 was upregulated in the same distribution as mPGES-1. COX-1 was present constitutively in all tissues.
COX-2/mPGES-1 are expressed in the wall of human cerebral aneurysms and more abundantly in ruptured aneurysms than non-ruptured. We speculate that the protective effect of aspirin against rupture of cerebral aneurysms may be mediated in part by inhibition of COX-2/mPGES-1
Aneurysm; mPGES-1; inflammation; COX-2; COX-1
Microsomal (m) prostaglandin (PG) E2 synthase (S)-1 catalyzes the formation of PGE2 from PGH2, a cyclooxygenase (COX) product that is derived from arachidonic acid. Previous studies in mice suggest that targeting mPGES-1 may be less likely to cause hypertension or thrombosis than COX-2 selective inhibition or deletion in vivo. Indeed, deletion of mPGES-1 retards atherogenesis and angiotensin II-induced aortic aneurysm formation. The role of mPGES-1 in the response to vascular injury is unknown.
Methods and Results
Mice were subjected to wire injury of the femoral artery. Both neointimal area and vascular stenosis were reduced significantly four weeks after injury in mPGES-1 knock out (KO) mice compared to wild type (WT) controls (65.6±5.7 vs 37.7±5.1×103 pixel area and 70.5±13.4% vs 47.7±17.4%, respectively; p < 0.01). Induction of tenascin C (TN-C) after injury, a pro-proliferative and promigratory extracellular matrix protein, was attenuated in the KOs. Consistent with in vivo rediversion of PG biosynthesis, mPGES-1 deleted vascular smooth muscle cells (VSMC) generated less PGE2, but more PGI2 and expressed reduced TN-C when compared with WT cells. Both suppression of PGE2 and augmentation of PGI2 attenuate TN-C expression, VSMC proliferation and migration in vitro.
Deletion of mPGES-1 in mice attenuates neointimal hyperplasia after vascular injury, in part by regulating TN-C expression. This raises for consideration the therapeutic potential of mPGES-1 inhibitors as adjuvant therapy for percutaneous coronary intervention.
Injury; percutaneous transluminal coronary angioplasty; prostacyclin; prostaglandins; vascular response
Rheumatoid arthritis (RA) is a chronic autoimmune disease which primarily affects the synovial joints leading to inflammation, pain and joint deformities. Nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids, both of which inhibit cyclooxygenase (COX), have been extensively used for treating RA patients. Prostaglandin E synthase (PGES) is a specific biosynthetic enzyme that acts downstream of COX and converts prostaglandin (PG) H2 to PGE2. Among PGES isozymes, microsomal PGES-1 (mPGES-1) has been shown to be induced in a variety of cells and tissues under inflammatory conditions. The induction of mPGES-1 in the synovial tissue of RA patients is closely associated with the activation of the tissue by proinflammatory cytokines. Although selective mPGES-1 inhibitors have not yet been widely available, mice lacking mPGES-1 (mPGES-1–/– mice) have been generated to evaluate the physiological and pathological roles of mPGES-1 in vivo. Recent studies utilizing mPGES-1–/– mice have demonstrated the significance of mPGES-1 in the process of chronic inflammation and evocation of humoral immune response in autoimmune arthritis models. These recent findings highlight mPGES-1 as a novel therapeutic target for the treatment of autoimmune inflammatory diseases, including RA. Currently, both natural and synthetic chemicals are being tested for inhibition of mPGES-1 activity to produce PGE2. The present review focuses on the recent advances in understanding the role of mPGES-1 in the pathophysiology of RA.
inflammation; microsomal prostaglandin E synthase-1; prostaglandin E2; rheumatoid arthritis; T-cell-dependent humoral immunity
Prostaglandin E synthase (PGES) including isoenzymes of membrane-associated PGES (mPGES)-1, mPGES-2, and cytosolic PGES (cPGES) is the recently identified terminal enzyme of the arachidonic acid cascade. PGES converts prostaglandin (PG)H2 to PGE2 downstream of cyclooxygenase (COX). We investigated the expression of PGES isoenzyme in articular chondrocytes from patients with osteoarthritis (OA). Chondrocytes were treated with various cytokines and the expression of PGES isoenzyme mRNA was analyzed by the reverse transcription–polymerase chain reaction and Northern blotting, whereas Western blotting was performed for protein expression. The subcellular localization of mPGES-1 was determined by immunofluorescent microscopy. Conversion of arachidonic acid or PGH2 to PGE2 was measured by enzyme-linked immunosorbent assay. Finally, the expression of mPGES-1 protein in OA articular cartilage was assessed by immunohistochemistry. Expression of mPGES-1 mRNA in chondrocytes was significantly induced by interleukin (IL)-1β or tumor necrosis factor (TNF)-α, whereas other cytokines, such as IL-4, IL-6, IL-8, IL-10, and interferon-γ, had no effect. COX-2 was also induced under the same conditions, although its pattern of expression was different. Expression of cPGES, mPGES-2, and COX-1 mRNA was not affected by IL-1β or TNF-α. The subcellular localization of mPGES-1 and COX-2 almost overlapped in the perinuclear region. In comparison with 6-keto-PGF1α and thromboxane B2, the production of PGE2 was greater after chondrocytes were stimulated by IL-1β or TNF-α. Conversion of PGH2 to PGE2 (PGES activity) was significantly increased in the lysate from IL-1β-stimulated chondrocytes and it was inhibited by MK-886, which has an inhibitory effect on mPGES-1 activity. Chondrocytes in articular cartilage from patients with OA showed positive immunostaining for mPGES-1. These results suggest that mPGES-1 might be important in the pathogenesis of OA. It might also be a potential new target for therapeutic strategies that specifically modulate PGE2 synthesis in patients with OA.
chondrocytes; interleukin-1β; osteoarthritis; prostaglandin E synthase; tumor necrosis factor-α
Microsomal prostaglandin E synthase-1 (mPGES-1) is a key enzyme that couples with cyclooxygenase-2 (COX-2) for the production of PGE2. Although COX-2 is known to mediate the growth and progression of several human cancers including hepatocellular carcinoma (HCC), the role of mPGES-1 in hepatocarcinogenesis is not well established. This study provides novel evidence for a key role of mPGES-1 in HCC growth and progression. Forced overexpression of mPGES-1 in two HCC cell lines (Hep3B and Huh7) increased tumor cell growth, clonogenic formation, migration and invasion, whereas knockdown of mPGES-1 inhibited these parameters, in vitro. In a SCID mouse tumor xenograft model, mPGES-1 overexpressed cells formed palpable tumors at earlier time points and developed larger tumors when compared to the control (p<0.01); in contrast, mPGES-1 knockdown delayed tumor development and reduced tumor size (p<0.01). Mechanistically, mPGES-1-induced HCC cell proliferation, invasion and migration involve PGE2 production and activation of early growth response 1 (EGR1) and β-catenin. Specifically, mPGES-1-derived PGE2 induces the formation of EGR1-β-catenin complex, which interacts with TCF4/LEF1 transcription factors and activates the expression of β-catenin downstream genes. Our findings depict a novel crosstalk between mPGES-1/PGE2 and EGR1/β-catenin signaling that is critical for hepatocarcinogenesis.
Microsomal prostaglandin E synthase-1 (mPGES-1); β-catenin; early growth response 1 (EGR1); hepatocellular carcinoma (HCC); liver
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.
In addition to cyclooxygenase-2 (COX-2) which is related to prostaglandin E2 synthesis, other enzymes such as cytosolic phospholipase A2 (cPLA2), microsomal prostaglandin E2 synthase-1 (mPGES-1), and 15-prostaglandin dehydrogenase (15-PGDH) have been suggested to be related to carcinogenesis of colorectal cancer (CRC). The aim of this study was to investigate the roles of cPLA2, COX-2, mPGES-1, and 15-PGDH in tumor progression.
Materials and Methods
cPLA2, COX-2, mPGES-1, 15-PGDH, and vascular endothelial growth factor (VEGF) expressions were immunohistochemically examined in 89 CRC, and their expressions were compared with each other or clinicopathologic parameters as well as VEGF as tumor progression parameters.
cPLA2 was expressed in 54.5%, COX-2 in 80.5%, mPGES-1 in 96.4%, 15-PGDH in 46.1%, and VEGF in 65.9%. The expression of cPLA2 correlated with VEGF expression. COX-2 expression was correlated with the depth of invasion, tumor stage, cPLA2, and VEGF expressions. Moreover, VEGF revealed the highest expression in the tissues positive for both cPLA2 and COX-2. Furthermore, 15-PGDH expression was inversely correlated with VEGF expression.
The present study demonstrates that cPLA2 and mPGES-1, in addition to COX-2, are constitutively overexpressed, and that 15-PGDH might be attenuated in colorectal cancer. Furthermore, cPLA2 and 15-PGDH as well as COX-2 could have an important role in tumor progression.
cPLA2; COX-2; mPGES-1; 15-PGDH; colorectal cancer
Microsomal prostaglandin E synthase-1 (mPGES-1) is an essential enzyme involved in a variety of diseases and is the most promising target for the design of next-generation anti-inflammatory drugs. In order to establish a solid structural base, we recently developed a model of mPGES-1 trimer structure by using available crystal structures of both microsomal glutathione transferase-1 (MGST1) and ba3-cytochrome c oxidase as templates. The mPGES-1 trimer model has been used, in the present study, to examine the detailed binding of mPGES-1 trimer with substrate PGH2 and cofactor GSH. Results obtained from the computational alanine scanning reveal the contribution of each residue at the protein-ligand interaction interface to the binding affinity, and the computational predictions are supported by the data obtained from the corresponding wet experimental tests. We have also compared our mPGES-1 trimer model with other available 3D models, including an alternative homology model and a low-resolution crystal structure, and found that our mPGES-1 trimer model based on the crystal structures of both MGST1 and ba3-cytochrome c oxidase is more reasonable than the other homology model of mPGES-1 trimer constructed by simply using a low-resolution crystal structure of MGST1 trimer alone as a template. The available low-resolution crystal structure of mPGES-1 trimer represents a closed conformation of the enzyme and, thus, is not suitable for studying mPGES-1 binding with ligands. Our mPGES-1 trimer model represents a reasonable open conformation of the enzyme and is, therefore, promising for studying mPGES-1 binding with ligands in future structure-based drug design targeting mPGES-1.
Prostaglandin E (PGE)2 produced by osteoblasts acts as a potent stimulator of bone resorption. Inflammatory bone loss is accompanied by osteoclast formation induced by bone-resorbing cytokines, but the mechanism of PGE2 production and bone resorption in vivo is not fully understood. Using cytosolic phospholipase A2α (cPLA2α)-null mice, we examined the role of cPLA2α in PGE2 synthesis and bone resorption. In bone marrow cultures, interleukin (IL)-1 markedly stimulated PGE2 production and osteoclast formation in wild-type mice, but not in cPLA2α-null mice. Osteoblastic bone marrow stromal cells induced the expression of cyclooxygenase (COX)-2 and membrane-bound PGE2 synthase (mPGES) in response to IL-1 and lipopolysaccharide (LPS) to produce PGE2. Osteoblastic stromal cells collected from cPLA2α-null mice also induced the expression of COX-2 and mPGES by IL-1 and LPS, but could not produce PGE2 due to the lack of arachidonic acid release. LPS administration to wild-type mice reduced femoral bone mineral density by increased bone resorption. In cPLA2α-null mice, however, LPS-induced bone loss could not be observed at all. Here, we show that cPLA2α plays a key role in PGE production by osteoblasts and in osteoclastic bone resorption, and suggest a new approach to inflammatory bone disease by inhibiting cPLA2α.
cPLA2α; bone loss; osteoclast; osteoblast; lipopolysaccharide
Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible enzyme that catalyzes the conversion of prostaglandin (PG)H2 to PGE2. Proinflammatory stimuli markedly increase levels of mPGES-1 expression both in vivo and in vitro. mPGES-1 knockout studies and animal models of inflammatory arthritis also provide a strong basis for the contribution of mPGES-1 in the increased local production of PGE2 observed in inflammatory arthritis. The focus of this article is to review some recent advances in our understanding of mechanisms specific to the regulation of inducible mPGES-1 in inflammatory arthritis.
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.
Prostaglandin E2 (PGE2) is a bioactive lipid that mediates a wide range of physiological effects and plays a central role in inflammation and cancer. PGE2 is generated from arachidonic acid by the sequential actions of the cyclooxygenases (COXs) and terminal synthases (PGES). Increased levels of COX-2, with a concomitant elevation of PGE2, are often found in colorectal cancers (CRC), providing the rationale for the use of COX-2 inhibitors for chemoprevention. Despite their proven efficacy in cancer prevention, however, COX-2 inhibitors exhibit dose-dependent toxicities that are mediated in part by their non-specific reduction of essential prostanoids, thus limiting their chemopreventive benefit. To achieve enhanced specificity, recent efforts have been directed towards targeting the inducible terminal synthase in the production of PGE2, microsomal PGES (mPGES-1). In the present study, we show that genetic deletion of mPGES-1 affords significant protection against carcinogen-induced colon cancer. mPGES-1 gene deletion results in an ~80% decrease in tumor multiplicity and up to a 90% reduction in tumor load in the distal colon of azoxymethane (AOM)-treated mice. Associated with the striking cancer suppression, we have identified a critical role for PGE2 in the control of immunoregulatory cell expansion (FoxP3-positive regulatory T cells) within the colon-draining mesenteric lymph nodes, providing a potential mechanism by which suppression of PGE2 may protect against CRC. These results provide new insights into how PGE2 controls anti-tumor immunity.
Colorectal cancer; microsomal prostaglandin synthase-1; prostaglandin E2; mucosal ulcer; regulatory T cells
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-PGF1α 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-PGF1α 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-Δ12,14prostaglandin 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-PGF1α 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.
Although prostaglandin E2 (PGE2), cyclooxygenase 2 (COX‐2), and microsomal prostaglandin E synthase 1 (mPGES‐1) are known to play a role in various inflammatory events, their roles in the pathogenesis of gastro‐oesophageal reflux disease are not known.
We examined the dynamics of COX‐1, COX‐2, mPGES‐1, mPGES‐2, cytosolic PGES (cPGES), and PGE2 synthetic activity in rat acid reflux oesophagitis and the effects of COX‐2 inhibitors on the severity of oesophagitis.
Acid reflux oesophagitis was induced by ligating the transitional region between the forestomach and the glandular portion and wrapping the duodenum near the pylorus. Rats were killed on day 3 (acute phase) or day 21 (chronic phase) after induction of oesophagitis.
Expression of COX‐2 and mPGES‐1 was markedly increased in oesophagitis while modest changes in COX‐1, cPGES, and mPGES‐2 expression were observed. COX‐2 and mPGES‐1 were colocalised in epithelial cells of the basal layer, as well as inflammatory and mesenchymal cells in the lamina propria and submucosa. COX‐2 inhibitors significantly reduced the severity of chronic oesophagitis but did not affect acute oesophageal lesions. COX‐2 inhibitors significantly inhibited the increase in PGE2 synthesis in oesophageal lesions on both days 3 and 21. Epithelial proliferation was significantly increased in the basal layer on day 21. Inflammatory cells and epithelial cells of the basal layer exhibited reactions for EP4 in oesophagitis.
PGE2 derived from COX‐2 and mPGES‐1 plays a significant role in the pathogenesis of chronic acid reflux oesophagitis, and possibly in basal hyperplasia and persistent inflammatory cell infiltration.
cyclooxygenase 2; microsomal prostaglandin E synthesis 1; prostaglandin E2; cyclooxygenase 2 inhibitors; reflux oesophagitis
PGE2 is a potent lipid mediator of pain and oedema found elevated in RA. Microsomal prostaglandin E synthase-1 (mPGES-1) is a terminal enzyme of the PGE2 pathway inducible by proinflammatory cytokines. mPGES-1 is markedly upregulated in RA synovial tissue despite antirheumatic treatments, suggesting that multiple inflammatory stimuli contribute to its induction. High-mobility group box chromosomal protein 1 (HMGB1) is known to induce inflammation both by direct interaction with TLR4 and by enhancement of other proinflammatory molecules signalling, through complex formation. The high expression of extracellular HMGB1 within the inflamed synovium, implies its pro-arthritogenic role in RA. We aimed to investigate the effects of IL-1β/HMGB1 complexes on mPGES-1 and other enzymes of the PGE2 pathway in synovial fibroblasts (SFs) from patients with arthritis. Furthermore, we studied the effect of COX-2 inhibition and IL-1RI antagonism on prostanoid and cytokine production by SFs. Stimulation of SFs with HMGB1 in complex with suboptimal amounts of IL-1β significantly increased mPGES-1 and COX-2 expressions as well as PGE2 production, as compared to treatment with HMGB1 or IL-1β alone. Furthermore, NS-398 reduced the production of IL-6 and IL-8, thus indicating that IL-1β/HMGB1 complexes modulate cytokine production in part through prostanoid synthesis. Treatment with IL-1RA completely abolished the induced PGE2 and cytokine production, suggesting an effect mediated through IL-1RI. IL-1β/HMGB1 complexes promote the induction of mPGES-1, COX-2 and PGE2 in SF. The amplification of the PGE2 biosynthesis pathway by HMGB1 might constitute an important pathogenic mechanism perpetuating inflammatory and destructive activities in rheumatoid arthritis.