Gene Deletion of Microsomal Prostaglandin E Synthase-1 Suppresses Chemically Induced Skin Carcinogenesis.

BACKGROUND/AIM: Microsomal prostaglandin (PG) E synthase-1 (mPGES-1) is a terminal enzyme in PGE2 synthesis and highly expressed in several cancers. In this study, to reveal the involvement of mPGES-1 in skin carcinogenesis, the effect of mPGES-1 deficiency on two-stage skin carcinogenesis in mice was investigated. MATERIALS AND METHODS: A two-stage skin carcinogenesis model using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter was applied on mPGES-1 knockout (KO) mice and littermate wild-type mice of a Balb/c genetic background. RESULTS: DMBA/TPA-induced skin carcinogenesis was suppressed in mPGES-1 KO mice. The induction of IL-17 and other inflammatory cytokines by TPA was also suppressed by mPGES-1 deficiency, although DMBA-induced apoptosis was not affected. CONCLUSION: mPGES-1 promotes chemically induced skin carcinogenesis and might play an important role in the TPA-induced promotion phase of the two-stage skin carcinogenesis model. mPGES-1 inhibition may be a therapeutic target for skin cancer.

Coordinated action of microsomal prostaglandin E synthase-1 and prostacyclin synthase on contact hypersensitivity.

Microsomal prostaglandin (PG) E synthase-1 (mPGES-1) and prostacyclin (PGI2) synthase (PGIS) are PG terminal synthases that work downstream of cyclooxygenase and synthesize PGE2 and PGI2, respectively. Although the involvement of PG receptors in acquired cutaneous immune responses was recently shown, the roles of these PG terminal synthases remain unclear. To identify the pathophysiological roles of mPGES-1 and PGIS in cutaneous immune systems, we applied contact hypersensitivity (CHS) to mPGES-1 and PGIS knockout (KO) mice as a model of acquired immune responses. Mice were treated with 1-fluoro-2,4-dinitrobenzene (DNFB) and evaluated for ear thickness and histopathological features. The results showed that the severity of ear swelling in both gene-deficient mice was much lower than that in wild-type (WT) mice. Histological examination of DNFB-treated ears showed that inflammatory cell infiltration and edema in the dermis were also less apparent in both genotypic mice. LC-MS analysis further showed that the increment in PGE2 levels in DNFB-treated ear tissue was reduced in mPGES-1 KO mice, and that 6-keto PGF1α (a stable metabolite of PGI2) was not detected in PGIS KO mice. Furthermore, we made bone marrow (BM) chimera and found that transplantation of WT mouse-derived BM cells restored the impaired CHS response in mPGES-1 KO mice but did not restore the response in PGIS KO mice. These results indicated that mPGES-1 in BM-derived cells and PGIS in non-BM-derived cells might play critical roles in DNFB-induced CHS. mPGES-1-derived PGE2 and PGIS-derived PGI2 might coordinately promote acquired cutaneous immune responses.

Involvement of prostacyclin synthase in high-fat-diet-induced obesity.

Prostacyclin (PGI2) synthase (PGIS) functions downstream of inducible cyclooxygenase COX-2 in the PGI2 biosynthetic pathway. Although COX-2 and PGI2 receptor (IP) are known to be involved in adipogenesis and obesity, the involvement of PGIS has not been fully elucidated. In this study, we examined the role of PGIS in adiposity by using PGIS-deficient mice. Although PGIS deficiency did not affect in vitro adipocyte differentiation, when fed a high-fat diet (HFD), PGIS knockout (KO) mice showed reductions in both body weight gain and epididymal fat mass relative to wild-type (WT) mice. PGIS deficiency might reduce HFD-induced obesity by suppressing PGI2 production. We further found that additional gene deletion of microsomal prostaglandin (PG) E synthase-1 (mPGES-1), one of the other PG terminal synthases that also functions downstream of COX-2, emphasized the metabolic phenotypes of PGIS-deficient mice. More marked reduction in obesity and improved insulin resistance were observed in PGIS/mPGES-1 double KO (DKO) mice. Since an additive increase in PGF2α level in epididymal fat was observed in DKO mice, mPGES-1 deficiency might affect adiposity by enhancing the production of PGF2α. Our immunohistochemical analysis further revealed that in adipose tissues, PGIS was expressed in vascular and stromal cells but not in adipocytes. These results suggested that PGI2 produced from PGIS-expressed stromal tissues might enhance HFD-induced obesity by acting on IP expressed in adipocytes. The balance of expressions of PG terminal synthases and the subsequent production of prostanoids might be critical for adiposity.

Gene Deletion of Calcium-Independent Phospholipase A2γ (iPLA2γ) Suppresses Adipogenic Differentiation of Mouse Embryonic Fibroblasts.

Adipogenic differentiation is a complex process by which fibroblast-like undifferentiated cells are converted into cells that accumulate lipid droplets. We here investigated the effect of gene deletion of calcium-independent phospholipase A2γ (iPLA2γ), a membrane-bound PLA2 enzyme, on adipogenic differentiation in mice. Since iPLA2γ knockout (KO) mice showed reduced fat volume and weight, we prepared mouse embryonic fibroblasts (MEF) from wild-type (WT) and iPLA2γ KO mice and examined the effect of iPLA2γ deletion on in vitro adipogenic differentiation. iPLA2γ increased during adipogenic differentiation in WT mouse-derived MEFs, and the differentiation was partially abolished in iPLA2γ KO-derived MEFs. In KO-derived MEFs, the inductions of peroxisome proliferator activator receptor γ (PPARγ) and CAAT/enhancer-binding protein α (C/EBPα) were also reduced during adipogenic differentiation, and the reductions in PPARγ and C/EBPα expressions and the defect in adipogenesis were restored by treatment with troglitazone, a PPARγ ligand. These results indicate that iPLA2γ might play a critical role in adipogenic differentiation by regulating PPARγ expression.

Long-chain acyl-CoA synthetase 4 participates in the formation of highly unsaturated fatty acid-containing phospholipids in murine macrophages.

Long-chain acyl-coenzyme A synthetases (ACSLs) are a family of enzymes that convert free long-chain fatty acids into their acyl-coenzyme A (CoA) forms. ACSL4, belonging to the ACSL family, shows a preferential use of arachidonic acid (AA) as its substrate and plays a role in the remodeling of AA-containing phospholipids by incorporating free AA. However, little is known about the roles of ACSL4 in inflammatory responses. Here, we assessed the roles of ACSL4 on the effector functions of bone marrow-derived macrophages (BMDMs) obtained from mice lacking ACSL4. Liquid chromatography-tandem mass spectrometry analysis revealed that various highly unsaturated fatty acid (HUFA)-derived fatty acyl-CoA species were markedly decreased in the BMDMs obtained from ACSL4-deficient mice compared with those in the BMDMs obtained from wild-type mice. BMDMs from ACSL4-deficient mice also showed a reduced incorporation of HUFA into phosphatidylcholines. The stimulation of BMDMs with lipopolysaccharide (LPS) elicited the release of prostaglandins (PGs), such as PGE2, PGD2 and PGF2α, and the production of these mediators was significantly enhanced by ACSL4 deficiency. In contrast, neither the LPS-induced release of cytokines, such as IL-6 and IL-10, nor the endocytosis of zymosan or dextran was affected by ACSL4 deficiency. These results suggest that ACSL4 has a crucial role in the maintenance of HUFA composition of certain phospholipid species and in the incorporation of free AA into the phospholipids in LPS-stimulated macrophages. ACSL4 dysfunction may facilitate inflammatory responses by an enhanced eicosanoid storm.

Calcium-independent phospholipase A2γ (iPLA2γ) and its roles in cellular functions and diseases.

Calcium-independent phospholipase A2γ (iPLA2γ)/patatin-like phospholipase domain-containing lipase 8 (PNPLA8) is one of the iPLA2 enzymes, which do not require Ca2+ ion for their activity. iPLA2γ is a membrane-bound enzyme with unique features, including the utilization of four distinct translation initiation sites and the presence of mitochondrial and peroxisomal localization signals. This enzyme is preferentially distributed in the mitochondria and peroxisomes and is thought to be responsible for the maintenance of lipid homeostasis in these organelles. Thus, both the overexpression and the deletion of iPLA2γ in vivo caused mitochondrial abnormalities and dysfunction. Roles of iPLA2γ in lipid mediator production and cytoprotection against oxidative stress have also been suggested by in vitro and in vivo studies. The dysregulation of iPLA2γ can therefore be a critical factor in the development of many diseases, including metabolic diseases and cancer. In this review, we provide an overview of the biochemical properties of iPLA2γ and then summarize the current understanding of the in vivo roles of iPLA2γ revealed by knockout mouse studies.

The group VIA calcium-independent phospholipase A2 and NFATc4 pathway mediates IL-1ß-induced expression of chemokines CCL2 and CXCL10 in rat fibroblasts.

Chemokines are secreted proteins that regulate cell migration and are involved in inflammatory and immune responses. Here, we sought to define the functional crosstalk between the lipid signaling and chemokine signaling. We obtained evidence that the induction of some chemokines is regulated by group VIA calcium-independent phospholipase A2 ß (iPLA2 ß) in IL-1ß-stimulated rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with IL-1ß elicited an increased release of chemotactic factor(s) for monocytic THP-1 cells into culture medium in a time-dependent manner. Inhibitor studies revealed that an intracellular PLA2 inhibitor, arachidonoyl trifluoromethyl ketone (AACOCF3 ), but not the cyclooxygenase inhibitor indomethacin, attenuated the release of chemotactic factor(s). The chemotactic activity was inactivated by treatment with either heat or proteinase K, suggesting this chemotactic factor(s) is a proteinaceous factor(s). We purified the chemotactic factor(s) from the conditioned medium of IL-1ß-stimulated 3Y1 cells using a heparin column and identified several chemokines, including CCL2 and CXCL10. The inducible expressions of CCL2 and CXCL10 were significantly attenuated by pretreatment with AACOCF3 . Gene silencing using siRNA revealed that the inductions of CCL2 and CXCL10 were attenuated by iPLA2 ß knockdown. Additionally, the transcriptional activation of nuclear factor of activated T-cell proteins (NFATs), but not nuclear factor-κB, by IL-1ß stimulation was markedly attenuated by the iPLA2 inhibitor bromoenol lactone, and NFATc4 knockdown markedly attenuated the IL-1ß-induced expression of both CCL2 and CXCL10. Collectively, these results indicated that iPLA2 ß plays roles in IL-1ß-induced chemokine expression, in part via NFATc4 signaling.

Cytosolic Prostaglandin E Synthase Is Involved in c-Fos Expression in Rat Fibroblastic 3Y1 Cells.

Cytosolic prostaglandin (PG) E synthase (cPGES/p23) plays a role in the biosynthesis of PGE2 and in the molecular chaperone machinery. Studies of knockout mice lacking cPGES/p23 have demonstrated that cPGES/p23 is essential in fetal mouse development. A cDNA microarray analysis revealed that a lack of cPGES/p23 decreases the expression of some immediate early genes, such as c-fos and activating transcription factor 3 (ATF3). Here we report the involvement of cPGES/p23 in c-Fos expression. A stable knockdown of cPGES/p23 in cultured fibroblasts not only reduced serum-induced c-Fos expression, but also decreased the phosphorylation of extracellular signal regulated kinase (ERK). These results suggest that cPGES/p23 is involved in the activation of ERK to promote c-Fos expression.

Regulatory Functions of Phospholipase A2.

Phospholipase A2 (PLA2) plays crucial roles in diverse cellular responses, including phospholipid digestion and metabolism, host defense and signal transduction. PLA2 provides precursors for generation of eicosanoids, such as prostaglandins (PGs) and leukotrienes (LTs), when the cleaved fatty acid is arachidonic acid, platelet-activating factor (PAF) when the sn-1 position of the phosphatidylcholine contains an alkyl ether linkage and some bioactive lysophospholipids, such as lysophosphatidic acid (lysoPA). As overproduction of these lipid mediators causes inflammation and tissue disorders, it is extremely important to understand the mechanisms regulating the expression and functions of PLA2. Recent advances in molecular and cellular biology have enabled us to understand the molecular nature, possible function, and regulation of a variety of PLA2 isozymes. Mammalian tissues and cells generally contain more than one enzyme, each of which is regulated independently and exerts distinct functions. Here we classify mammalian PLA2s into three large groups, namely, secretory (sPLA2), cytosolic (cPLA2), and Ca2+-independent PLA2s, on the basis of their enzymatic properties and structures and focus on the general undestanding of the possible regulatory functions of each PLA2 isozyme. In particular, the roles of type II sPLA2 and cPLA2 in lipid mediator generation are discussed.

Genetic-deletion of Cyclooxygenase-2 Downstream Prostacyclin Synthase Suppresses Inflammatory Reactions but Facilitates Carcinogenesis, unlike Deletion of Microsomal Prostaglandin E Synthase-1.

Prostacyclin synthase (PGIS) and microsomal prostaglandin E synthase-1 (mPGES-1) are prostaglandin (PG) terminal synthases that function downstream of inducible cyclooxygenase (COX)-2 in the PGI2 and PGE2 biosynthetic pathways, respectively. mPGES-1 has been shown to be involved in various COX-2-related diseases such as inflammatory diseases and cancers, but it is not yet known how PGIS is involved in these COX-2-related diseases. Here, to clarify the pathophysiological role of PGIS, we investigated the phenotypes of PGIS and mPGES-1 individual knockout (KO) or double KO (DKO) mice. The results indicate that a thioglycollate-induced exudation of leukocytes into the peritoneal cavity was suppressed by the genetic-deletion of PGIS. In the PGIS KO mice, lipopolysaccharide-primed pain nociception (as assessed by the acetic acid-induced writhing reaction) was also reduced. Both of these reactions were suppressed more effectively in the PGIS/mPGES-1 DKO mice than in the PGIS KO mice. On the other hand, unlike mPGES-1 deficiency (which suppressed azoxymethane-induced colon carcinogenesis), PGIS deficiency up-regulated both aberrant crypt foci formation at the early stage of carcinogenesis and polyp formation at the late stage. These results indicate that PGIS and mPGES-1 cooperatively exacerbate inflammatory reactions but have opposing effects on carcinogenesis, and that PGIS-derived PGI2 has anti-carcinogenic effects.

Role of microsomal prostaglandin E synthase-1 (mPGES-1)-derived prostaglandin E2 in colon carcinogenesis.

Nonsteroidal anti-inflammatory drugs, especially selective cyclooxygenase-2 (COX-2) inhibitors, are among the most promising chemopreventive agents for colorectal cancer. However, recent clinical trials have indicated that these inhibitors pose a significantly increased cardiovascular risk. Microsomal prostaglandin E (PGE) synthase-1 (mPGES-1) and mPGES-1-derived PGE2 have gained attention recently as alternative targets to COX-2 for colorectal cancer chemoprevention and chemotherapy. In this review, we summarize the current understanding of the roles of mPGES-1, a PGE2-inactivating enzyme (15-hydroxyprostagladin dehydrogenase), and PGE2 specific receptors (EPs) in colon carcinogenesis.

Group VIB calcium-independent phospholipase A2 (iPLA2γ) regulates platelet activation, hemostasis and thrombosis in mice.

In platelets, group IVA cytosolic phospholipase A2 (cPLA2α) has been implicated as a key regulator in the hydrolysis of platelet membrane phospholipids, leading to pro-thrombotic thromboxane A2 and anti-thrombotic 12-(S)-hydroxyeicosatetranoic acid production. However, studies using cPLA2α-deficient mice have indicated that other PLA2(s) may also be involved in the hydrolysis of platelet glycerophospholipids. In this study, we found that group VIB Ca2+-independent PLA2 (iPLA2γ)-deficient platelets showed decreases in adenosine diphosphate (ADP)-dependent aggregation and ADP- or collagen-dependent thromboxane A2 production. Electrospray ionization mass spectrometry analysis of platelet phospholipids revealed that fatty acyl compositions of ethanolamine plasmalogen and phosphatidylglycerol were altered in platelets from iPLA2γ-null mice. Furthermore, mice lacking iPLA2γ displayed prolonged bleeding times and were protected against pulmonary thromboembolism. These results suggest that iPLA2γ is an additional, long-sought-after PLA2 that hydrolyzes platelet membranes and facilitates platelet aggregation in response to ADP.

Role of long-chain acyl-coenzyme A synthetases in the regulation of arachidonic acid metabolism in interleukin 1ß-stimulated rat fibroblasts.

Acyl coenzyme A synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs and play an important role in fatty acid metabolism. Here we show the role of ACSL isozymes in interleukin (IL)-1ß-induced arachidonic acid (AA) metabolism in rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with triacsin C, an ACSL inhibitor, markedly enhanced the IL-1ß-induced prostaglandin (PG) biosynthesis. Small interfering RNA-mediated knockdown of endogenous Acsl4 expression increased significantly the release of AA metabolites, including PGE2, PGD2, and PGF2α, compared with replicated control cells, whereas knockdown of Acsl1 expression reduced the IL-1ß-induced release of AA metabolites. Experiments with double knockdown of Acsl4 and intracellular phospholipase A2 (PLA2) isozymes revealed that cytosolic PLA2α, but not calcium-independent PLA2s, is involved in the Acsl4 knockdown-enhanced PG biosynthesis. Electrospray ionization mass spectrometry of cellular phospholipids bearing AA showed that the levels of some, if not all, phosphatidylcholine (PC) and phosphatidylinositol species in Acsl4 knockdown cells were decreased after IL-1ß stimulation, while those in control cells were not so obviously decreased. In Acsl1 knockdown cells, the levels of some AA-bearing PC species were reduced even in the unstimulated condition. Collectively, these results suggest that Acsl isozymes play distinct roles in the control of AA remodeling in rat fibroblasts: Acsl4 acts as the first step of enzyme for AA remodeling following IL-1ß stimulation, and Acsl1 is involved in the maintenance of some AA-containing PC species.

Microsomal prostaglandin E synthase-1 is induced in alzheimer's disease and its deletion mitigates alzheimer's disease-like pathology in a mouse model.

Epidemiological studies have suggested that long-term use of nonsteroidal anti-inflammatory drugs that inhibit cyclooxygenase (COX) activity can moderate the onset or progression of Alzheimer's disease (AD). Thus it has been suggested that prostaglandin E2 (PGE2 ), a major end-product of COX, may play a pathogenic role in AD, but the involvement of PGE synthase (PGES), a terminal enzyme downstream from COX, has not been fully elucidated. Here we found that, among three PGES enzymes, only microsomal PGES-1 (mPGES-1) is induced, and its expression is associated with ß-amyloid (Aß) plaques in the cerebral cortex in human AD patients and in Tg2576 mice, a transgenic AD mouse model. Furthermore, to investigate whether mPGES-1 contributes to AD-like pathology, we bred mPGES-1-deficient mice with Tg2576 mice. We found that mPGES-1 deletion reduced the accumulation of microglia around senile plaques and attenuated learning impairments in Tg2576 mice. These results indicated that mPGES-1 is induced in the AD brain and thus plays a role in AD pathology. Blockage of mPGES-1 could form the basis for a novel therapeutic strategy for patients with AD.

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

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

Involvement of the constitutive prostaglandin E synthase cPGES/p23 in expression of an initial prostaglandin E2 inactivating enzyme, 15-PGDH.

We previously showed that cytosolic prostaglandin (PG) E synthase (cPGES/p23) which isomerizes PGH(2) to PGE(2), is essential for fetal mouse development. Embryonic fibroblasts derived from cPGES/p23 knockout mice generated higher amounts of PGE(2) in culture supernatants than wild-type-derived cells. In order to elucidate this apparent conflict that absence of PGE(2) synthetic enzyme caused facilitation of PGE(2) biosynthesis, we examined expression of the PGE(2) degrading enzyme in embryonic fibroblasts. We report here that embryonic fibroblasts deficient in cPGES/p23 decreased the expression of the PGE(2) degrading enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which catalyzes the inactivating conversion of the PGE(2) 15-OH to a 15-keto group, compared with that of wild-type. In addition, rat fibroblastic 3Y1 cells harboring cPGES/p23 siRNA exhibited lower 15-PGDH expression than mock-transfected cells. Furthermore, forcible expression of cPGES/p23 in 3Y1 cells resulted in facilitation of 15-PGDH promoter activity. These results suggest that the PGE(2)-inactivating pathway is controlled by the PGE(2) biosynthetic enzyme, cPGES/p23.

Mitochondrial dysfunction and reduced prostaglandin synthesis in skeletal muscle of Group VIB Ca2+-independent phospholipase A2gamma-deficient mice.

Group VIB Ca(2+)-independent phospholipase A(2)γ (iPLA(2)γ) is a membrane-bound iPLA(2) enzyme with unique features, such as the utilization of distinct translation initiation sites and the presence of mitochondrial and peroxisomal localization signals. Here we investigated the physiological functions of iPLA(2)γ by disrupting its gene in mice. iPLA(2)γ-knockout (KO) mice were born with an expected Mendelian ratio and appeared normal and healthy at the age of one month but began to show growth retardation from the age of two months as well as kyphosis and significant muscle weakness at the age of four months. Electron microscopy revealed swelling and reduced numbers of mitochondria and atrophy of myofilaments in iPLA(2)γ-KO skeletal muscles. Increased lipid peroxidation and the induction of several oxidative stress-related genes were also found in the iPLA(2)γ-KO muscles. These results provide evidence that impairment of iPLA(2)γ causes mitochondrial dysfunction and increased oxidative stress, leading to the loss of skeletal muscle structure and function. We further found that the compositions of cardiolipin and other phospholipid subclasses were altered and that the levels of myoprotective prostanoids were reduced in iPLA(2)γ-KO skeletal muscle. Thus, in addition to maintenance of homeostasis of the mitochondrial membrane, iPLA(2)γ may contribute to modulation of lipid mediator production in vivo.

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

Prostaglandin E synthase (PGES), which converts cyclooxygenase (COX)-derived prostaglandin H(2) (PGH(2)) to PGE(2), 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 PGE(2) 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.

Microsomal prostaglandin E synthase-1 in both cancer cells and hosts contributes to tumour growth, invasion and metastasis.

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 mice lacking cPGES/p23, a constitutively expressed PGE2 synthetic enzyme, are peri-natally lethal.

Cytosolic prostaglandin (PG) E synthase (cPGES) is constitutively expressed in various cells and regulates cyclooxygenase (COX)-1-dependent immediate PGE(2) generation. Its primary structure is identical to co-chaperone p23, a heat shock protein 90 (Hsp90)-binding protein. We have revealed that Hsp90 regulated both cPGES/p23 and its client protein kinase CK2. In this study, in order to examine the role of cPGES/p23 in vivo, we generated mice deficient in cPGES/p23 by a targeted disruption of exons 2 and 3, containing Tyr9, which is essential for catalytic activity. Heterozygotes are viable, fertile, and appear normal, despite a decrease in cPGES/p23 protein level. A generation of offsprings derived from intercrosses of cPGES/p23 homozygous mice revealed that 109, 247, and 10 pups were wild type, heterozygous, and homozygous, respectively; however, all homozygotes died at birth. The absence of viable null mutants, with heterozygotes and wild-type offspring obtained at a ratio of approximately 2:1, indicated that homozygosity for the cPGES/p23 null mutant leads to peri-natal lethality. Embryos homozygous for cPGES/p23-null had lower body weights than wild-type embryos, and abnormal morphology of skin and lungs. Moreover, the PGE(2) content in the lungs of cPGES/p23-null embryos was lower than that of the wild type. These results indicate that cPGES-derived PGES is involved in the normal development of mouse embryonic lung.