GLIA 56:844–855 (2008)

Regulation of Prostaglandin E2 Synthase Expression in Activated Primary Rat Microglia: Evidence for Uncoupled Regulation of mPGES-1 and COX-2 ANTONIO CARLOS PINHEIRO DE OLIVEIRA,1 EDUARDO CANDELARIO-JALIL,1,2 1 1 € HARSHARAN S. BHATIA,1 KLAUS LIEB,1,3 MICHAEL HULL, AND BERND L. FIEBICH * 1 Department of Psychiatry, University of Freiburg Medical School, Freiburg, Germany 2 Department of Neurology, University of New Mexico, Albuquerque, New Mexico 3 Department of Psychiatry, University Medical Center Mainz, Mainz, Germany

KEY WORDS neuroinflammation; phosphatidylinositol-3-kinase; transduction; prostanoids

signal

ABSTRACT Prostaglandin E2 (PGE2) is among the most important mediators involved in neuroinflammatory processes. The final step of its synthesis is regulated by enzymes termed prostaglandin E2 synthases (PGES). Three PGES are known, cytosolic (c)PGES, membrane-associated (m)PGES-1 and mPGES-2. The expression of mPGES-1 is induced by inflammatory stimuli such as lipopolysaccharide (LPS), interleukin (IL)-1b, and tumor necrosis factor (TNF)-a. Although some roles of mPGES-1 have already been suggested, its function in the CNS and the signaling pathways involved in its upregulation are poorly understood. In this study, we examined the regulation of mPGES-1 in primary rat microglia and the signaling pathways involved in its expression. Whereas the expression of cPGES and mPGES-2 was not stimulated by LPS, low doses of LPS (0.1–1 ng/mL) sufficiently stimulated mPGES-1 mRNA expression. A corresponding protein synthesis, however, was obtained only with higher doses (10–100 ng/mL). The LPS-induced increase of mPGES-1 was inhibited by different signaling pathway inhibitors, such as SP600125, LY294002, GF109203X, and SC-514, suggesting the involvement of c-Jun N-terminal kinase (JNK), phosphatidylinositol 3-kinase (PI-3K)/Akt, protein kinase C (PKC) pathways, and the nuclear factor (NF)-jB, respectively. In contrast to other reports, LPSinduced mPGES-1 synthesis was not invariably coupled to the synthesis of COX-2, since inhibition of PI-3K with LY294002 decreased mPGES-1 but increased COX-2 levels. This detailed view of the intracellular signaling pathways involved in mPGES-1 expression in activated microglia opens a new avenue in the search for novel potential therapeutic targets to reduce neuroinflammation, and demonstrates that mPGES-1 expression is not strictly coupled to the expression of COX-2. V2008 Wiley-Liss, Inc. C

et al., 2005; Dickson et al., 1993; Hoozemans et al., 2006; McGeer et al., 1988; Minghetti, 2005; Sherman et al., 1992). At such lesion sites, activated microglia release several types of inflammatory mediators (Liu and Hong, 2003). Among these mediators, prostaglandin E2 (PGE2) is of major importance for the initiation, propagation, and modulation of brain inflammation. Cyclooxygenase (COX)-2 expression in the brain is elevated in AD and ischemia (Bazan et al., 2002; Colangelo et al., 2002; Ho et al., 1999, 2001; Iadecola et al., 1999; Kitamura et al., 1999; Oka and Takashima, 1997; Pasinetti and Aisen, 1998; Yokota et al., 2003), and COX-2 protein content in the brain correlates with the severity of amyloidosis and clinical dementia (Ho et al., 2001). PGE2 is accumulated in the cerebrospinal fluid and/or at lesion sites in HIV-dementia and in probable AD patients (Griffin et al., 1994; Montine et al., 1999). However, the role of COX-2 in AD is still unclear. Although epidemiological data indicates that nonsteroidal anti-inflammatory drugs might have a therapeutic potential in the treatment of AD, most studies failed to show beneficial effects (Hoozemans and O’Banion, 2005). Agents reducing PGE2 synthesis exhibit neuroprotective activity in certain neuroinflammation models (Lim et al., 2000; Nakayama et al., 1998; Teismann et al., 2003). Thus, the characterization of COX activities and subsequent PG generation in the brain, as well as their potential roles in amyloidosis, is receiving a great deal of attention. This inflammatory response may damage neurons and exacerbate the pathological processes underlying the disease. In contrast, PGE2 has also been suggested to reveal anti-inflammatory properties. Exogenous PGE2 protects neuronal cell death induced by LPS through reduction of NO released by microglia and reactive oxygen species from neurons (Kim et al., 2002). Direct administration of PGE2 into the brain has also been shown to reduce

INTRODUCTION Localized inflammatory responses in the brain parenchyma have been associated with the pathogenesis and progression of numerous neurological disorders, including infection, ischemia, multiple sclerosis, Alzheimer’s disease, and Parkinson’s disease (Andersen, 2004; Craft C 2008 V

Wiley-Liss, Inc.

*Correspondence to: Bernd L. Fiebich, Ph.D., Department of Psychiatry, University of Freiburg Medical School, Hauptstr. 5, D - 79104 Freiburg, Germany. E-mail: bernd.fi[email protected] Received 7 November 2007; Accepted 28 January 2008 DOI 10.1002/glia.20658 Published online 27 March 2008 in Wiley InterScience (www.interscience.wiley. com).

EXPRESSION AND REGULATION OF MPGES-1 IN MICROGLIA

microglial activation and TNF-a expression in the brain parenchyma induced by intraperitoneal LPS injection (Zhang and Rivest, 2001). In the biosynthetic pathway leading to PGE2, arachidonic acid released from membrane phospholipids by cytosolic or secretory phospholipases is converted to PGH2 by COX-1 or COX-2 and is then isomerized to PGE2 by terminal prostaglandin E synthases (PGES). To date, three PGESs have been characterized, the microsomal PGESs (mPGES-1 and mPGES-2) and the cytosolic PGES (cPGES) (Jakobsson et al., 1999; Tanikawa et al., 2002; Tanioka et al., 2000; Watanabe et al., 1999). Among all PGESs, mPGES-1 has received the most attention, as this enzyme is induced by proinflammatory stimuli, downregulated by anti-inflammatory glucocorticoids, and functionally coupled with COX-2 in marked preference to COX-1 (Jakobsson et al., 1999; Mancini et al., 2001; Murakami et al., 2000). In comparison, cPGES (the heat shock protein-associated protein p23) is constitutively and ubiquitously expressed and is selectively coupled with COX-1 (Tanioka et al., 2000). Expression of mPGES-1 is postulated to be of major importance in various pathophysiological events in which COX-2-derived PGE2 plays a crucial role. Because of its strong preferential coupling with COX-2, mPGES-1 is probably the most important PGES in inflammation-associated PGE2 synthesis (Jakobsson et al., 1999; Mancini et al., 2001; Murakami et al., 2000). mPGES-1 has the most terminal position in the PGE2-synthesizing cascade, the highest magnitude of upregulation among all PGE2-synthesizing enzymes studied, and a long duration of mRNA expression. Because of the withdrawal from the market of most of the COX-2 inhibitors, specific inhibition of mPGES-1 is believed to be a potential therapeutic approach to prevent the synthesis of PGE2 but not of other prostanoids. Therefore, mPGES-1 is an attractive target for antiinflammatory therapy. The fact that mPGES-1 is strongly upregulated along with COX-2 makes mPGES1 an attractive pharmacological target. Data on PGES expression or regulation in the brain or in brain-derived cells are very limited up to now. A relatively recent study has demonstrated the induction of mPGES-1 mRNA by treatment of rat astrocytes with Ab (Satoh et al., 2000), suggesting that the Ab-induced elevation of PGE2 secretion may be regulated directly by

Abbreviations

AD COX cPGES IL JNK LPS MAPK mPGES NF-jB PI-3K PG PKC PPAR TLR TNF

Alzheimer’s disease cyclooxygenase cytosolic prostaglandin E synthase interleukin c-Jun N-terminal kinase lipopolysaccharide mitogen-activated protein kinase membrane-associated prostaglandin E synthase nuclear factor jB phosphatidylinositol-3-kinase prostaglandin protein kinase C peroxisome proliferator activated-receptor Toll-like receptor tumor necrosis factor.

845

mPGES-1. Interestingly, a recent report (Ikeda-Matsuo et al., 2005) has demonstrated that in neuron-glial mixed cultures, mPGES-1 was co-induced with COX-2 specifically in microglia, but not in astrocytes, oligodendrocytes or neurons. In microglia-enriched cultures, the induction of mPGES-1, the activity of PGES and the production of PGE2 were preceded by the induction of mPGES-1 mRNA. These results suggest that the activation of microglia contributes to PGE2 production through the concerted de novo synthesis of mPGES-1 and COX-2, and emphasize the importance of mPGES-1 in microglial PGE2 production. Only a few studies addressed the effects of drugs on mPGES-1 expression in microglia (Bernardo et al., 2003; Candelario-Jalil et al., 2007; Greco et al., 2003). However, a detailed and specific investigation of the signaling pathways that contribute to the regulation of mPGES-1 in microglia is still lacking. Considering the evidences showing that mPGES-1 and COX-2 are co-induced and work together in the production of PGE2 during inflammation, it is important to establish the signaling pathways that control their productions. Although many studies have delineated the pathways of COX-2 synthesis in activated microglia, less is known about the regulation of mPGES-1 in these cells. Although the main role of PGE2 in brain inflammation is still controversial, it is of significant importance to establish the regulation of the enzymes responsible for its production. In the present study, we show the induction of mPGES-1 after microglia activation and investigate the signaling pathways that control the production of this enzyme after inflammatory stimuli.

MATERIALS AND METHODS Materials SP600125 (Anthra(1,9-cd)pyrazol-6(2H)-one 1,9-Pyrazoloanthrone), an inhibitor of JNK; GF109203X (2-[1(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide; G€ o 6983 (2-[1-(3-dimethylaminopropyl)-5methoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide) and G€ o 6976 (12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole), inhibitors of PKC; SB 202190 (4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole), an inhibitor of p38 mitogen-activated protein kinase (MAPK); PD 98059 (20 Amino-30 -methoxyflavone), an inhibitor of p42/44 MAPK; were obtained from Calbiochem (Bad Soden, Germany). SC-514 (5-(Thien-3-yl)-3-aminothiophene-2-carboxamide), an IjB kinase 2 (IKK2) inhibitor, was purchased from Calbiochem (San Diego, CA) and LY294002 (2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride), a PI-3K inhibitor, was from Tocris (Ellisville, MO). Stock solutions (10–25 mM) were prepared in dimethyl sulfoxide (DMSO) and stored at 220°C. Further dilutions were carried out in DMSO. All compounds, used at the given concentrations, do not affect the viability of the cells as observed through the CellTiter-GloÒ luminescent cell viability assay kit (Promega, Mannheim, Germany), which measures metabolic ATP levels (data not shown). GLIA

846

DE

OLIVEIRA ET AL.

Cell Culture

Western Blot Analysis

Primary microglial cell cultures were established from cerebral cortices of one-day neonatal Wistar rats as previously described (Akundi et al., 2005; Seregi et al., 1984). Briefly, forebrains were minced and gently dissociated by repeated pipetting in Hank’s balanced salt solution. Cells were collected by centrifugation, resuspended in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum and antibiotics and cultured on 10-cm cell culture dishes (Falcon, 5 3 105 cells/plate) in 5% CO2 at 37°C. Medium was prepared taking extreme care to avoid lipopolysaccharide contamination (GebickeHaerter et al., 1989). Floating microglia were harvested from 14- to 30-day-old mixed (astrocyte-microglia) primary cultures and re-seeded into 35-mm cell culture dishes in fresh complete medium to give pure microglial cultures (2 3 104 cells/dish). Microglial cultures were washed 1 h after seeding to remove non-adherent cells. The purity of the microglial culture was >98% as previously determined by immunofluorescence and cytochemical analysis (Gebicke-Haerter et al., 1989).

After the respective experimental set up, microglial cells were washed with phosphate buffered saline (PBS) and lysed in 1.3 3 SDS (sodium dodecyl sulfate)containing sample buffer without DTT or bromophenol blue containing 100 lM orthovanadate (Laemmli, 1970). Lysates were homogenized by repeated passage through a 26-gauge needle. Protein contents were measured using the bicinchoninic acid (BCA) method (kit obtained from Pierce, distributed by KFC Chemikalien, M€ unchen, Germany). Bovine serum albumin (BSA) was used a protein standard at concentrations ranging from 0.2 to 4 lg/lL; and the optical density was read at 570 nm using a microplate reader. Before electrophoresis, bromophenol blue and DTT (final concentration, 10 mM) were added to the samples. For mPGES-1 and COX-2 immunoblotting, 30–50 lg of protein from each sample was subjected to SDS-PAGE (polyacrylamide gel electrophoresis) on a 15% gel under reducing conditions. Proteins were then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA) by semi-dry blotting. The membrane was blocked for 1 or 2 h at room temperature using Rotiblock (Roth, Karlsruhe, Germany) or 5% blocking milk (BioRad, M€ unchen, Germany), respectively, before the overnight incubation at 4°C with the primary antibody. Primary antibodies were goat anti-COX-2 (M-19, Santa Cruz, Heidelberg, Germany) diluted 1:500 in Tris-buffered saline (TBS) containing 0.1% Tween 20 (Merck, Darmstadt, Germany) and 1% bovine serum albumin (BSA, Sigma), rabbit anti-mPGES-1 (Cayman, 1:500), rabbit anti-actin (Sigma 1:5,000). After extensive washing (three times for 15 min each in TBS containing 0.1% Tween 20), proteins were detected with horseradish peroxidase (HRP)-coupled rabbit anti-goat IgG (Santa Cruz, 1:100,000) or HRP-coupled donkey anti-rabbit (GE Healthcare, Freiburg Germany, 1:25,000) using chemiluminescence (ECL) reagents (GE Healthcare). All Western blot experiments were carried out at least three times.

RNA Extraction and RT-PCR Analysis Total RNA was isolated using the guanidine isothiocyanate method (Chomczynski and Sacchi, 1987). Two microgram of total RNA was reverse transcribed using M-MLV reverse transcriptase and random hexamers (Promega, Mannheim, Germany). One microlitre of the resulting cDNA was amplified using Taq DNA polymerase (Promega), dNTPs (Invitek, Berlin, Germany) and primers specific for rat mPGES-1 (forward: 50 -ATG ACT TCC CTG GGT TTG GTG ATG GAG-30 , reverse: 50 -ACA GAT GGT GGG CCA CTT CCC AGA-30 , annealing temperature 65°C, 35 cycles, amplicon size: 459 bp); rat mPGES-2 (forward: 50 -ACT TCC ACT CCC TGC CCT AT-30 and reverse, 50 -CGG TAC ACG TTG GGA GAG AT-30 , annealing temperature 59°C, 35 cycles, amplicon size: 394 bp); rat cPGES (forward: 50 -ACC ATG CAG CCT GCT TCT GC-30 and reverse, 50 -CAT GAC TGG CCG GAT TCT CC-30 , annealing temperature 59°C, amplicon size: 240 bp); rat COX-2 (forward, 50 -TGC GAT GCT CTT CCG AGC TGT GCT-30 and reverse, 50 -TCA GGA AGT TCC TTA TTT CCT TTC-30 , annealing temperature 55°C, 35 cycles, amplicon size: 479 bp); rat b-actin (forward: 50 -ATG GAT GAC GAT ATC GCT-30 , reverse: 50 -ATG AGG TAG TCT GTC AGG T30 , 48°C, 30 cycles, amplicon size: 569 bp) or rat S12 (forward: 50 -ACG TCA ACA CTG CTC TAC A-30 , reverse: 50 CTT TGC CAT AGT CCT TAA C-30 , 56°C, 30 cycles, amplicon size: 312 bp), that were designed using the Primer Select software (DNA Star, Madison, WI) and synthesised through an in-house facility (Dr. Gabor Igloi, Institute for Biology III, Freiburg, Germany). All PCR amplifications included a final 10-min extension at 72°C. The products were analyzed on a 2% agarose gel. Contamination by genomic DNA was identified by substituting total RNA instead of cDNA in the reaction mixture using S12 or bactin primers. GLIA

ELISA Supernatants were harvested, centrifuged at 10,000g for 10 min and levels of prostaglandin E2 or PGD2 in the media were measured by enzyme immunoassay (EIA) (Biotrend, K€ oln, Germany and Cayman Chemicals, Ann Arbor, MI, respectively) according to the manufacturer’s instructions. Standards from 39 to 2,500 pg/mL were used; sensitivity of the assay was 36.2 pg/mL.

Statistical Analysis At least three independent experiments were used for data analysis. Original data were converted into %-values of LPS control and mean 6 S.E.M. were calculated.

EXPRESSION AND REGULATION OF MPGES-1 IN MICROGLIA

847

Fig. 1. mPGES-1 and COX-2 expression in microglial cells is induced by LPS at the mRNA (A-C) and at the protein (D-G) levels. (A) Microglial cells were stimulated with LPS (10 ng/mL) for different times (1, 2, 4, 8, 12, 24, 48, and 72 h; Lanes 2–9, respectively). Control cells were left untreated for 1 or 72 h (Lanes 1 and 10, respectively). (B and C) Microglial cells were stimulated with different doses of LPS

(0.1–100 ng/mL) for 4 h. (D and E) Microglial cells were stimulated with LPS (10 ng/mL) for different times (4–48 h) and the immunoreactivities for mPGES-1 and COX-2 were evaluated using western blotting. In panels F and G, microglial cells were stimulated with different doses of LPS (0.1–100 ng/mL) for 48 h. *P < 0.05 with respect to untreated control.

Values were compared using t-test (two groups) or oneway ANOVA with post-hoc Student-Newman-Keuls test (multiple comparisons).

mRNA expression started after 2 h of stimulation with LPS (10 ng/mL) and reached a peak between 24 and 48 h (Fig. 1A). COX-2 mRNA was induced earlier and remained constant until 48 h (Fig. 1A). The low dose of 0.1 ng/mL LPS induced mPGES-1 and COX-2 mRNA in rat primary microglia, maximal mRNA expression was achieved starting at 1 ng/mL (Fig. 1B,C). No alteration in the levels of mPGES-2 and cPGES was observed (Fig. 1B). mPGES-1 protein was observed after 24-h stimulation with LPS, with maximal levels at 48 h. In contrast to

RESULTS Induction of mPGES-1 and COX-2 by LPS in Primary Rat Microglia We first demonstrated that LPS induced mPGES-1 and COX-2 mRNA in primary rat microglia. mPGES-1

GLIA

848

DE

OLIVEIRA ET AL.

Fig. 2. Effect of PKC inhibitors, GF109203X, G€ o 6976, and G€ o 6983, on the expression of mPGES-1 and COX-2 in primary activated rat microglia. (A) Semi-quantitative analysis of the effect of GF109203X (10 (M) on mPGES-1 and COX-2 mRNA expression. (B) Quantitative densitometric analysis of mPGES-1 and COX-2 mRNA expression normalized to b-actin control. RT-PCR analysis was performed after 4 h of incubation with LPS. (C) Immunoblot analysis of protein levels of COX-2, mPGES-1 and b-actin in LPS-activated microglia treated with GF109203X (10 lM), G€ o 6976 (0.1 lM), and G€ o 6983 (1 lM). (D) Quantitative densitometric analysis of mPGES-1 and COX-2 protein expression normalized to b-actin loading control. *P < 0.05 with respect to LPS control.

the long-lasting accumulation of mPGES-1, COX-2 protein levels remained constant after 24 h of LPS stimulation (Fig. 1D,E). Significant increase in mPGES-1 and COX-2 immunoreactivity was obtained using 1 ng/mL LPS and peaking was found with 10–100 ng/mL (Fig. 1F,G). Therefore, the dose of 10 ng/mL of LPS was used in further experiments for studying the regulation of mPGES-1 and COX-2.

Regulation of mPGES-1 and COX-2 in Primary Activated Microglia Several inhibitors of second messenger pathways were tested for their ability to suppress LPS-induced mPGES1 expression. For all the experiments, mPGES-1 and COX-2 mRNA levels were evaluated at 4–24 h and protein immunoreactivity at 48 h, respectively. All the inhibitors were added 30 min before the stimulation with LPS. None of the inhibitors showed toxicity at the doses used as evaluated by the ability of the cells to produce ATP (CellTiter-GloÒ luminescent cell viability GLIA

assay kit (Promega, Mannheim, Germany, data not shown).

Effect of PKC Inhibitors on the Expression of mPGES-1 and COX-2 in LPS-Activated Rat Microglia We have previously shown that PKC is involved in the regulation of COX-2 in activated microglia (Akundi et al., 2005). To investigate whether comparable effects are observed on mPGES-1 expression, we studied the effects of different PKC inhibitors on mPGES-1 in LPS-treated rat microglia. The non-specific PKC inhibitor, GF203190X (10 lM), inhibited the up-regulation of mPGES-1 and COX2 mRNA after 4 h of stimulation with LPS (Fig. 2A,B). GF203109X also prevented LPS-induced mPGES-1 and COX-2 protein immunoreactivity (Fig. 2C,D). To address the role of the A-group (classical) PKC isotypes (a, b, and g) in mPGES-1 regulation, we used G€ o 6976 and G€ o 6983. A dramatic decrease in mPGES-1 and COX-2 immunoreactivity was observed after pre-treatment with G€ o 6976 (0.1 lM) and G€ o 6983 (1 lM),

EXPRESSION AND REGULATION OF MPGES-1 IN MICROGLIA

849

Fig. 3. Effect of a JNK inhibitor, SP600125, on the expression of mPGES-1 and COX-2 in primary activated rat microglia. (A) Semiquantitative analysis of the effect of SP600125 (10 lM) on mPGES-1 and COX-2 mRNA expression. (B) Quantitative densitometric analysis of mPGES-1 and COX-2 mRNA expression normalized to b-actin control. RT-PCR analysis was performed after 4 h of incubation with LPS.

(C) Immunoblot analysis of protein levels of COX-2, mPGES-1 and bactin in LPS-activated microglia treated with SP600125 (0.1–10 lM). (D) Quantitative densitometric analysis of mPGES-1 and COX-2 protein expression normalized to b-actin loading control. *P < 0.05 with respect to LPS control.

although these drugs did not abolish the expression of both enzymes (Fig. 2C,D). PKC blockade produced a similar degree of inhibition of both mPGES-1 and COX2 protein expression.

Effect of NF-jB Inhibition on the Expression of mPGES-1 and COX-2 in LPS-Activated Rat Microglia

Effect of the JNK Inhibitor SP600125 on the Expression of mPGES-1 and COX-2 in LPS-Activated Rat Microglia Many studies have already demonstrated an important role of MAPK cascades in the expression of inflammatory mediators in activated microglia (Akundi et al., 2005; Fiebich et al., 2002). To investigate whether inhibition of one member of these kinases, JNK, leads to the inhibition of mPGES-1, we evaluated the effect of SP600125 (0.1–10 lM) on the mPGES-1 expression. The highest dose of SP600125 was not able to reduce the up-regulation of mPGES-1 and COX-2 mRNA at 4 h of stimulation with LPS (Fig. 3A,B). However, mPGES-1 and COX-2 immunoreactivities were reduced by SP600125 (1–10 lM) after 48 h of LPS stimulation (Fig. 3C,D), suggesting that the effect of JNK on mPGES-1 and COX-2 might occur at a post-transcriptional level. Similar to what we described before for COX-2 (Akundi et al., 2005), inhibition of p38 and p42/ 44 MAPK by the respective inhibitors SB 202190 and PD 98059, resulted in a complete inhibition of mPGES-1 mRNA expression (data not shown).

Many transcription factors are involved in inflammatory processes. One of the most studied ones is the NF-jB, which is important in the up-regulation of many inflammatory mediators (Schmitz et al., 2001). To investigate the effect of NF-jB inhibition on mPGES-1 and COX-2 in LPS-stimulated primary microglia, we used the selective IKK2 inhibitor SC514. At the highest dose (25 lM), SC514 did not significantly reduce LPS-induced mPGES1 and COX-2 mRNA expression (Fig. 4A,B). This was the highest non-toxic dose for the cells, as estimated by the ATP cell viability assay (data not shown). However, COX2 protein synthesis was reduced to
60% of the nontreated cells with the doses of 10 and 25 lM (Fig. 4C,D), and the immunoreactivity for mPGES-1 was almost abolished with the highest dose (25 lM, Fig. 4C,D). This is the first evidence that the inhibition of NF-jB signaling by direct inhibition of IKK2 leads to a dramatic down-regulation of mPGES-1 protein in activated rat microglia. This effect may be due to a delayed effect, since the NF-jB inhibitor did not significantly reduced mPGES-1 and COX-2 mRNA expression at 4 h (Fig. 4A,B). Interestingly, the lowest dose of SC514 (1 lM) reduces LPS-induced PGE2 release after 24 and 48 h of stimulation, and the production of this prostanoid is abolished at 10 and 25 lM (Fig. 4E). The production of PGD2 was also reduced to basal concentrations (Fig. 4F). GLIA

850

DE

OLIVEIRA ET AL.

Fig. 4. Effect of SC514, an IKK2 inhibitor, on the expression of mPGES-1 and COX-2, and on the PGE2 and PGD2 levels in primary activated rat microglia. (A) Semi-quantitative analysis of the effect of SC514 (25 lM) on mPGES-1 and COX-2 mRNA expression. (B) Quantitative densitometric analysis of mPGES-1 and COX-2 mRNA expression normalized to b-actin control. RT-PCR analysis was performed after 4 h of incubation with LPS. (C) Immunoblot analysis of protein levels of

COX-2, mPGES-1 and b-actin in LPS-activated microglia treated with SC514 (1–25 lM). (D) Quantitative densitometric analysis of mPGES-1 and COX-2 protein expression normalized to b-actin loading control. Panels E and F: Effect of SC514 (1–25 lM) on PGE2 and PGD2 production after 24 and 48 h of LPS stimulation in rat primary microglia. *P < 0.05 with respect to LPS control.

Effect of PI-3K Inhibition on the Expression of mPGES-1 and COX-2 in LPS-Activated Rat Microglia

increasing the levels of other COX-2-dependent prostanoids. To confirm this hypothesis, we measured the levels of PGE2 and PGD2 after LPS treatment in presence of LY294002. As shown in Figure 5C,D, LPS-induced both prostaglandins, addition of 25 lM LY294002 led to an inhibition of PGE2 with a parallel increase in PGD2 after 24 h of LPS treatment. After 48 h of stimulation with LPS, incubation with LY294002 (10 and 25 lM) led to an increase in PGE2 and PGD2 content (Fig. 5C,D), suggesting a production of PGE2 by the other PGESs.

To further investigate the signaling pathways of mPGES-1 and COX-2 induction in primary activated rat microglia, we used the well-established PI-3K inhibitor, LY294002. The highest dose (25 lM) of LY294002 reduced mPGES-1 (4–24 h) induced by LPS (1 ng/ml), but increased the COX-2 mRNA expression (8–24 h, data not shown). This dose of LY294002 also completely abolished LPS-induced immunoreactivity of mPGES-1, evaluated 48 h after LPS stimulation. However, the same dose led to an increase in COX-2 synthesis (Fig. 5A,B). Inhibition of mPGES-1 expression with increased COX-2 levels as shown for the inhibition of PI-3K, suggests a decrease of PGE2 without affecting or even GLIA

DISCUSSION In the present study, we investigated the regulation of mPGES-1 and COX-2 in activated microglia. We demonstrated here the involvement of JNK, PKC, NF-jB, p38

EXPRESSION AND REGULATION OF MPGES-1 IN MICROGLIA

851

Fig. 5. Effect of a PI-3K inhibitor, LY294002, on the expression of mPGES-1 and COX-2, and on the PGE2 and PGD2 levels in primary activated rat microglia. (A) Immunoblot analysis of protein levels of COX-2, mPGES-1 and b-actin in LPS-activated microglia treated with LY294002 (1–25 lM). (B) Quantitative densitometric analysis of

mPGES-1 and COX-2 protein expression normalized to b-actin loading control. (C and D) Effect of LY294002 (1–25 lM) on PGE2 and PGD2 levels after 24 and 48 h of LPS stimulation in rat primary microglia. *P < 0.05 with respect to LPS control.

and p42/44 MAPKs in the regulation of LPS-induced mPGES-1 in microglia. These pathways have also been demonstrated to be involved in COX-2 expression (Akundi et al., 2005; Bauer et al., 1997). Therefore, these findings are in line with other studies reporting a coupled regulation of mPGES-1 and COX-2 (Catley et al., 2003; Pham et al., 2006). However, in contrast to these reports, we provide here evidence of an uncoupled regulation of mPGES-1 and COX-2 in microglia. Inhibition of PI-3K with LY294002 led to an inhibition of mPGES-1 but increased COX-2, challenging the wellaccepted regulatory coupling of the two enzymes. A great deal of attention has been given to mPGES-1 due to its end-terminal position in the cascade to produce PGE2 during inflammatory processes. Recently, it has been demonstrated that mPGES-1 knockout mice showed reduced infarction, edema and apoptotic cell death after cerebral ischemia as compared with the wild-type mice (Ikeda-Matsuo et al., 2006). In our study, LPS strongly induced mPGES-1 in primary rat microglia (see Fig. 1), confirming data from previous studies (Ajmone-Cat et al., 2003; Bernardo et al., 2003; Candelario-Jalil et al., 2007; Greco et al., 2003; IkedaMatsuo et al., 2005; Zhang et al., 2006). Few attempts have been done in order to elucidate the regulation of mPGES-1 and its activity in microglia. It has been

demonstrated that paracetamol has no effect on its activity and expression (Greco et al., 2003), and that, 15d-PGJ2 increased expression of both mPGES-1 and COX-2 (Bernardo et al., 2003). Interestingly, PGE2 is reduced due to an inhibitory effect of 15d-PGJ2 on COX-2 activity (Bernardo et al., 2003). In activated microglia, dexamethasone also potently downregulated mPGES-1 (Ikeda-Matsuo et al., 2005). However, there are no reports addressing signal transduction pathways involved in the regulation of mPGES-1 in primary microglial cells. We first evaluated the effect of PKC inhibition on the regulation of LPS-induced mPGES-1 and COX-2 (see Fig. 2). PKC has been associated with the production of NO (Fiebich et al., 1998; McMillian et al., 1997) and COX-2 (Akundi et al., 2005; Bauer et al., 1997) in activated rat microglia. Here, we present the first evidence that inhibition of PKC leads to decreased expression of mPGES-1 in activated rat microglia. The specific PKC isoform(s) involved in mPGES-1 expression is presently unknown. Our current data support the notion that PKC mediates the concerted induction of mPGES-1 and COX-2 in activated microglia (Akundi et al., 2005). Recently, the regulation of mPGES-1 by PKC has also been demonstrated in different peripheral cells (Kawao et al., 2005; Pham et al., 2006; Subbaramaiah et al., GLIA

852

DE

OLIVEIRA ET AL.

2004). However, the role of PKC in mPGES-1 expression is still controversial, since not all studies showed that inhibition of PKC results in mPGES-1 downregulation (Yucel-Lindberg et al., 2006). MAPKs have been strongly implicated in inflammatory processes. For example, ERK and p38 MAPK cascades are important in the regulation of COX-2 expression in activated microglia (Akundi et al., 2005; Egger et al., 2003). In the present study, we demonstrate that ERK and p38 MAPK are also involved in mPGES-1 induction (data not shown). We further demonstrated a common involvement of JNK in the regulation of both mPGES-1 and COX-2 (see Fig. 3). It has been demonstrated that LPS induces phosphorylation of the JNK isoforms in microglia and that inhibition of JNKs reduced the LPS induction of the AP-1 target genes like COX-2 (Waetzig et al., 2005). We observed down-regulation of COX-2 by JNK inhibition, which is in line with results from a recent report (Yang et al., 2006). Furthermore, we showed that JNK inhibition down-regulates mPGES-1 protein. However, this downregulation of mPGES-1 by SP600125 was not seen at the mRNA level. Although we did not perform experiments to further elucidate the different effects of SP600125 at the mRNA and protein levels of mPGES-1 and COX-2, involvement of mRNA stabilization of both enzymes is likely, as previously demonstrated (Degousee et al., 2006; Nieminen et al., 2005, 2006). In addition to regulate mPGES-1 mRNA stability, JNK enzymes may also participate in the regulation of mPGES-1 mRNA translation and/or the regulation of mPGES-1 protein stability, as JNK enzymes have been shown to stabilize many proteins (Kim et al., 2002; Pages et al., 2000; Toh et al., 2004). The transcription factor NF-jB is among the most well-known transcription factors that regulate the expression of inflammatory mediators. Importantly, inhibition of NF-jB activation suppresses LPS-induced COX-2 protein expression and prostanoids generation (D’Acquisto et al., 1997). Blockade of NF-jB signaling results in decreased mPGES-1 expression in peripheral cells exposed to pro-inflammatory stimuli (G
omezHern
andez et al., 2006; Moon et al., 2005). Here we showed that inhibition of IKK2 by SC514 leads to a drastic inhibition of mPGES-1 and COX-2 expression, and PGE2 and PGD2 production in LPS-activated rat microglia (Fig. 4C–F). By using a selective IKK2 inhibitor, we provide for the first time evidence of the involvement of IKK2 in mPGES-1 expression induced by LPS. We found that SC514 was not as effective on COX-2 as it was on mPGES-1, suggesting a possible different contribution in the regulation of the two enzymes. In the present study, the inhibitors of JNK, PKC, IKK2, p38, and p42/44 MAPKs, inhibited the expression of both mPGES-1 and COX-2, indicating a coupling between these two enzymes in relation to some signaling pathways in the rat activated microglia. Although we observed a similar regulation of mPGES-1 and COX-2 after inhibition of some signaling pathways, this was not observed after inhibition of PI-3K. GLIA

PI-3K has been linked to different pathological conditions and can be activated by different stimuli including LPS in different cells (Fukao and Koyasu, 2003). Pharmacological blockade of PI-3K, e.g. using LY294002, suppresses the progression of inflammation in a mouse model of rheumatoid arthritis (Camps et al., 2005). However, its role in inflammation is still controversial, since its inhibition can lead to increase in some inflammatory mediators (Aksoy et al., 2005). Here we demonstrated that the PI-3K inhibitor LY294002 inhibited the mPGES-1 mRNA and protein expression but increased COX-2 mRNA and protein (Fig. 5A,B). A specific inhibition of mPGES-1 with an increased COX-2 expression, as described here by inhibiting PI-3K, would lead to a reduction in PGE2 synthesis without an alteration or even with an augmentation of other prostanoids. Our data demonstrating that the PI3K inhibitor LY294002 prevented PGE2 synthesis after 24 h of LPS-stimulation, but increased PGD2 release (Fig. 5C,D), supports the specific direct involvement of PI-3K in mPGES-1 down-regulation but not of COX-2. After 48-h stimulation, mPGES-1 was abolished and COX-2 was up-regulated by LY294002. At this time point, the pattern of PGD2 and PGE2 production differed from that observed at 24 h of treatment. PGD2 content was increased by LY294002 at the doses of 10 and 25 lM, and PGE2 was increased with 10 lM of LY294002. This result is in accordance with the protein data, since the dose of 10 lM of LY294002 induced a slight increase in COX-2 immunoreactivity, but had no effect on mPGES-1 protein levels (Fig. 5A,B). Because of the fact that PGE2 is not reduced by 25 lM of LY294002, even when mPGES-1 is downregulated, it is possible that other PGES isoforms such as cPGES and mPGES-2 are responsible for the production of PGE2, even when these isoforms are not upregulated by LPS (Fig. 1B). The increased COX-2 expression and activity would lead to increased availability of PGH2, the substrate for all PGESs. Another interesting finding was the induction of PGD2, but not PGE2, by LY294002, in the absence of LPS (Fig. 5D). This effect might be due to an upregulation of COX-2 and/or an increase in PGD synthase. Similar to our results, Monick et al. (2002) reported that blockade of PI-3K with LY294002 leads to an increase in COX-2 expression and PGE2 release in human alveolar macrophages stimulated with LPS. The authors suggest that PI-3K inhibition may enhance p38 MAPK activation and COX-2 mRNA stability. However, this is not a plausible explanation to our results, since p38 MAPK seems also to participate in the regulation of mPGES-1. An enhancement of p38 MAPK phosphorylation leads to an upregulation in mPGES-1 mRNA (data not shown). Recently, Bage ˚ et al. (2007) demonstrated that PGE2 production is not reduced by silencing mPGES-1 in IL1b or TNF-a-stimulated gingival fibroblasts. To explain the lack of reduction on PGE2 production in the presence of mPGES-1 siRNA, the authors speculate that COX-2 might be the rate-limiting enzyme responsible for the production of PGE2, as suggested also by others (Degousee et al., 2003; Mancini et al., 2007; Sevigny

EXPRESSION AND REGULATION OF MPGES-1 IN MICROGLIA

et al., 2006). More important, the authors also demonstrated that MK-886, a compound reported to decrease mPGES-1 activity, reduces the expression of mPGES-1 but upregulates COX-2 (Bage ˚ et al., 2007). We recently demonstrated that resveratrol inhibits PGE2 release and mPGES-1 expression, but not expression of COX-2 in LPS-stimulated primary microglia. (Candelario-Jalil et al., 2007). In contrast to the effects of LY294002 as shown here, resveratrol does not increase COX-2 expression, but inhibits COX activity and the production of free radicals, which in turn, contribute to reduced PGE2 production. Other authors also studied the effect of PI-3K blockade on COX-2 expression. Recently, Choi et al. (2006) showed that LY294002, but not wortmannin, another PI-3K inhibitor, increased COX-2 mRNA expression by IL-1b or TNF-a in human monocytic THP-1 cells. In addition, LY294002, but not wortmannin, also elevated PGE2 production induced by IL-1b. It has been suggested by the authors, that pharmacological inhibition of PI-3K enhances the NF-jB-activating pathways by IL1b through augmentation of IKK activation. Only little is known about the role of PI-3K in microglia activation. Jang et al. (2005) have demonstrated that LY294002 inhibits the phosphorylation of Akt and p70S6K, and also COX-2 induced by catalase in BV2 microglial cells. However, the different cells and stimuli used may explain the difference to the results that we obtained. It has been shown that LY294002 inhibits the production of nitric oxide production in LPS stimulated rat primary microglia and down-regulates IL-6 and TNF-a in BV2 microglial cells (Kim et al., 2004). The principal role of PI-3K inhibition in microglia activation can not be addressed considering these first results, since LY294002 can induce both PGE2 and PGD2 at later time points. Although PGE2 is believed to have a predominantly deleterious effect, the role of PGD2 is also a topic of debate. Activation of PGD2 receptors is believed to induce pro-inflammatory effects (Bate et al., 2006). However, it is known that PGD2 is rapidly converted to a number of metabolites including 15d-PGJ2 (Straus and Glass, 2001), a cyclopentanone PG that is an agonist of PPARg, which anti-inflammatory properties has been widely demonstrated. The increase in PGD2 could reduce or exacerbate the inflammatory conditions. The protective or deleterious effect of PI-3K inhibition in neuroinflammation must be addressed in future studies. The major conclusion of this investigation is that the regulation of mPGES-1 and COX-2 are not strictly coupled to each other. We provide evidence that inhibition of mPGES-1 might not be enough to reduce PGE2 synthesis if the expression of COX-2 is increased, as it is the case in activated microglia after PI-3K inhibition.

ACKNOWLEDGMENTS The skilful technical assistance of Ulrike G€ otzingerBerger is greatfully acknowledged. Antonio Carlos Pinheiro de Oliveira was supported by the CAPES Founda-

853

tion (Bras
ılia, Brazil). Eduardo Candelario-Jalil was supported by a research fellowship from the Alexander von Humboldt Foundation (Bonn, Germany). All authors read and approved the final manuscript.

REFERENCES Ajmone-Cat MA, Nicolini A, Minghetti L. 2003. Prolonged exposure of microglia to lipopolysaccharide modifies the intracellular signaling pathways and selectively promotes prostaglandin E2 synthesis. J Neurochem 87:1193–203. Aksoy E, Vanden Berghe W, Detienne S, Amraoui Z, Fitzgerald KA, Haegeman G, Goldman M, Willems F. 2005. Inhibition of phosphoinositide 3-kinase enhances TRIF-dependent NF-kappa B activation and IFN-beta synthesis downstream of Toll-like receptor 3 and 4. Eur J Immunol 35:2200–2209. Akundi RS, Candelario-Jalil E, Hess S, Hull M, Lieb K, GebickeHaerter PJ, Fiebich BL. 2005. Signal transduction pathways regulating cyclooxygenase-2 in lipopolysaccharide-activated primary rat microglia. Glia 51:199–208. Andersen JK. 2004. Oxidative stress in neurodegeneration: Cause or consequence? Nat Med 10 (Suppl):S18–S25. Bage T, Modeer T, Kawakami T, Quezada HC, Yucel-Lindberg T. 2007. Regulation of prostaglandin E synthases: Effects of siRNA-mediated inhibition of microsomal prostaglandin E synthase-1. Biochim Biophys Acta 1773:1589–1598. Bate C, Kempster S, Williams A. 2006. Prostaglandin D2 mediates neuronal damage by amyloid-beta or prions which activates microglial cells. Neuropharmacology 50:229–237. Bauer MK, Lieb K, Schulze-Osthoff K, Berger M, Gebicke-Haerter PJ, Bauer J, Fiebich BL. 1997. Expression and regulation of cyclooxygenase-2 in rat microglia. Eur J Biochem 243:726–731. Bazan NG, Colangelo V, Lukiw WJ. 2002. Prostaglandins and other lipid mediators in Alzheimer’s disease. Prostaglandins Other Lipid Mediat 68/69:197–210. Bernardo A, Ajmone-Cat MA, Levi G, Minghetti L. 2003. 15-deoxydelta12,14-prostaglandin J2 regulates the functional state and the survival of microglial cells through multiple molecular mechanisms. J Neurochem 87:742–51. Camps M, Ruckle T, Ji H, Ardissone V, Rintelen F, Shaw J, Ferrandi C, Chabert C, Gillieron C, Francon B, Martin T, Gretener D, Perrin D, Leroy D, Vitte PA, Hirsch E, Wymann MP, Cirillo R, Schwarz MK, Rommel C. 2005. Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis. Nat Med 11:936–943. Candelario-Jalil E, de Oliveira AC, Graf S, Bhatia HS, Hull M, Munoz E, Fiebich BL. 2007. Resveratrol potently reduces prostaglandin E2 production and free radical formation in lipopolysaccharide-activated primary rat microglia. J Neuroinflammation 4:25. Catley MC, Chivers JE, Cambridge LM, Holden N, Slater DM, Staples KJ, Bergmann MW, Loser P, Barnes PJ, Newton R. 2003. IL-1betadependent activation of NF-kappaB mediates PGE2 release via the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase. FEBS Lett 547:75–79. Choi EK, Jang HC, Kim JH, Kim HJ, Kang HC, Paek YW, Lee HC, Lee SH, Oh WM, Kang IC. 2006. Enhancement of cytokine-mediated NFkappaB activation by phosphatidylinositol 3-kinase inhibitors in monocytic cells. Int Immunopharmacol 6:908–915. Chomczynski P, Sacchi N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159. Colangelo V, Schurr J, Ball MJ, Pelaez RP, Bazan NG, Lukiw WJ. 2002. Gene expression profiling of 12633 genes in Alzheimer hippocampal CA1: Transcription and neurotrophic factor down-regulation and up-regulation of apoptotic and pro-inflammatory signaling. J Neurosci Res 70:462–473. Craft JM, Watterson DM, Van Eldik LJ. 2005. Neuroinflammation: A potential therapeutic target. Expert Opin Ther Targets 9:887–900. D’Acquisto F, Iuvone T, Rombola L, Sautebin L, Di Rosa M, Carnuccio R. 1997. Involvement of NF-kappaB in the regulation of cyclooxygenase-2 protein expression in LPS-stimulated J774 macrophages. FEBS Lett 418:175–178. Degousee N, Angoulvant D, Fazel S, Stefanski E, Saha S, Iliescu K, Lindsay TF, Fish JE, Marsden PA, Li RK, et al. 2006. c-Jun N-terminal kinase-mediated stabilization of microsomal prostaglandin E2 synthase-1 mRNA regulates delayed microsomal prostaglandin E2 synthase-1 expression and prostaglandin E2 biosynthesis by cardiomyocytes. J Biol Chem 281:16443–16452.

GLIA

854

DE

OLIVEIRA ET AL.

Degousee N, Martindale J, Stefanski E, Cieslak M, Lindsay TF, Fish JE, Marsden PA, Thuerauf DJ, Glembotski CC, Rubin BB. 2003. MAP kinase kinase 6-p38 MAP kinase signaling cascade regulates cyclooxygenase-2 expression in cardiac myocytes in vitro and in vivo. Circ Res 92:757–764. Dickson DW, Lee SC, Mattiace LA, Yen SH, Brosnan C. 1993. Microglia and cytokines in neurological disease, with special reference to AIDS and Alzheimer’s disease. Glia 7:75–83. Egger T, Schuligoi R, Wintersperger A, Amann R, Malle E, Sattler W. 2003. Vitamin E (alpha-tocopherol) attenuates cyclo-oxygenase 2 transcription and synthesis in immortalized murine BV-2 microglia. Biochem J 370:459–467. Fiebich BL, Butcher RD, Gebicke-Haerter PJ. 1998. Protein kinase Cmediated regulation of inducible nitric oxide synthase expression in cultured microglial cells. J Neuroimmunol 92:170–178. Fiebich BL, Lieb K, Engels S, Heinrich M. 2002. Inhibition of LPSinduced p42/44 MAP kinase activation and iNOS/NO synthesis by parthenolide in rat primary microglial cells. J Neuroimmunol 132: 18–24. Fukao T, Koyasu S. 2003. PI3K and negative regulation of TLR signaling. Trends Immunol 24:358–363. Gebicke-Haerter PJ, Bauer J, Schobert A, Northoff H. 1989. Lipopolysaccharide-free conditions in primary astrocyte cultures allow growth and isolation of microglial cells. J Neurosci 9:183–194. Gomez-Hernandez A, Martin-Ventura JL, Sanchez-Galan E, Vidal C, Ortego M, Blanco-Colio LM, Ortega L, Tunon J, Egido J. 2006. Overexpression of COX-2, Prostaglandin E synthase-1 and prostaglandin E receptors in blood mononuclear cells and plaque of patients with carotid atherosclerosis: Regulation by nuclear factor-kappaB. Atherosclerosis 187:139–149. Greco A, Ajmone-Cat MA, Nicolini A, Sciulli MG, Minghetti L. 2003. Paracetamol effectively reduces prostaglandin E2 synthesis in brain macrophages by inhibiting enzymatic activity of cyclooxygenase but not phospholipase and prostaglandin E synthase. J Neurosci Res 71:844–852. Griffin DE, Wesselingh SL, McArthur JC. 1994. Elevated central nervous system prostaglandins in human immunodeficiency virus-associated dementia. Ann Neurol 35:592–597. Ho L, Pieroni C, Winger D, Purohit DP, Aisen PS, Pasinetti GM. 1999. Regional distribution of cyclooxygenase-2 in the hippocampal formation in Alzheimer’s disease. J Neurosci Res 57:295–303. Ho L, Purohit D, Haroutunian V, Luterman JD, Willis F, Naslund J, Buxbaum JD, Mohs RC, Aisen PS, Pasinetti GM. 2001. Neuronal cyclooxygenase 2 expression in the hippocampal formation as a function of the clinical progression of Alzheimer disease. Arch Neurol 58:487–492. Hoozemans JJ, O’Banion MK. 2005. The role of COX-1 and COX-2 in Alzheimer’s disease pathology and the therapeutic potentials of nonsteroidal anti-inflammatory drugs. Curr Drug Targets CNS Neurol Disord 4:307–315. Hoozemans JJ, Veerhuis R, Rozemuller JM, Eikelenboom P. 2006. Neuroinflammation and regeneration in the early stages of Alzheimer’s disease pathology. Int J Dev Neurosci 24:157–165. Iadecola C, Forster C, Nogawa S, Clark HB, Ross ME. 1999. Cyclooxygenase-2 immunoreactivity in the human brain following cerebral ischemia. Acta Neuropathol (Berl) 98:9–14. Ikeda-Matsuo Y, Ikegaya Y, Matsuki N, Uematsu S, Akira S, Sasaki Y. 2005. Microglia-specific expression of microsomal prostaglandin E2 synthase-1 contributes to lipopolysaccharide-induced prostaglandin E2 production. J Neurochem 94:1546–1558. Ikeda-Matsuo Y, Ota A, Fukada T, Uematsu S, Akira S, Sasaki Y. 2006. Microsomal prostaglandin E synthase-1 is a critical factor of strokereperfusion injury. Proc Natl Acad Sci USA 103:11790–11795. Jakobsson PJ, Thoren S, Morgenstern R, Samuelsson B. 1999. Identification of human prostaglandin E synthase: A microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target. Proc Natl Acad Sci USA 96:7220–7225. Jang BC, Paik JH, Kim SP, Shin DH, Song DK, Park JG, Suh MH, Park JW, Suh SI. 2005. Catalase induced expression of inflammatory mediators via activation of NF-kappaB, PI3K/AKT, p70S6K, and JNKs in BV2 microglia. Cell Signal 17:625–633. Kawao N, Nagataki M, Nagasawa K, Kubo S, Cushing K, Wada T, Sekiguchi F, Ichida S, Hollenberg MD, MacNaughton WK, et al. 2005. Signal transduction for proteinase-activated receptor-2-triggered prostaglandin E2 formation in human lung epithelial cells. J Pharmacol Exp Ther 315:576–589. Kim EJ, Kwon KJ, Park JY, Lee SH, Moon CH, Baik EJ. 2002. Neuroprotective effects of prostaglandin E2 or cAMP against microglial and neuronal free radical mediated toxicity associated with inflammation. J Neurosci Res 70:97–107. Kim GY, Mercer SE, Ewton DZ, Yan Z, Jin K, Friedman E. 2002. The stress-activated protein kinases p38 alpha and JNK1 stabilize p21(Cip1) by phosphorylation. J Biol Chem 277:29792–29802.

GLIA

Kim WK, Hwang SY, Oh ES, Piao HZ, Kim KW, Han IO. 2004. TGFbeta1 represses activation and resultant death of microglia via inhibition of phosphatidylinositol 3-kinase activity. J Immunol 172:7015– 7023. Kitamura Y, Shimohama S, Koike H, Kakimura J, Matsuoka Y, Nomura Y, Gebicke-Haerter PJ, Taniguchi T. 1999. Increased expression of cyclooxygenases and peroxisome proliferator-activated receptor-gamma in Alzheimer’s disease brains. Biochem Biophys Res Commun 254:582–586. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. Lim GP, Yang F, Chu T, Chen P, Beech W, Teter B, Tran T, Ubeda O, Ashe KH, Frautschy SA, et al. 2000. Ibuprofen suppresses plaque pathology and inflammation in a mouse model for Alzheimer’s disease. J Neurosci 20:5709–5714. Liu B, Hong JS. 2003. Role of microglia in inflammation-mediated neurodegenerative diseases: Mechanisms and strategies for therapeutic intervention. J Pharmacol Exp Ther 304:1–7. Mancini JA, Blood K, Guay J, Gordon R, Claveau D, Chan CC, Riendeau D. 2001. Cloning, expression, and up-regulation of inducible rat prostaglandin e synthase during lipopolysaccharide-induced pyresis and adjuvant-induced arthritis. J Biol Chem 276:4469–4475. Mancini A, Jovanovic DV, He QW, Di Battista JA. 2007. Site-specific proteolysis of cyclooxygenase-2: A putative step in inflammatory prostaglandin E(2) biosynthesis. J Cell Biochem 101:425–441. McGeer PL, Itagaki S, Boyes BE, McGeer EG. 1988. Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson’s and Alzheimer’s disease brains. Neurology 38:1285–1291. McMillian MK, Vainio PJ, Tuominen RK. 1997. Role of protein kinase C in microglia-induced neurotoxicity in mesencephalic cultures. J Neuropathol Exp Neurol 56:301–307. Minghetti L. 2005. Role of inflammation in neurodegenerative diseases. Curr Opin Neurol 18:315–321. Monick MM, Robeff PK, Butler NS, Flaherty DM, Carter AB, Peterson MW, Hunninghake GW. 2002. Phosphatidylinositol 3-kinase activity negatively regulates stability of cyclooxygenase 2 mRNA. J Biol Chem 277:32992–33000. Montine TJ, Sidell KR, Crews BC, Markesbery WR, Marnett LJ, Roberts LJ II, Morrow JD. 1999. Elevated CSF prostaglandin E2 levels in patients with probable AD. Neurology 53:1495–1498. Moon Y, Glasgow WC, Eling TE. 2005. Curcumin suppresses interleukin 1beta-mediated microsomal prostaglandin E synthase 1 by altering early growth response gene 1 and other signaling pathways. J Pharmacol Exp Ther 315:788–795. Murakami M, Naraba H, Tanioka T, Semmyo N, Nakatani Y, Kojima F, Ikeda T, Fueki M, Ueno A, Oh S, et al. 2000. Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2. J Biol Chem 275:32783–32792. Nakayama M, Uchimura K, Zhu RL, Nagayama T, Rose ME, Stetler RA, Isakson PC, Chen J, Graham SH. 1998. Cyclooxygenase-2 inhibition prevents delayed death of CA1 hippocampal neurons following global ischemia. Proc Natl Acad Sci USA 95:10954–10959. Nieminen R, Lahti A, Jalonen U, Kankaanranta H, Moilanen E. 2006. JNK inhibitor SP600125 reduces COX-2 expression by attenuating mRNA in activated murine J774 macrophages. Int Immunopharmacol 6:987–996. Nieminen R, Leinonen S, Lahti A, Vuolteenaho K, Jalonen U, Kankaanranta H, Goldring MB, Moilanen E. 2005. Inhibitors of mitogen-activated protein kinases downregulate COX-2 expression in human chondrocytes. Mediators Inflamm 2005:249–255. Oka A, Takashima S. 1997. Induction of cyclo-oxygenase 2 in brains of patients with Down’s syndrome and dementia of Alzheimer type: Specific localization in affected neurones and axons. Neuroreport 8:1161–1164. Pages G, Berra E, Milanini J, Levy AP, Pouyssegur J. 2000. Stress-activated protein kinases (JNK and p38/HOG) are essential for vascular endothelial growth factor mRNA stability. J Biol Chem 275:26484– 26491. Pasinetti GM, Aisen PS. 1998. Cyclooxygenase-2 expression is increased in frontal cortex of Alzheimer’s disease brain. Neuroscience 87:319–324. Pham H, Shafer LM, Slice LW. 2006. CREB-dependent cyclooxygenase2 and microsomal prostaglandin E synthase-1 expression is mediated by protein kinase C, calcium. J Cell Biochem 98:1653–1666. Satoh K, Nagano Y, Shimomura C, Suzuki N, Saeki Y, Yokota H. 2000. Expression of prostaglandin E synthase mRNA is induced in betaamyloid treated rat astrocytes. Neurosci Lett 283:221–223. Schmitz ML, Bacher S, Kracht M. 2001. I kappa B-independent control of NF-kappa B activity by modulatory phosphorylations. Trends Biochem Sci 26:186–190. Seregi A, Keller M, Jackisch R, Hertting G. 1984. Comparison of the prostanoid synthesizing capacity in homogenates from primary neuronal and astroglial cell cultures. Biochem Pharmacol 33:3315–3318.

EXPRESSION AND REGULATION OF MPGES-1 IN MICROGLIA Sevigny MB, Li CF, Alas M, Hughes-Fulford M. 2006. Glycosylation regulates turnover of cyclooxygenase-2. FEBS Lett 580:6533–6. Sherman MP, Griscavage JM, Ignarro LJ. 1992. Nitric oxide-mediated neuronal injury in multiple sclerosis. Med Hypotheses 39:143–146. Straus DS, Glass CK. 2001. Cyclopentenone prostaglandins: New insights on biological activities and cellular targets. Med Res Rev 21:185–210. Subbaramaiah K, Yoshimatsu K, Scherl E, Das KM, Glazier KD, Golijanin D, Soslow RA, Tanabe T, Naraba H, Dannenberg AJ. 2004. Microsomal prostaglandin E synthase-1 is overexpressed in inflammatory bowel disease. Evidence for involvement of the transcription factor Egr-1. J Biol Chem 279:12647–12658. Tanikawa N, Ohmiya Y, Ohkubo H, Hashimoto K, Kangawa K, Kojima M, Ito S, Watanabe K. 2002. Identification and characterization of a novel type of membrane-associated prostaglandin E synthase. Biochem Biophys Res Commun 291:884–889. Tanioka T, Nakatani Y, Semmyo N, Murakami M, Kudo I. 2000. Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis. J Biol Chem 275:32775–32782. Teismann P, Tieu K, Choi DK, Wu DC, Naini A, Hunot S, Vila M, Jackson-Lewis V, Przedborski S. 2003. Cyclooxygenase-2 is instrumental in Parkinson’s disease neurodegeneration. Proc Natl Acad Sci USA 100:5473–5478. Toh WH, Siddique MM, Boominathan L, Lin KW, Sabapathy K. 2004. c-Jun regulates the stability and activity of the p53 homologue, p73. J Biol Chem 279:44713–44722.

855

Waetzig V, Czeloth K, Hidding U, Mielke K, Kanzow M, Brecht S, Goetz M, Lucius R, Herdegen T, Hanisch UK. 2005. c-Jun N-terminal kinases (JNKs) mediate pro-inflammatory actions of microglia. Glia 50:235–246. Watanabe K, Kurihara K, Suzuki T. 1999. Purification and characterization of membrane-bound prostaglandin E synthase from bovine heart. Biochim Biophys Acta 1439:406–414. Yang MS, Ji KA, Jeon SB, Jin BK, Kim SU, Jou I, Joe E. 2006. Interleukin-13 enhances cyclooxygenase-2 expression in activated rat brain microglia: Implications for death of activated microglia. J Immunol 177:1323–1329. Yokota O, Terada S, Ishizu H, Ishihara T, Ujike H, Nakashima H, Nakashima Y, Kugo A, Checler F, Kuroda S. 2003. Cyclooxygenase-2 in the hippocampus is up-regulated in Alzheimer’s disease but not in variant Alzheimer’s disease with cotton wool plaques in humans. Neurosci Lett 343:175–179. Yucel-Lindberg T, Olsson T, Kawakami T. 2006. Signal pathways involved in the regulation of prostaglandin E synthase-1 in human gingival fibroblasts. Cell Signal 18:2131–2142. Zhang J, Fujii S, Wu Z, Hashioka S, Tanaka Y, Shiratsuchi A, Nakanishi Y, Nakanishi H. 2006. Involvement of COX-1 and up-regulated prostaglandin E synthases in phosphatidylserine liposome-induced prostaglandin E2 production by microglia. J Neuroimmunol 172:112– 120. Zhang J, Rivest S. 2001. Anti-inflammatory effects of prostaglandin E2 in the central nervous system in response to brain injury and circulating lipopolysaccharide. J Neurochem 76:855–864.

GLIA