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GLIA 51:13–21 (2005)

Involvement of A2 and Lipoxygenase in Lipopolysaccharide-Induced Inducible Nitric Oxide Synthase Expression in Glial Cells

1,2 1 1 2,3 1 JE-SEONG WON, YEONG-BIN IM, MUSHFIQUDDIN KHAN, AVTAR K. SINGH, AND INDERJIT SINGH * 1Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 2Department of Pathology, Medical University of South Carolina, Charleston, South Carolina 3Laboratory Medicine Service, Ralph H. Johnson Veterans Administration Medical Center, Charleston, South Carolina, South Carolina

KEY WORDS In addition to iNOS gene induction, the activation of k PLA2; COX; 5-LO; 12-LO; iNOS; NF B; nitric oxide; glial (PLA2) and accumulation of arachido- cells nic acid (AA) in those disease conditions are another hallmark of neuroinflammation (Huterer et al., 1995; Stephenson et al., 1996; Arai et al., 2001). Three groups ABSTRACT of PLA are involved in AA generation, depending on The present study underlines the importance of phospholi- 2 their cellular localization and functional regulation: pase A2 (PLA2)- and lipoxygenase (LO)-mediated signaling processes in the regulation of inducible nitric oxide secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2), and cal- synthase (iNOS) gene expression. In glial cells, lipopolysac- cium-independent PLA2 (iPLA2). AA produced by these charide (LPS) induced the activities of PLA2 (-inde- PLA2 has been known to regulate various signal trans- pendent PLA2; iPLA2 and cytosolic PLA2; cPLA2) as well as duction pathways directly via activation of protein gene expression of iNOS. The inhibition of cPLA2 by methyl kinase C (PKC) (Murakami and Routtenberg, 1985), arachidonyl fluorophosphates (MAFP) or antisense oligomer intracellular Ca2þ level increase (Ferrante et al., 1999), against cPLA2 and inhibition of iPLA2 by bromoenol lactone neutral sphingomyelinase (Robinson et al., 1997), and k reduced the LPS-induced iNOS gene expression and NF B mitogen-activated protein kinases (ERK1/2, p38, and activation. In addition, the inhibition of LO by nordihydro- JNK) (Cui and Douglas, 1997; Rizzo and Carlo-Stella, guaiaretic acid (NDGA; general LO inhibitor) or MK886 (5- LO inhibitor), but not (12-LO inhibitor), comple- 1996; Hii et al., 1995, 1998) or indirectly via generation tely abrogated the LPS-induced iNOS expression. Because of various . NDGA could abrogate the LPS-induced activation of NFkB, In mammalian cells, the release of AA constitutes the while MK886 had no effect on it, LO-mediated inhibition of rate-limiting step in the biosynthesis of eicosanoids iNOS gene induction by LPS may involve an NFkB-depen- including (PGs), (LTs), and dent or -independent (by 5-LO) pathway. In contrast to LO, platelet-activating factor (PAF), all of which act as potent however, the (COX) may not be involved in inflammatory mediators. Among these, PGs that play a the regulation of LPS-mediated induction of iNOS gene role as an intercellular or intracellular messenger because COX inhibition by indomethacin (general COX (Bjorkman, 1998; Dubois et al., 1998; Vane et al., 1998) inhibitor), SC560 (COX-1 inhibitor), and NS398 (COX-2 inhibitor) affected neither the LPS-induced iNOS expres- are implicated in various physiological or pathophysiolo- sion nor activation of NFkB. These results indicate a role gical processes, such as smooth muscle tone, vascular permeability, cellular proliferation, and inflammatory/ for cPLA2 and iPLA2 in LPS-mediated iNOS gene induction in glial cells and the involvement of LO in these reac- immune function (Harris et al., 2002). Currently, two tions. ' 2005 Wiley-Liss, Inc. isoforms of responsible for generation of PGs have been identified; cyclooxygenase-1 (COX-1) is consti- tutively expressed in most tissue, COX-2 is primarily considered an inducible gene product whose synthesis INTRODUCTION can be upregulated by mitogenic or inflammatory sti- muli (Dubois et al., 1998). In addition to PGs, leuko- Recent research has focused on the regulation of the trienes (LTs) are potent mediators that have inducible nitric oxide (NO) synthase (iNOS) isoform. The iNOS produces large quantities of NO over prolonged Grant sponsor: National Institutes of Health; Grant number: NS-22576; Grant periods of time. In central nervous system, NO produced number: NS-34741; Grant number: NS-37766. by iNOS is a major pathophysiological mediator in many *Correspondence to: Inderjit Singh, Department of Pediatrics, Medical Univer- inflammatory neurodegenerative diseases, such as ische- sity of South Carolina, 96 Jonathan Lucas Street, Clinical Science Building, Room 316, Charleston, SC 29425. E-mail: [email protected] mia, Alzheimer’s disease, and multiple sclerosis, in Received 7 July 2004; Accepted 28 December 2004 which astrocyte- and microglia-derived NO contribute to DOI 10.1002/glia.20178 oligodendrocyte degeneration and neuronal death (Iade- Published online 18 March 2005 in Wiley InterScience (www.interscience.wiley. cola et al., 1997; Liu et al., 2002; Minagar et al., 2002). com).

# 2005 Wiley-Liss, Inc. 14 WON ET AL. important roles in the regulation of both normal cells were incubated with serum-free DMEM medium for function and the inflammatory process. The synthesis of 24 h before incubation with LPS and other agents. LTs is initiated by the enzymes belong to the family of lipoxygenases (LO). Currently, three isoforms of LO have been identified: 5-LO, 15-LO, and 12-LO (Nathoo Assay of cPLA2 and iPLA2 Activity et al., 2004). Recent studies have document the involve- ment of 5-LO in the biosynthesis of leukotrienes, which To assess cPLA2 activity, the cells were washed once constitute mediators of anaphylactic and inflammatory with phosphate-buffered saline (PBS), suspended in buf- disorders (Engels and Nijkamp, 1998). In addition, an fer composed of 10 mM Tris-HCl (pH 7.4) and 150 mM involvement of 12/15-LO in atherogenesis has been NaCl (Tris-buffered saline [TBS]), and lysed by sonica- reported (Feinmark and Cornicelli, 1997; Kuhn and tion. The resulting lysates were incubated in 250 ml of buf- Chan, 1997). However, the role of PLA2, COX, and LO fer comprising 100 mM Tris-HCl (pH 9.0), 4 mM CaCl2, in lipid-mediated signaling in inflammatory disease is and 2 mM 1-palmitoyl-2-[14C]arachidonyl-glyceropho- not well understood. sphoethanolamine (NEN Life Science Products, Boston, The involvement of PLA2-dependent pathway in the MA) as the substrate at 378C for 30 min. To assess iPLA2 regulation of iNOS gene expression has been studied activity, the lysates (prepared in 10 mM HEPES (pH 7.5) recently. AA generation by group IIA secretary PLA2 containing 1 mM EDTA, 1 mM dithiothreitol (DTT), and (Baek et al., 2001) and the production of prostaglandins 0.34 M ), were incubated in 250 ml of buffer com- (PGs) by COX-2 (Chen et al., 1999) are reported to be posed of 100 mM HEPES (pH 7.5), 5 mM EDTA, 0.4 mM positive regulator for iNOS gene expression in macro- Triton X-100, 0.1 mM ATP, and 10 mM 1-palmitoyl-2- 14 phages. However, even if the inductions of PLA2 activity [ C]arachidonyl-glycerophosphoethanolamine at 408C and AA accumulation are observed in various neuroin- for 30 min (Ackermann et al., 1995; Balsinde et al., 1997). flammatory disease conditions (Huterer et al., 1995; Ste- The [14C]-AA released was extracted and the associated phenson et al., 1996; Arai et al., 2001), less attention radioactivity was counted. has been paid to the their involvement, as well as AA metabolism, in the regulation of gene expression of inflammatory mediators including iNOS in the CNS. In Assay for NO Production and Induction the present study, we examined the effect of AA metabo- of iNOS lism on the induction of iNOS gene expression in glial cells and also sought to determine the involvement of The cells were cultured in 12-well plastic tissue cul- NFkB in AA-metabolism-mediated regulation of iNOS ture plates; 24 h after the LPS treatment, production of gene expression. We report that activation of cPLA2 and NO was determined by an assay of the culture superna- iPLA2, and AA metabolites of 5-LO pathway are tant for nitrite by using Griess reagent and NaNO2 as a involved in LPS-induced iNOS gene expression via both standard (Green et al., 1982). For iNOS protein, cells activation of NFkB-dependent and -independent path- stimulated with LPS or other drugs for 12 h washed ways in glial cells. with cold TBS (20 mM Trizma base, and 137 mM NaCl, pH 7.5), lysed in 1 sodium dodecyl sulfate (SDS) sam- ple loading buffer (62.5 mM Trizma base, 2% w/v SDS, 10% ), sonicated briefly, and centrifuged at MATERIALS AND METHODS 15,000g for 5 min. The resulting supernatant was used Isolation and Maintenance of Primary Rat for the iNOS Western immunoblot assay. All samples Astrocytes and C6 Rat Glioma Cells were boiled for 3 min with 0.1 vol of 10% b-mercap- toethanol and 0.5% bromophenol blue mix; 50 mg of total Astrocytes were prepared from 1–2-day-old rat cerebral cellular protein was resolved by sodium dodecyl sulfate- tissue as described by McCarthy and DeVellis (1980). polyacrylamide gel electrophoresis (SDS-PAGE). After Cells were maintained in Dulbecco’s modified Eagle’s blotting to polyvinylidene difluoride (PVDF) filter, the medium (DMEM) containing 3 g/L of , 5% fetal iNOS bands were detected according to standard proto- bovine serum (FBS), and 10 mg/ml gentamicin on a 75-cm2 col by using anti-iNOS rabbit polyclonal antibody (BD 1 polystyrene flask (Costar , Corning, NY). After 10 days of Transduction Laboratories, cat. no: 610332 Franklin culture, astrocytes were separated from microglia and oli- Lakes, NJ). godendrocytes by shaking for 24 h in an orbital shaker at 240 rpm. To ensure the complete removal of all the oligo- Isolation of Total RNA and Northern Blot Assay dendrocytes and microglia before subculturing, the shak- ing was repeated twice after a gap of 1 or 2 days. Cells The isolation and Northern blot assay was performed were trypsinized and subcultured and further incubated as described previously (Won et al., 2003). Five mg of total for 2 days for stabilization. C6 rat glioma cells (obtained RNA was electrophoresed on 1% agarose- from American Type Cell Culture [ATCC)] were main- gels and transferred onto nylon Hybond-N hybridization tained in DMEM containing 3 g/L of glucose, 10% FBS, membrane sheets (Amersham Pharmacia Biotech, Piscat- and 10 mg/ml gentamicin. All cultured cells were main- away, NJ). After ultraviolet (UV)-cross linking, the mem- tained at 378Cin5%CO2/95% air. At 80% confluence, the branes were prehybridized at 688C in prehybridization PLA2 AND LO IN iNOS EXPRESSION 15 buffer (5 SSC, 50% formamide, 0.02% SDS, 0.1% sodium (Roche Molecular Biochemicals). Protein–DNA com- N-lauroyl sarcosine, and 2% blocking reagent), and then plexes were resolved from protein-free DNA in 5% polya- digoxigenin (DIG)-labeled iNOS antisense-cRNA (1–893 crylamide gels at room temperature in 50 mM Tris, pH bp) probes were added to prehybridization buffer and 8.3, 0.38 M glycine, and 2 mM EDTA, and electroblotted incubated overnight. The membranes were washed three onto positively charged nylon membranes. Chemilumi- times in 2 SSC and 0.1% SDS at room temperature nescence detection for DIG-labeled probes was per- (RT), and in 0.1 SSC and 0.1% SDS at 688C. After equili- formed as described in Northern blot analysis. brating the membranes in maleic acid buffer (100 mM maleic acid and 150 mM NaCl, pH 7.5), the membranes were blocked in maleic acid buffer containing 1% blocking reagent (Roche Molecular Biochemicals, Indianapolis, IN). Nuclear Run-On Assay Chemiluminescent autoradiography detection was per- 6 formed as suggested by the manufacturer (Roche Molecular The cells (5 10 ) were pelleted at 500g for 5 min at Biochemicals) using alkaline phosphatase-conjugated 48C, and then washed in 10 ml of ice-cold TBS. The cells anti-DIG F fragment (Roche Molecular Biochemicals) were placed in 2 ml of lysis buffer (10 mM Tris-HCl, pH 1ab and CSPD (disodium 3-(4-methoxyspiro{1,2-dioxetane- 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40) for 3 min on 3,20-(50-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl ice, and the nuclei were obtained by centrifugation at phosphate; Roche Molecular Biochemicals). 1,000g for 5 min at 48C. The nuclei were washed twice in 5 ml lysis buffer without NP-40. The nuclear pellet was suspended in 120 ml nuclear freezing buffer (50 mM Tris- HCl, pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA). Knockdown of cPLA2 Using Antisense 8 DNA Oligomer The nuclei were either used directly or stored at 70 C. If frozen, nuclei were thawed on ice before use. The nuclei m For the transfection, C6 rat glioma cells were cultured on were mixed with 30 lof5 run-on buffer (25 mM Tris- 24-well plates. At 80% of confluence, phosphorothioated HCl, pH 8, 12.5 mM MgCl2, 750 mM KCl, 5 mM DTT, 5 mM EDTA, 2 mM each of ATP, GTP, and CTP and UTP, cPLA2 antisense DNA oligomer (Gs TsGCTGATAAGGATCT- sAsT) directed against codons 4–9 of the rat cytosolic, calcium- and 20 nM DIG-11-UTP). The mixture was incubated for 30 min at 308C, and then 15 ml RNase-free DNase I (Pro- dependent PLA2 (GeneBank ID: 19424223) and its sequence m scramble mixture (GsAsTAGAAGCTGTTTCAsCsT) were mega, Madison, WI; 1 U/ l) was added and incubated for transfected using OligofectaminTM (Invitrogen, Carlsbad, CA) another 15 min. The nuclear RNA was extracted by using according to manufacturer’s instructions. Trizol (Invitrogen). After washing RNA pellet twice with 75% ethanol, it was dissolved in 50 ml of formamide and further diluted with prehybridization buffer. For slot blot- ting, linearized iNOS (Won et al., 2003) and cyclophilin Gel Shift Analysis (Takahashi et al., 1989) plasmid DNA (5 mg per sample) were dissolved 20 ml with TE and further dilute with 180 7 mlof6 SSC. After denaturation by boiling for 10 min, Nuclear extract from C6 cells (1 10 cells) were pre- þ pared using previously published method (Dignam et al., the DNA solution was applied to N -nylon membrane 1983) with slight modification. Cells were harvested, (Amersham-Pharmacia) using a slot blot apparatus (Bio- washed twice with ice-cold TBS, and lysed in 1 ml of Rad, Hercules, CA). After UV cross-linking, each mem- buffer A [10 mM KCl, 2 mM MgCl2, 0.5 mM DTT, pro- brane was prehybridized with prehybridization buffer for tease inhibitor cocktail (Sigma Chemical Co., St. Louis, 2 h at 608C and hybridized with sample probe solution MO), and 0.1% Nonidet P-40 (NP-40) in 10 mM HEPES, overnight at 608C. The washing and chemiluminescence pH 7.9] for 10 min on ice. After centrifugation at 1,000g, detection for DIG-labeled probes was performed as the pelleted nuclei were washed with buffer A without described in Northern blot analysis. NP-40, and resuspended in 40 ml of buffer B [25% (v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and protease inhibitor cocktail (Sigma) in 20 mM HEPES, pH 7.9] for 30 min on ice. The lysates Transient Transfection and Reporter were centrifuged at 15,000g for 15 min and the superna- Gene Assay tants containing the nuclear proteins were stored at 708C until use; 10 mg of nuclear proteins was used for The cells (3 105 cells/well) were cultured in 6-well the electrophoretic mobility shift assay for detection of plates for 36 h before the transfection. Transfection was NFkB DNA binding activity. The DNA-protein binding performed with 2 mgofkB repeat SEAP reporter construct reactions were performed at room temperature for 20 (pNFkB-SEAP, BD Bioscience, CA) and 0.2 mg of pCMV-b- min in 10 mM Trizma base (pH 7.9), 50 mM NaCl, 5 Gal (Stratagene, La Jolla, CA). One day after transfec- mM MgCl2, 1 mM EDTA, 1 mM DTT, 1 mg poly (dI-dC), tion, the cells were placed in serum-free media overnight. 5% (v/v) glycerol, and 0.3 pmol of NFkB (Santa Cruz After appropriate treatment, the media was used for the Biotechnology, Santa Cruz, CA) labeled with DIG- detection of level of SEAP, and the cells were washed with ddUTP, using terminal deoxynucleotidyl transferase PBS, scraped, and then resuspended with 100 ml of lysis 16 WON ET AL.

Fig. 1. Lipopolysaccharide (LPS) induces the enzymatic activities of cPLA2 and iPLA2 in glial cells. C6 rat glioma cells were stimulated with lipopolysaccharide (LPS; 1 mg/ml) and calcium-dependent cytosolic PLA2 (cPLA2) and calcium-independent PLA2 (iPLA2) activity was determined at various time points as described in Materials and Methods. buffer (40 mM of Tricine pH 7.8, 50 mM of NaCl, 2 mM of EDTA, 1 mM of MgSO4, 5 mM of DTT, and 1% of Triton X- 100) for the detection of b-galactosidase. Detection of SEAP and b-galactosidase was performed using Great EscAPeTM SEAP chemiluminescence detection kit and b- gal Detection Kit (BD Bioscience). The emitted light and optical absorbance was measured using Spectra Max/ Gemini XG (Molecular Devices, Sunnyvale, CA) and Spec- traMax 190 (Molecular Devices), respectively.

Statistical Analysis

All values shown in the figures are expressed as means 6 standard error of mean (SEM) of n determina- tions, obtained on at least three independent experi- ments. The results were examined by one- and two-way analysis of variance (ANOVA). Individual group means were then compared with the Bonferroni test. A P-value of <0.05 was considered significant. Fig. 2. Lipopolysaccharide (LPS)-induced iNOS gene expression is mediated by cPLA2 and iPLA2. To examine the involvement of cPLA2 and iPLA2 in iNOS gene expression, C6 rat glioma cells (A–C) and pri- RESULTS mary astrocytes (D) were treated with vehicle (VHC; dimethylsulfox- ide), MAFP (methyl arachidonyl fluorophosphonate; cPLA2 and iPLA2 LPS Induces Enzymatic Activities of cPLA2 inhibitor) and BEL (bromoenol lactone; iPLA2 specific inhibitor) for 30 m and iPLA2 in Glial Cells min before lipopolysaccharide (LPS; 1 g/ml) treatment. To quantify NO production (A,D) and iNOS protein (C,D), nitrite level in media and iNOS proteins were determined 24 h after LPS treatment as described The involvement of cPLA2 and iPLA2 in various inflam- in Materials and Methods. For the detection of iNOS mRNA, total RNA matory conditions has been suggested in various cells was extracted 6 h after LPS treatment, and 5 mg of total RNA was used for detection of iNOS mRNA. The equal amounts of CPN (cyclophilin) types. To address the role of these enzymes in the regula- mRNA verify that equal amounts of RNA were loaded. Vertical bars tion of iNOS gene expression, we examined the effect of indicate standard error of mean (*P < 0.05, **P < 0.01, ***P < 0.001 LPS (1 mg/ml) in the regulation of activation of cPLA and compared to control group; þP < 0.05, þþP < 0.01, þþþP < 0.001 2 compared to LPS-treated group). iPLA2 in C6 rat glioma cells. As depicted in Figure 1, LPS increased enzyme activities of cPLA2 and iPLA2 within 30 min. These activities continued to increase until 3 h fol- fluorophosphonate (MAFP, a cPLA2 inhibitor) or bromoe- lowed by a slow decline. Similar results were observed in nol lactone (BEL, iPLA2 specific inhibitor) before LPS three sets of independent experiments by using arachido- treatment. As shown in Figure 2, the LPS-induced NO nyl thiophosphatidylcholine as a colorimetric substrate production and iNOS mRNA and protein expression were (data not shown). inhibited by MAFP (Fig. 2A,C) and BEL (Fig. 2B,C) in C6 rat glioma cells as well as in primary cultured astrocytes (Fig. 2D). LPS-Induced iNOS Gene Expression Since MAFP is known to inhibit the activity of iPLA2 as Is Mediated by cPLA2 and iPLA2 well as cPLA2 (Lio et al., 1996), we confirmed the role of cPLA2 in the regulation of iNOS gene expression by using To examine the involvement of LPS-mediated induction antisense DNA oligomer against cPLA2 mRNA. As shown of cPLA2 and iPLA2 activities in iNOS gene regulation, in Figure 3A, the knock-down of cPLA2 protein by anti- the cells were preincubated with methyl arachidonyl sense DNA inhibited LPS-mediated increases in NO pro- PLA2 AND LO IN iNOS EXPRESSION 17 fic inhibitor), or baicalein (BCL, a 12-LO specific inhibitor, 5,6,7-trihydroxyflavone) before LPS treatment. As shown in Figure 5, the LPS-induced NO production and iNOS mRNA and protein expression were inhibited by NDGA at a dose-dependent manner in C6 rat glioma cells and primary cultured astrocytes. Furthermore, MK886, but not BCL, inhibited LPS-induced iNOS expression, sug- gesting that 5-LO is one of LO which mediates LPS sig- naling to iNOS gene expression.

Inhibitors of PLA2 or LO Inhibit Fig. 3. Effect of antisense DNA oligomer against cPLA2 and propra- iNOS Expression Via Inhibition nolol on LPS-induced NO production and iNOS protein expression. A: To of Transcriptional Activity confirm the involvement of cPLA2 in iNOS gene regulation, cells were preincubated with antisense DNA oligomer against cPLA2 (AS-cPLA2)or its sequence scramble mixture (SC-cPLA2) before lipopolysaccharide To identify whether the inhibition of iNOS expression (LPS; 1 mg/ml) treatment. B: To confirm the role of BEL (bromoenol lac- tone; an inhibitor of iPLA2 and phosphatidate phosphohydrolase) in by the inhibitors of PLA2 or LO is mediated by the inhibi- iNOS gene expression, cells were pretreated with vehicle (VHC; phos- tion of transcription activity of iNOS mRNA, nuclei run phate-buffered saline) and propranolol (phosphatidate phosphohydrolase specific inhibitor; 200 mM) 30 min before LPS treatment. on assay was performed. As shown in Figure 6, the activa- tion of transcription of iNOS mRNA by LPS is downregu- lated by the preincubation with MAFP, BEL, NDGA, and duction and iNOS protein expression. This result suggests MK886 similar to the level of mRNA accumulation that cPLA2 is involved in the regulation of LPS-induced depicted in Figures 2 and 5, suggesting that the inhibition iNOS gene expression. of iNOS mRNA level by those inhibitors resulted from the As BEL is known to inhibit phosphatidate phosphohy- inhibition of its transcriptional activity. drolase (PAP), we examined the effect of PAP inhibition on NO production and iNOS protein expression. As shown in Figure 3B, 150 mM propranolol, a PAP inhibitor, further PLA2/LO-Mediated LPS-Induced iNOS k enhanced LPS-induced NO production and iNOS protein Expression via NF B-Dependent expression. This result indicates that BEL inhibits iNOS or -Independent Pathways production via inhibition of iPLA rather than PAP. 2 NFkB is an essential transcription factor in the acti- vation of iNOS gene in rodents as well as humans (Low- enstein et al., 1993; Beck and Sterzel, 1996; Marks- Are Not Involved Konczalik et al., 1998). To define the role of NFkBin in LPS-Induced iNOS Gene Expression PLA2- or LO-mediated inhibition of iNOS expression, the effects of inhibitors of those enzymes on LPS- Possible involvement of COX in the upregulation of iNOS induced increase in NFkB DNA binding activity and its gene expression has been suggested in various cell types transactivity were examined. As shown in Figure 7, (Minghetti et al., 1997; Chen et al., 1999; Hori et al., 2001). MAFP, BEL, and NDGA inhibited LPS-induced activa- To address the involvement of COX in the regulation of tion of NFkB DNA binding activity and its transactivity iNOS expression in glial cells, the cells were preincubated (kB-luciferase reporter gene activity), suggesting that with indomethacin (INDO, a general inhibitor of COX), PLA and LO are involved in NFkB-mediated iNOS gene NS389 (a COX-2 specific inhibitor), or SC560 (a COX-1-spe- 2 regulation. Although, MK886 inhibited the iNOS expres- cific inhibitor) before LPS treatment. As shown in Figure 4, sion (Fig. 5) but it had no effect on NFkB DNA binding the LPS-induced NO production and iNOS mRNA and pro- activity as well as its transactivity, suggesting that 5-LO tein expression were not affected by any of the COX inhibi- may mediate iNOS gene expression via NFkB-indepen- tors (INDO, NS389, or SC560) in C6 rat glioma cells and in dent pathways. primary cultured astrocytes, suggesting that COX are not involved in the regulation of iNOS expression in glial cells. DISCUSSION

Lipoxygenases Mediate LPS-Induced These studies document the involvement of cPLA2 and iNOS Gene Expression iPLA2-mediated signaling pathways in the induction of iNOS expression by LPS, and indicate LO as one of the Growing evidence suggests certain leukotrienes have a mediators that may regulate PLA2-mediated iNOS gene critical role in the signaling cascade of inflammatory gene induction by activation of NFkB-dependent or indepen- expression (Sala et al., 1998). To address the involvement dent pathways in glial cells. These conclusions are based of LO in the regulation of iNOS expression in glial cells, on the following observations: (1) addition of cPLA2 or the cells were preincubated with nordihydroguaiaretic iPLA2 inhibitor and decoy of cPLA2 mRNA inhibited the acid (NDGA, a general LO inhibitor), MK886 (5-LO speci- LPS-induced iNOS gene expression; and (2) addition of 18 WON ET AL.

Fig. 4. Lipopolysaccharide (LPS)-induced iNOS gene expression is not mediated by COX. To examine the involvement of COX in iNOS gene expression, C6 rat glioma cells (A–C) and primary astrocytes (D) were treated with vehicle (VHC; dimethylsulfoxide), INDO (indomethacin, general COX inhibitor), NS398 (COX-2 inhibitor), and SC560 (COX-1 inhibitor) for 30 min before lipopolysaccharide (LPS; 1 mg/ml) treatment. To quantify NO production (A,D) and iNOS protein (C,D), nitrite level in media and iNOS proteins were deter- mined 24 h after LPS treatment as described in Materi- als and Methods. For the detection of iNOS mRNA, total RNA was extracted 6 h after LPS treatment, and 5 mg of total RNA was used for detection of iNOS mRNA. The equal amounts of CPN (cyclophilin) mRNA verify that equal amounts of RNA were loaded. Vertical bars indicate standard error of mean (*P < 0.05, **P < 0.01, ***P < 0.001 compared to control group; þP < 0.05, þþP < 0.01, þþþP < 0.001 compared to LPS-treated group). general inhibitor of LO (NDGA) and 5-LO specific inhibi- interleukin-1b (IL-1b) (McHowat and Liu, 1997). In tor, but none of inhibitors for COX (INDO, NS398, or macrophages, iPLA2 is upregulated by reactive oxygen SC560) and 12-LO used, inhibited LPS-induced iNOS species (Martinez and Moreno, 2001). In addition, iPLA2 gene expression. Finally, LPS-induced activation of NFkB inhibitor prevents double strand RNA- and virus-induced was inhibited by the inhibitor of cPLA2, iPLA2 and gen- iNOS and COX-2 expression in macrophages (Maggi eral inhibitor of LO, but not by specific inhibitor of 5-LO. et al., 2002; Steer et al., 2003). In the present study, we Previously, the possible involvement of cPLA2 in TNF- demonstrated the possible involvement of iPLA2 in LPS- a-mediated pathway leading to mediated NFkB activation and iNOS gene expression in nuclear translocation of NFkB and to NFkB-activated glial cells by using bromoenol lactone (BEL). Although gene expression in a keratinocyte cell line has been docu- BEL is a potent (1,000-fold more than cPLA2), selective mented by using cPLA2 inhibitor, AACOCF3 (Thommesen inhibitor for iPLA2, it is also known to inhibit magne- et al., 1998). Consistent with this study, we also observed sium-dependent PAP in mouse P388D1 macrophages that cPLA2-inhibitor (MAFP) inhibited NFkB activation (Balsinde and Dennis, 1996). However, enhancement of and iNOS gene expression in glial cells. However, since LPS-induced NO production and iNOS protein expression both inhibitors (AACOCF3 and MAFP) also inhibit the by 150 mM propranolol, a PAP inhibitor at high concentra- activity of iPLA2 (but not sPLA2) (Balsinde and Dennis, tion (>100 mM) (Billah et al., 1989; Bourgoin et al., 1990; 1996; Lio et al., 1996), it is unclear whether LPS-induced Kanaho et al., 1991; Thompson et al., 1991), indicates that iNOS gene expression is mediated by cPLA2 or iPLA2. BEL-mediated inhibition of iNOS gene expression may be Therefore, the observed inhibition of NO production and mediated by the inhibition of iPLA2 rather than PAP. The iNOS protein expression by antisense DNA oligomer mechanism by which LPS stimulates iPLA2 activation against cPLA2 mRNA in this study documents the role of has yet to be determined. Although iPLA2 contains con- cPLA2 in LPS-mediated iNOS expression in glial cells. sensus sites for a number of serine kinases (Ma and Turk, Recently, the role of iPLA2 in pathophysiological signal- 2001), LPS-mediated phosphorylation and activation of ing cascades has been described. In rat cardiac ventricu- iPLA2 has not been described so far. Moreover, the lar myocytes, iPLA2 is activated during exposure to mechanism of iPLA2-mediated regulation of NFkB path- PLA2 AND LO IN iNOS EXPRESSION 19

Fig. 5. Lipopolysaccharide (LPS)-induced iNOS gene expression is mediated by LO. To examine the involvement of lipoxygenases (LO) in iNOS gene expression, C6 rat glioma cells (A–C) and primary astrocytes (D) were treated with vehicle (VHC; dimethylsulfoxide), NDGA (nordihydroguaiaretic acid, general LO inhibitor), MK886 (5-LO inhibitor), and BCL (baicalein, 12-LO inhibitor) for 30 min before lipo- polysaccharide (LPS; 1 mg/ml) treatment. To quantify NO production (A,D) and iNOS protein (C,D), nitrite level in media and iNOS proteins were determined 24 h after LPS treatment as described in Materials and Methods. For the detection of iNOS mRNA, total RNA was extracted 6 h after LPS treatment, and 5 mg of total RNA was used for detection of iNOS mRNA. The equal amounts of CPN (cyclophilin) mRNA verify that equal amounts of RNA were loaded. Vertical bars indicate standard error of mean (*P < 0.05, **P < 0.01, ***P < 0.001 compared to control group; þP < 0.05, þþP < 0.01, þþþP < 0.001 compared to LPS-treated group). ways in response to LPS should be examined in future general inhibitor of LO (NDGA) inhibited LPS-induced study. activation of NFkB as well as iNOS gene expression. It has been reported that cytokine induces COX-2 Since NDGA can also interfere weakly with PLA2 and expression and followed PGE2 production mediate upre- COX activities (Aktan et al., 1993; Meade et al., 1993) and gulation of iNOS gene expression in macrophages via act as an antioxidant (Ramasamy et al., 1999), the inhibi- activating cAMP pathway (Chen et al., 1999). However, tory effect of NDGA on iNOS gene expression and NFkB no involvement of COX in iNOS gene regulation was activation induced by LPS may be via inhibiting PLA2 observed in glial cells in the present study. The differ- activities or its anti-oxidative properties. Moreover, these ences between these cell types for the role of COX-2 in data indicate the possible involvement of other NDGA- iNOS gene expression remain unclear, but the undetect- sensitive enzymes that regulate of iNOS expression via able COX-2 protein level in resting glial cells and the regulation of NFkB activation. The involvement of leuko- opposite role of cAMP in the regulation of iNOS expres- trienes in the regulation of iNOS gene expression has sion between in macrophages and glial cells may been reported previously in various cell types. In rat vas- account, at least in part, for these differences (Minghetti cular smooth muscle cells, the 12-LO product, 12-HETE, et al., 1997; Pahan et al., 1997; Chen et al., 1999; Won dose-dependently enhanced nitrite production induced by et al., 2001). IL-1b (Shimpo et al., 2000). Furthermore, BCL (12-LO It has been shown that a significant amount of cPLA2 inhibitor) inhibits iNOS gene expression in macrophages resides within the nucleus (Sierra-Honigmann et al., (Chen et al., 2001; Hashimoto et al., 2003). However, we 1996). Moreover, 5-LO translocates to a perinuclear site did not observe the involvement of 12-LO in iNOS gene in stimulated cells (Peters-Golden and McNish, 1993). expression or in NFkB activation in C6 rat glioma cells as Therefore, in activated cells cPLA2 may be ideally posi- well as primary astrocytes. Therefore, the involvement of tioned to provide free to 5-LO in the 12-LO in iNOS gene expression is believed to be a cell nucleus. In this study, we observed that the MK886 (an type specific event. inhibitor of 5-LO) inhibited the LPS-induced iNOS gene In conclusion, our results document that PLA2 and LO expression without altering NFkB activation. However, play an essential role in the activation of iNOS gene 20 WON ET AL. REFERENCES

Ackermann EJ, Conde-Frieboes K, Dennis EA. 1995. Inhibition of 2þ macrophage Ca -independent phospholipase A2 by bromoenol lac- tone and trifluoromethyl ketones. J Biol Chem 270:445–450. Aktan S, Aykut C, Yegen BC, Okar I, Ozkutlu U, Ercan S. 1993. The effect of nordihydroguaiaretic acid on C4 and prostaglan- din E2 production following different reperfusion periods in rat brain after forebrain ischemia correlated with morphological changes. Pros- Fig. 6. Inhibitors of PLA2 or LO inhibit iNOS expression via inhibi- tion of transcription level. To identify whether the inhibition of iNOS taglandins Leukot Essent Fatty Acids 49:633–641. Arai K, Ikegaya Y, Nakatani Y, Kudo I, Nishiyama N, Matsuki N. 2001. expression by the inhibitors of PLA2 or LO is mediated by the inhibition of transcription activity of iNOS mRNA, nuclei run-on assays were per- Phospholipase A2 mediates ischemic injury in the hippocampus: a formed. C6 rat glioma cells were treated with vehicle (VHC; dimethylsulf- regional difference of neuronal vulnerability. Eur J Neurosci 13: oxide), MAFP (methyl arachidonyl fluorophosphonate; cPLA and iPLA 2319–2323. 2 2 Baek SH, Lim JH, Park DW, Kim SY, Lee YH, Kim JR, Kim JH. 2001. inhibitor, 10 mM) and BEL (bromoenol lactone; iPLA2 specific inhibitor, 1 mM), NDGA (nordihydroguaiaretic acid, general LO inhibitor, 20 mM), Group IIA secretory phospholipase A2 stimulates inducible nitric and MK886 (5-LO inhibitor, 10 mM) for 30 min before lipopolysaccharide oxide synthase expression via ERK and NF-kappaB in macrophages. (LPS; 1 mg/ml) treatment. At 3 h after LPS treatment, the nuclei were iso- Eur J Immunol 31:2709–2717. lated from cells and nuclear run-on assay was performed as described in Balsinde J, Dennis EA. 1996. Bromoenol lactone inhibits magnesium- Materials and Methods. The equal amounts of CPN (cyclophilin) mRNA dependent phosphatidate phosphohydrolase and blocks triacylglycerol verify that equal amounts of mRNA were loaded. biosynthesis in mouse P388D1 macrophages. J Biol Chem 271:31937– 31941. Balsinde J, Balboa MA, Dennis EA. 1997. Antisense inhibition of group 2þ VI Ca -independent phospholipase A2 blocks remodeling in murine P388D1 macrophages. J Biol Chem 272:29317– 29321. Beck KF, Sterzel RB. 1996. Cloning and sequencing of the proximal promoter of the rat iNOS gene: activation of NFkappaB is not suffi- cient for transcription of the iNOS gene in rat mesangial cells. FEBS Lett 394:263–267. Billah MM, Eckel S, Mullmann TJ, Egan RW, Siegel MI. 1989. Phos- phatidylcholine by determines phosphati- date and levels in chemotactic peptide-stimulated human neutrophils. Involvement of phosphatidate phosphohydrolase in sig- nal transduction. J Biol Chem 264:17069–17077. Bjorkman DJ. 1998. The effect of and nonsteroidal anti-inflam- matory drugs on prostaglandins. Am J Med 105:8S–12S. Bourgoin S, Plante E, Gaudry M, Naccache PH, Borgeat P, Poubelle PE. 1990. Involvement of a phospholipase D in the mechanism of action of granulocyte-macrophage colony-stimulating factor (GM- CSF): priming of human neutrophils in vitro with GM-CSF is asso- Fig. 7. PLA /LO-mediated lipopolysaccharide (LPS)-induced iNOS 2 ciated with accumulation of and diradylglycerol. J expression via NFkB-dependent or -independent pathways. To define Exp Med 172:767–777. the role of NFkB in PLA2-(A) or LO- (B) mediated inhibition of iNOS expression, the effects of inhibitors of those enzymes on LPS-induced Chen CC, Chiu KT, Sun YT, Chen WC. 1999. Role of the cyclic AMP- increase in NFkB DNA binding activity and its transactivity were A pathway in lipopolysaccharide-induced nitric oxide examined. For the reporter gene assay, pNFkB-SEAP (secreted alkaline synthase expression in RAW 264.7 macrophages. Involvement of phosphatase) and pCMV-b-Gal (b-galactosidase) were co-transfected to cyclooxygenase-2. J Biol Chem 274:31559–31564. C6 rat glioma cells and incubated overnight. The cells were treated Chen YC, Shen SC, Chen LG, Lee TJ, Yang LL. 2001. Wogonin, bai- with vehicle (VHC; dimethylsulfoxide), MAFP (methyl arachidonyl calin, and baicalein inhibition of inducible nitric oxide synthase and fluorophosphonate; cPLA and iPLA inhibitor, 10 mM) and BEL (bro- cyclooxygenase-2 gene expressions induced by nitric oxide synthase 2 2 inhibitors and lipopolysaccharide. Biochem Pharmacol 61:1417– moenol lactone; iPLA2 specific inhibitor, 1 mM), NDGA (nordihydro- guaiaretic acid, general LO inhibitor, 20 mM), and MK886 (5-LO 1427. inhibitor, 10 mM) for 30 min before lipopolysaccharide (LPS; 1 mg/ml) Cui XL, Douglas JG. 1997. Arachidonic acid activates c-jun N-terminal treatment. For the reporter gene assay, the activity of SEAP and b-Gal kinase through NADPH oxidase in rabbit proximal tubular epithelial were measured 12 h after LPS treatment as described in Materials and cells. Proc Natl Acad Sci USA 94:3771–3776. Methods, and SEAP activities were normalized by b-Gal activity (top). Dignam JD, Lebovitz RM, Roeder RG. 1983. Accurate transcription For the gel shift assay, the nuclear proteins were extracted ½ h after initiation by RNA polymerase II in a soluble extract from isolated LPS treatment. mammalian nuclei. Nucleic Acids Res 11:1475–1489. Dubois RN, Abramson SB, Crofford L, Gupta RA, Simon LS, Van De Putte LB, Lipsky PE. 1998. Cyclooxygenase in biology and disease. k FASEB J 12:1063–1073. expression via NF B-dependent and -independent path- Engels F, Nijkamp FP. 1998. Pharmacological inhibition of leukotriene ways. With this background, future studies will address actions. Pharm World Sci 20:60–65. (1) the involvement of cPLA , iPLA and 5-LO in other Feinmark SJ, Cornicelli JA. 1997. Is there a role for 15-lipoxygenase in 2 2 atherogenesis? Biochem Pharmacol 54:953–959. proinflammatory gene regulation, (2) the mechanism of Ferrante A, Hii CST, Huang ZH, Rathjen DA. 1999. The neutrophils: iPLA2-mediated regulation of NFkB activation, and (3) the new outlook for old cells. London: Imperial College Press. mechanism of 5-LO-mediated regulation of iNOS expres- Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannen- baum SR. 1982. Analysis of nitrate, nitrite, and [15N]nitrate in biolo- sion without altering NFkBactivity. gical fluids. Anal Biochem 126:131–138. Harris SG, Padilla J, Koumas L, Ray D, Phipps RP. 2002. Prostaglan- dins as modulators of immunity. Trends Immunol 23:144–150. Hashimoto T, Kihara M, Yokoyama K, Fujita T, Kobayashi S, Matsush- ACKNOWLEDGMENTS ita K, Tamura K, Hirawa N, Toya Y, Umemura S. 2003. Lipoxygenase products regulate nitric oxide and inducible nitric oxide synthase pro- duction in interleukin-1beta stimulated vascular smooth muscle cells. The authors are grateful to Dr. Anne G. Gilg for proof- Hypertens Res 26:177–184. reading this manuscript and to Ms. Joyce Bryan and Hii CS, Ferrante A, Edwards YS, Huang ZH, Hartfield PJ, Rathjen DA, Poulos A, Murray AW. 1995. Activation of mitogen-activated protein Ms. Hope Terry for laboratory assistance and secretarial kinase by arachidonic acid in rat liver epithelial WB cells by a pro- assistance, respectively. tein kinase C-dependent mechanism. J Biol Chem 270:4201–4204. PLA2 AND LO IN iNOS EXPRESSION 21

Hii CS, Huang ZH, Bilney A, Costabile M, Murray AW, Rathjen DA, microglial cultures is downregulated by exogenous E2 Der CJ, Ferrante A. 1998. Stimulation of p38 phosphorylation and and by cyclooxygenase inhibitors. Glia 19:152–160. activity by arachidonic acid in HeLa cells, HL60 promyelocytic leu- Murakami K, Routtenberg A. 1985. Direct activation of purified protein kemic cells, and human neutrophils. Evidence for cell type-specific kinase C by unsaturated fatty acids (oleate and arachidonate) in the activation of mitogen-activated protein kinases. J Biol Chem 273: absence of and Ca2þ. FEBS Lett 192:189–193. 19277–19282. Nathoo N, Barnett GH, Golubic M. 2004. The cascade: possi- Hori M, Kita M, Torihashi S, Miyamoto S, Won KJ, Sato K, Ozaki H, ble role in gliomas and meningiomas. J Clin Pathol 57:6–13. Karaki H. 2001. Upregulation of iNOS by COX-2 in muscularis resi- Pahan K, Namboodiri AM, Sheikh FG, Smith BT, Singh I. 1997. dent macrophage of rat intestine stimulated with LPS. Am J Physiol Increasing cAMP attenuates induction of inducible nitric-oxide Gastrointest Liver Physiol 280:G930–938. synthase in rat primary astrocytes. J Biol Chem 272:7786–7791. Huterer SJ, Tourtellotte WW, Wherrett JR. 1995. Alterations in the Peters-Golden M, McNish RW. 1993. Redistribution of 5-lipoxygenase activity of A in postmortem white matter from 2 and cytosolic phospholipase A2 to the nuclear fraction upon macro- patients with multiple sclerosis. Neurochem Res 20:1335–1343. phage activation. Biochem Biophys Res Commun 196:147–153. Iadecola C, Zhang F, Casey R, Nagayama M, Ross ME. 1997. Delayed Ramasamy S, Drummond GR, Ahn J, Storek M, Pohl J, Parthasarathy reduction of ischemic brain injury and neurological deficits in mice lack- S, Harrison DG. 1999. Modulation of expression of endothelial nitric ing the inducible nitric oxide synthase gene. J Neurosci 17:9157–9164. oxide synthase by nordihydroguaiaretic acid, a phenolic antioxidant Kanaho Y, Kanoh H, Saitoh K, Nozawa Y. 1991. Phospholipase D activa- in cultured endothelial cells. Mol Pharmacol 56:116–123. tion by platelet-activating factor, , and formyl-methio- Rizzo MT, Carlo-Stella C. 1996. Arachidonic acid mediates interleukin- nyl-leucyl-phenylalanine in rabbit neutrophils. Phospholipase D 1 and tumor necrosis factor-alpha- induced activation of the c-jun activation is involved in enzyme release. J Immunol 146: 3536–3541. amino-terminal kinases in stromal cells. Blood 88:3792–3800. Kuhn H, Chan L. 1997. The role of 15-lipoxygenase in atherogenesis: Robinson BS, Hii CS, Poulos A, Ferrante A. 1997. Activation of neutral pro- and antiatherogenic actions. Curr Opin Lipidol 8:111–117. sphingomyelinase in human neutrophils by polyunsaturated fatty Lio YC, Reynolds LJ, Balsinde J, Dennis EA. 1996. Irreversible inhibi- 2þ acids. Immunology 91:274–280. tion of Ca -independent phospholipase A2 by methyl arachidonyl Sala A, Zarini S, Bolla M. 1998. Leukotrienes: lipid bioeffectors of fluorophosphonate. Biochim Biophys Acta 1302:55–60. inflammatory reactions. Biochemistry (Mosc) 63:84–92. Liu B, Gao HM, Wang JY, Jeohn GH, Cooper CL, Hong JS. 2002. Role Shimpo M, Ikeda U, Maeda Y, Ohya K, Murakami Y, Shimada K. 2000. of nitric oxide in inflammation-mediated neurodegeneration. Ann NY Effects of aspirin-like drugs on nitric oxide synthesis in rat vascular Acad Sci 962:318–331. smooth muscle cells. Hypertension 35:1085–1091. Lowenstein CJ, Alley EW, Raval P, Snowman AM, Snyder SH, Russell SW, Murphy WJ. 1993. Macrophage nitric oxide synthase gene: two Sierra-Honigmann MR, Bradley JR, Pober JS. 1996. ‘‘Cytosolic’’ phos- upstream regions mediate induction by interferon gamma and lipopo- pholipase A2 is in the nucleus of subconfluent endothelial cells but lysaccharide. Proc Natl Acad Sci USA 90:9730–974. confined to the cytoplasm of confluent endothelial cells and redistri- Ma Z, Turk J. 2001. The molecular biology of the group VIA Ca2þ-inde- butes to the nuclear envelope and cell junctions upon histamine sti- mulation. Lab Invest 74:684–695. pendent phospholipase A2. Prog Nucleic Acid Res Mol Biol 67:1–33. Maggi LB, Jr., Moran JM, Scarim AL, Ford DA, Yoon JW, McHowat J, Steer SA, Moran JM, Maggi LB Jr, Buller RM, Perlman H, Corbett JA. Buller RM, Corbett JA. 2002. Novel role for calcium-independent 2003. Regulation of cyclooxygenase-2 expression by macrophages in response to double-stranded RNA and viral infection. J Immunol 170: phospholipase A2 in the macrophage antiviral response of inducible nitric-oxide synthase expression. J Biol Chem 277: 38449–38455. 1070–1076. Marks-Konczalik J, Chu SC, Moss J. 1998. Cytokine-mediated tran- Stephenson DT, Lemere CA, Selkoe DJ, Clemens JA. 1996. Cytosolic scriptional induction of the human inducible nitric oxide synthase phospholipase A2 (cPLA2) immunoreactivity is elevated in Alzhei- gene requires both activator protein 1 and nuclear factor kappaB- mer’s disease brain. Neurobiol Dis 3:51–63. binding sites. J Biol Chem 273:22201–22208. Takahashi N, Hayano T, Suzuki M. 1989. Peptidyl-prolyl cis-trans iso- Martinez J, Moreno JJ. 2001. Role of Ca2þ-independent phospholipase merase is the cyclosporin A-binding protein cyclophilin. Nature 337: 473–475. A2 on arachidonic acid release induced by reactive oxygen species. Arch Biochem Biophys 392:257–262. Thommesen L, Sjursen W, Gasvik K, Hanssen W, Brekke OL, Skattebol McCarthy KD, de Vellis J. 1980. Preparation of separate astroglial and L, Holmeide AK, Espevik T, Johansen B, Laegreid A. 1998. Selective oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol inhibitors of cytosolic or secretory phospholipase A2 block TNF- 85:890–902. induced activation of transcription factor nuclear factor-kappa B and expression of ICAM-1. J Immunol 161:3421–3430. McHowat J, Liu S. 1997. Interleukin-1beta stimulates phospholipase A2 activity in adult rat ventricular myocytes. Am J Physiol 272(Pt 1): Thompson NT, Bonser RW, Garland LG. 1991. -coupled phos- C450–456. pholipase D and its inhibition. Trends Pharmacol Sci 12:404–408. Meade EA, Smith WL, DeWitt DL. 1993. Differential inhibition of prosta- Vane JR, Bakhle YS, Botting RM. 1998. Cyclooxygenases 1 and 2. Annu glandin endoperoxide synthase (cyclooxygenase) isozymes by aspirin Rev Pharmacol Toxicol 38:97–120. and other non-steroidal anti-inflammatory drugs. J Biol Chem 268: Won JS, Lee JK, Suh HW. 2001. Forskolin inhibits expression of induci- 6610–6614. ble nitric oxide synthase mRNA via inhibiting the mitogen activated Minagar A, Shapshak P, Fujimura R, Ownby R, Heyes M, Eisdorfer C. protein kinase in C6 cells. Brain Res Mol Brain Res 89:1–10. 2002. The role of macrophage/microglia and astrocytes in the patho- Won JS, Im YB, Key L, Singh I, Singh AK. 2003. The involvement of genesis of three neurologic disorders: HIV-associated dementia, Alz- glucose metabolism in the regulation of inducible nitric oxide heimer disease, and multiple sclerosis. J Neurol Sci 202:13–23. synthase gene expression in glial cells: possible role of glucose-6- Minghetti L, Nicolini A, Polazzi E, Creminon C, Maclouf J, Levi G. phosphate dehydrogenase and CCAAT/enhancing binding protein. 1997. Inducible nitric oxide synthase expression in activated rat J Neurosci 23:7470–7478.