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Inhibition of Phosphatidylinositol 3-Kinase Induces Nitric-oxide Synthase in Lipopolysaccharide- or Cytokine-stimulated C6 Glial Cells*

Open AccessPublished:March 12, 1999DOI:https://doi.org/10.1074/jbc.274.11.7528
      Nitric oxide (NO) produced by inducible nitric-oxide synthase (iNOS) in different cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of demyelinating and neurodegenerative diseases. The present study underlines the importance of phosphatidylinositol 3-kinase (PI 3-kinase) in the expression of iNOS in C6 glial cells and rat primary astrocytes. Bacterial lipopolysaccharide (LPS) or interleukin-1β (IL-1β) was unable to induce the expression of iNOS and the production of NO in rat C6 glial cells. Similarly, wortmannin and LY294002, compounds that inhibit PI 3-kinase, were also unable to induce the expression of iNOS and the production of NO. However, a combination of wortmannin or LY294002 with LPS or IL-1β induced the expression of iNOS and the production of NO in C6 glial cells. Consistent with the induction of iNOS, wortmannin also induced iNOS promoter-derived chloramphenicol acetyltransferase activity in LPS- or IL-1β-treated C6 glial cells. The expression of iNOS by LPS in C6 glial cells expressing a dominant-negative mutant of p85α, the regulatory subunit of PI 3-kinase, further supports the conclusion that inhibition of PI 3-kinase provides a necessary signal for the induction of iNOS. Next we examined the effect of wortmannin on the activation of mitogen-activated protein (MAP) kinase and nuclear factor NF-κB in LPS- or IL-1β-stimulated C6 glial cells. In contrast to the inability of LPS and IL-1β alone to induce the expression of iNOS, both LPS and IL-1β individually stimulated MAP kinase activity and induced DNA binding and transcriptional activity of NF-κB. Wortmannin alone was unable to activate MAP kinase and NF-κB. Moreover, wortmannin had no effect on LPS- or IL-1β-mediated activation of MAP kinase and NF-κB, suggesting that wortmannin induced the expression of iNOS in LPS- or IL-1β-stimulated C6 glial cells without modulating the activation of MAP kinase and NF-κB. Similar to C6 glial cells, wortmannin also stimulated LPS-mediated expression of iNOS and production of NO in astrocytes without affecting the LPS-mediated activation of NF-κB. Taken together, the results from specific chemical inhibitors and dominant-negative mutant expression studies demonstrate that apart from the activation of NF-κB, inhibition of PI 3-kinase is also necessary for the expression of iNOS and production of NO.
      Nitric oxide (NO),
      The abbreviations used are: NO, nitric oxide; NOS, nitric-oxide synthase(s); iNOS, inducible NOS; LPS, lipopolysaccharide; IL-1β, interleukin-1β; IFN-γ, interferon-γ; NF-κB, nuclear factor κB; MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase kinase; PI 3-kinase, phosphatidylinositol 3-kinase; DMEM, Dulbecco's modified Eagle's medium; CAT, chloramphenicol acetyltransferase; Erk, extracellular signal-regulated kinase; l-NMA, l-N-methylarginine
      1The abbreviations used are: NO, nitric oxide; NOS, nitric-oxide synthase(s); iNOS, inducible NOS; LPS, lipopolysaccharide; IL-1β, interleukin-1β; IFN-γ, interferon-γ; NF-κB, nuclear factor κB; MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase kinase; PI 3-kinase, phosphatidylinositol 3-kinase; DMEM, Dulbecco's modified Eagle's medium; CAT, chloramphenicol acetyltransferase; Erk, extracellular signal-regulated kinase; l-NMA, l-N-methylarginine
      a vascular and neuronal messenger and a cytotoxic and cytostatic agent, is enzymatically formed from l-arginine by the enzyme nitric-oxide synthase (NOS). The NOS are basically divided into two forms. One constitutive form present in neurons (nNOS) and endothelial cells (eNOS) is a calcium-dependent enzyme, and the inducible form (iNOS) present in macrophages and astrocytes is regulated at the transcriptional level in response to stimuli (e.g. cytokine/lipopolysaccharide (LPS)) and does not require calcium for its activity (
      • Jaffrey S.R.
      • Snyder S.H.
      ,
      • Nathan C.
      ). Although the NO produced by iNOS accounts for the bactericidal and tumoricidal properties of macrophages, it is also of particular importance in the pathophysiologies of inflammatory neurological diseases including demyelinating disorders (e.g. multiple sclerosis, experimental allergic encephalopathy, X-adrenoleukodystrophy) and in ischemia and traumatic injuries associated with infiltrating macrophages and the production of proinflammatory cytokines (
      • Mitrovic B.
      • Ignarro L.J.
      • Montestruque S.
      • Smoll A.
      • Merril J.E.
      ,
      • Bo L.
      • Dawson T.M.
      • Wesselingh S.
      • Mork S.
      • Choi S.
      • Kong P.A.
      • Hanley D.
      • Trapp B.D.
      ,
      • Merrill J.E.
      • Ignarro L.J.
      • Sherman M.P.
      • Melinek J.
      • Lane T.E.
      ,
      • Koprowski H.
      • Zheng Y.M.
      • Heber-Katz E.
      • Fraser N.
      • Rorke L.
      • Fu Z.F.
      • Hanlon C.
      • Dietzshold B.
      ,
      • Cross A.H.
      • Misko T.P.
      • Lin R.F.
      • Hickey W.F.
      • Trotter J.L.
      • Tilton R.G.
      ,
      • Hooper D.C.
      • Bagsra O.
      • Marini J.C.
      • Zborek A.
      • Ohnishi S.T.
      • Kean R.
      • Champion J.M.
      • Sarker A.B.
      • Bobroski L.
      • Farber J.L.
      • Akaike T.
      • Maeda H.
      • Koprowski H.
      ). It is now increasingly clear that glial cells in the central nervous system also produce NO in response to the induction of iNOS by bacterial LPS and a series of cytokines including interleukin-1β (IL-1β), tumor necrosis factor-α, and interferon-γ (IFN-γ). Astrocytes in the healthy brain do not express iNOS; however, after ischemic, traumatic, neurotoxic, or inflammatory damage the reactive astrocytes express iNOS in the mouse, rat, and human (
      • Hu S.X.
      • Sheng W.S.
      • Peterson P.K.
      • Chao C.C.
      ,
      • Galea E.
      • Feinstein D.L.
      • Reis D.J.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ). NO derived from both astrocytes and macrophages is assumed to contribute to oligodendrocyte degeneration in demyelinating diseases and neuronal death during ischemia and trauma (
      • Mitrovic B.
      • Ignarro L.J.
      • Montestruque S.
      • Smoll A.
      • Merril J.E.
      ,
      • Bo L.
      • Dawson T.M.
      • Wesselingh S.
      • Mork S.
      • Choi S.
      • Kong P.A.
      • Hanley D.
      • Trapp B.D.
      ,
      • Merrill J.E.
      • Ignarro L.J.
      • Sherman M.P.
      • Melinek J.
      • Lane T.E.
      ).
      Characterization of the intracellular pathways required to transduce the signal from the cell surface to the nucleus for the induction of iNOS is an active area of investigation. Identification of the DNA binding site for nuclear factor (NF)-κB in the promoter region of iNOS (
      • Xie Q.-W.
      • Kashiwabara Y.
      • Nathan C.
      ) and inhibition of iNOS induction by inhibitors of NF-κB activation have established an essential role of NF-κB activation in the induction of iNOS (
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Kwon G.
      • Corbett J.A.
      • Rodi C.P.
      • Sullivan P.
      • McDaniel M.L.
      ). Suppression of NF-κB and inhibition of iNOS expression (
      • Feinstein D.L.
      • Galea E.
      • Cermak J.
      • Chugh P.
      • Lyandvert L.
      • Reis D.J.
      ,
      • Nishiya T.
      • Uehara T.
      • Nomura Y.
      ) by inhibitors of tyrosine kinase in different cell types suggest the possible involvement of tyrosine phosphorylation in the activation of NF-κB and the induction of iNOS. Inhibition of LPS- and cytokine-induced activation of NF-κB and induction of iNOS by inhibitors of the mevalonate pathway and Ras farnesyl protein transferase also indicate that Ras may be involved in the activation of NF-κB and the induction of iNOS (
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ). Again, increasing cAMP and protein kinase A activity has been shown to inhibit the activation of NF-κB and the induction of iNOS possibly because of the inhibition of Raf-1 (
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ). Recently we have also observed that PD98059, an inhibitor of mitogen-activated protein (MAP) kinase kinase (MEK), the kinase responsible for the activation of MAP kinase, inhibits the LPS-induced activation of NF-κB and the induction of iNOS in astrocytes, suggesting the possible involvement of the MAP kinase pathway in the LPS- and proinflammatory cytokine-mediated induction of iNOS (
      • Pahan K.
      • Sheikh F.G.
      • Khan M.
      • Namboodiri A.M.S.
      • Singh I.
      ). Taken together, these studies suggest that any alteration of the Ras-Raf-MEK-MAP kinase signal transduction pathway alters the activation of NF-κB and so the induction of iNOS in astrocytes and C6 glial cells.
      In this paper we present evidence that the signal mediated by inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) induces/stimulates the expression of iNOS in LPS- or cytokine-stimulated C6 glial cells and rat primary astrocytes and that the signal is not mediated via MAP kinase and NF-κB. Specific inhibitors of PI 3-kinase (wortmannin and LY294002) and expression of the dominant-negative mutant of p85α, the regulatory subunit of PI 3-kinase, induced the expression of iNOS in LPS- or cytokine-stimulated C6 glial cells or stimulated the expression of iNOS in rat primary astrocytes without modulating the LPS- or cytokine-mediated activation of MAP kinase and NF-κB, suggesting that apart from the activation of NF-κB by LPS or cytokines, the inhibition of PI 3-kinase also provides an essential signal for the expression of iNOS and production of NO in C6 glial cells and astrocytes.

      MATERIALS AND METHODS

      Reagents

      Recombinant rat IFN-γ, DMEM/F-12, fetal bovine serum, Hanks' balanced salt solution, and NF-κB DNA-binding protein detection kit were from Life Technologies, Inc. Human IL-1β was from Genzyme. LPS (Escherichia coli) and pyrrolidine dithiocarbamate were purchased from Sigma. Phosphatidylinositol and phosphatidylserine were purchased from Matreya Inc. Wortmannin, LY294002, and antibodies against the regulatory subunit of PI 3-kinase (p85α) were obtained from Calbiochem. Antibodies against mouse macrophage iNOS were obtained from Transduction Laboratories, [γ-32P]ATP (3,000 Ci/mmol) was from Amersham Pharmacia Biotech.

      Induction of NO Production in Astrocytes and C6 Glial Cells

      Astrocytes were prepared from rat cerebral tissue as described by McCarthy and DeVellis (
      • McCarthy K.
      • DeVellis J.
      ). Cells were maintained in DMEM/F-12 medium containing 10% fetal bovine serum. After 10 days of culture astrocytes were separated from microglia and oligodendrocytes by shaking for 24 h in an orbital shaker at 240 rpm. The shaking was repeated twice more after a gap of 1 or 2 weeks before subculturing to ensure the complete removal of all of the oligodendrocytes and microglia. Cells were trypsin treated, subcultured, and stimulated with LPS or different cytokines in serum-free DMEM/F-12. C6glial cells obtained from ATCC were also maintained and induced with different stimuli as above.

      Assay for NO Synthesis

      Synthesis of NO was determined by an assay of the culture supernatant for nitrite, a stable reaction product of NO with molecular oxygen. Briefly, 400 μl of culture supernatant was allowed to react with 200 μl of Griess reagent (
      • Feinstein D.L.
      • Galea E.
      • Roberts S.
      • Berquist H.
      • Wang H.
      • Reis D.J.
      ) and incubated at room temperature for 15 min. The optical density of the assay samples was measured spectrophotometrically at 570 nm. Fresh culture media served as the blank in all experiments. Nitrite concentrations were calculated from a standard curve derived from the reaction of NaNO2 in the assay.

      Immunoblot Analysis for iNOS

      After a 24-h incubation in the presence or absence of different stimuli, cells were scraped off, washed with Hanks' buffer, and homogenized in 50 mmTris-HCl (pH 7.4) containing protease inhibitors (1 mmphenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml antipain, 5 μg/ml pepstatin A, and 5 μg/ml leupeptin). After electrophoresis the proteins were transferred onto a nitrocellulose membrane, and the iNOS band was visualized by immunoblotting with antibodies against mouse macrophage iNOS and 125I-labeled protein A (
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ).

      RNA Isolation and Northern Blot Analysis

      Cells were taken out of the culture dishes directly by adding Ultraspec-II RNA reagent (Biotecx Laboratories Inc.), and total RNA was isolated according to the manufacturer's protocol. For Northern blot analyses, 20 μg of total RNA was electrophoresed on 1.2% denaturing formaldehyde-agarose gels, electrotransferred to Hybond nylon membrane (Amersham Pharmacia Biotech), and hybridized at 68 °C with 32P-labeled cDNA probe using Express Hyb hybridization solution (CLONTECH) as described by the manufacturer. The cDNA probe was made by polymerase chain reaction amplification using two primers (forward primer: 5′-CTC CTT CAA AGA GGC AAA AAT A-3′; reverse primer: 5′-CAC TTC CTC CAG GAT GTT GT-3′) (
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ,
      • Geller D.A.
      • Lowenstein C.J.
      • Shapiro R.A.
      • Nussler A.K.
      • DiSilvio M.
      • Wang S.C.
      • Nakayama D.K.
      • Simmons R.L.
      • Snyder S.H.
      • Billiar T.R.
      ). After hybridization, the filters were washed two or three times in solution I (2 × SSC, 0.05% SDS) for 1 h at room temperature followed by solution II (0.1 × SSC, 0.1% SDS) at 50 °C for another hour. The membranes were then dried and exposed to x-ray films (Kodak). The same filters were stripped and rehybridized with probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative mRNA content for iNOS (iNOS/GAPDH) was measured after scanning the bands with a Bio-Rad (model GS-670) imaging densitometer.

      Construction of Reporter Plasmid, Transfection, and Assay of Chloramphenicol Acetyltransferase (CAT) Activity

      The CAT under the control of the iNOS promoter was created by subcloning a 1.5-kilobase promoter from pGEM-NOS at the SphI and SalI restriction sites of pCAT-basic vector (Promega). The full-length promoter (
      • Eberhardt W.
      • Kunz D.
      • Hummel R.
      • Pfeilschifler J.
      ) was amplified by using two primers (forward: 5′-GAG AGT GTG CAA GTA TTT GTA GGA G-3′ and reverse: 5′-AAG GTG GCT GAG AAG TTT CA-3′) from rat genomic DNA and cloned in pGEM-T vector (Promega) to produce pGEM-NOS. The clone was confirmed by restriction mapping and sequencing. The cells were transfected with 2 μg of reporter plasmid by using the Lipotaxi (Stratagene) method, as has been described in manufacturer's protocol. 24 h after transfection, cells were treated with different stimuli for 14 h and harvested. The radioisotopic method was used to assay CAT activity using a kit (Promega) as described by manufacturer's protocol.

      Expression of the Dominant-negative Mutant of p85α in C6 Glial Cells

      In the dominant-negative form of p85α, 35 amino acids in the inter-SH2 region from residues 479–513 of wild type p85α, important for binding the p110 subunit of PI 3-kinase, are deleted, and two other amino acids (Ser-Arg) are inserted in this deleted position. The engineering of the construct and description of the vector driving the expression of the proteins have been published previously (
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jackson R.
      • Hawkin P.T.
      • Dhand R.
      • Clark A.E.
      • Holman G.D.
      • Waterfied M.D.
      • Kasuga M.
      ). C6 glial cells were transfected with either the dominant-negative form of p85α or an empty vector by Lipotaxi following manufacturer's protocol. 24 h after transfection, cells were treated with different stimuli.

      Assay of PI 3-Kinase

      After stimulation in serum-free DMEM/F-12 cells were lysed with ice-cold lysis buffer containing 1% v/v Nonidet P-40, 100 mm NaCl, 20 mm Tris (pH 7.4), 10 mm iodoacetamide, 10 mm NaF, 1 mm sodium orthovanadate, 1 mmphenylmethylsulfonyl chloride, 1 μg/ml leupeptin, 1 μg/ml antipain, 1 μg/ml aprotinin, and 1 μg/ml pepstatin A. Lysates were incubated at 4 °C for 15 min followed by centrifugation at 13,000 ×g for 15 min. The supernatant was precleared with protein G-Sepharose beads (Amersham Pharmacia Biotech) for 1 h at 4 °C followed by the addition of 1 μg/ml p85α monoclonal antibody. After a 2-h incubation at 4 °C, protein G-Sepharose beads were added, and the resulting mixture was further incubated for 1 h at 4 °C. The immunoprecipitates were washed twice with lysis buffer, once with phosphate-buffered saline, once with 0.5 m LiCl and 100 mm Tris (pH 7.6), once in water, and once in kinase buffer (5 mm MgCl2, 0.25 mm EDTA, 20 mm HEPES (pH 7.4)). PI 3-kinase activity was determined as described (
      • Ward S.G.
      • Reif K.
      • Ley S.
      • Fry M.J.
      • Waterfield M.D.
      • Cantrell D.A.
      ) using a lipid mixture of 100 μl of 0.1 mg/ml PI and 0.1 mg/ml phosphatidylserine dispersed by sonication in 20 mm HEPES (pH 7.0) and 1 mm EDTA. The reaction was initiated by the addition of 20 μCi of [γ-32P]ATP (3,000 Ci/mmol; NEN Life Science Products) and 100 μm ATP and terminated after 15 min by the addition of 80 μl of 1n HCl and 200 μl of chloroform:methanol (1:1). Phospholipids were separated by TLC and visualized by exposure to iodine vapor and autoradiography (
      • Ward S.G.
      • Reif K.
      • Ley S.
      • Fry M.J.
      • Waterfield M.D.
      • Cantrell D.A.
      ).

      Assay of MAP Kinase

      Cells were lysed directly with 2 × SDS sample buffer, and the lysates were boiled, electrophoresed in 4–20% gradient gels, transferred onto nitrocellulose membranes, and immunoblotted with phospho-specific MAP kinase antibody (New England Biolabs). Phospho-specific p44/42 MAP kinase antibody detects p42 and p44 MAP kinase (Erk1 and Erk2) only when activated by phosphorylation at Tyr-204.

      Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay

      Nuclear extracts from stimulated or unstimulated astrocytes (1 × 107 cells) were prepared using the method of Dignam et al. (
      • Dignam D.
      • Lebovitz R.M.
      • Roeder R.G.
      ) with slight modification. Cells were harvested, washed twice with ice-cold phosphate-buffered saline, and lysed in 400 μl of buffer A (10 mm HEPES (pH 7.9), 10 mm KCl, 2 mm MgCl2, 0.5 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml pepstatin A, and 5 μg/ml leupeptin) containing 0.1% Nonidet P-40 for 15 min on ice, vortexed vigorously for 15 s, and centrifuged at 14,000 rpm for 30 s. The pelleted nuclei were resuspended in 40 μl of buffer B (20 mm HEPES (pH 7.9), 25% (v/v) glycerol, 0.42 mNaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm dithiothreitol, 1 mmphenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml pepstatin A, and 5 μg/ml leupeptin). After 30 min on ice, lysates were centrifuged at 14,000 rpm for 10 min. Supernatants containing the nuclear proteins were diluted with 20 μl of modified buffer C (20 mm HEPES (pH 7.9), 20% (v/v) glycerol, 0.05 mKCl, 0.2 mm EDTA, 0.5 mm dithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride) and stored at −70 °C until use. Nuclear extracts were used for the electrophoretic mobility shift assay using the NF-κB DNA-binding protein detection system kit (Life Technologies, Inc.) according to the manufacturer's protocol.

      Assay of Transcriptional Activity of NF-κB

      To assay the transcriptional activity of NF-κB, cells were transfected with pNF-κB-Luc, an NF-κB-dependent reporter construct (obtained from Stratagene), using the Lipotaxi method. 24 h after transfection, cells were treated with different stimuli for 4 h. Total cell extracts were used to measure luciferase activity in a scintillation counter (Beckman LS 3801) (
      • Nguyen V.T.
      • Morange M.
      • Bensaude O.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ) using an assay kit from Stratagene.

      Cell Viability

      The cytotoxic effects of all of the inhibitors were determined by measuring the metabolic activity of cells with the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay.

      RESULTS

      Inhibitors of PI 3-Kinase (Wortmannin and LY294002) Induce the Expression of iNOS and Production of NO in LPS-stimulated Rat C6 Glial Cells

      We investigated the effect of specific inhibitors of PI 3-kinase (wortmannin and LY294002) on the induction of iNOS and production of NO in C6 glial cells. C6glial cells were cultured in serum-free DMEM/F-12 in the presence of LPS and inhibitors of PI 3-kinase. Consistent with previous observations (
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Feinstein D.L.
      • Galea E.
      • Cermak J.
      • Chugh P.
      • Lyandvert L.
      • Reis D.J.
      ,
      • Feinstein D.L.
      • Galea E.
      • Roberts S.
      • Berquist H.
      • Wang H.
      • Reis D.J.
      ,
      • Dobashi K.
      • Pahan K.
      • Chahal A.
      • Singh I.
      ), the bacterial LPS or cytokines alone did not induce the production of NO in C6 glial cells (Table I). Wortmannin and LY294002 alone were also unable to induce the production of NO; however, addition of these inhibitors along with LPS induced the production of NO as nitrite by about 8–10-fold (Table I). Inhibition of this NO production by arginase, an enzyme that degrades the substrate (l-arginine) of NOS, and l-NMA, a competitive inhibitor of NOS activity, suggests that wortmannin- or LY294002-induced NO production in LPS-stimulated C6 glial cells is dependent on NOS-mediated arginine metabolism (Table I). To understand the mechanism of the PI 3-kinase inhibitor-induced production of NO in LPS-treated C6 glial cells, we examined the effect of these inhibitors on the protein and mRNA level of iNOS. Fig. 1 shows that wortmannin dose-dependently induced the production of NO (Fig. 1A) and the expression of iNOS protein (Fig. 1B) in LPS-treated C6 glial cells. The lowest dose of wortmannin found to induce the production of NO and the expression of iNOS protein was 50 nm. At 300 nm, the production of NO and the expression of iNOS protein were found to be maximum (Fig. 1). Similar to the effect of wortmannin in LPS-treated C6 glial cells, wortmannin also induced the production of NO in IL-1β-treated C6 cells (Fig. 2A). However, wortmannin was unable to induce the production of NO in IFN-γ-treated cells. Consistent with the effect of wortmannin on the production of NO, wortmannin was also able to induce the expression of iNOS protein (Fig. 2B) and mRNA (Fig. 2C) in LPS- or IL-1β-treated cells. Although LPS, IL-1β, and IFN-γ individually were unable to induce the expression of iNOS and the production of NO, different combinations of these stimuli (e.g. LPS + IL-1β; LPS + IFN-γ) induced the production of NO (Fig. 2A) and the expression of iNOS protein (Fig. 2B) and mRNA (Fig. 2C). To understand the effect of wortmannin on the transcription of the iNOS gene, C6 cells were transfected with a construct containing the iNOS promoter fused to the CAT gene. Activation of this promoter was measured after stimulating the cells with LPS or cytokines in the presence or absence of wortmannin. Consistent with the effect of wortmannin on the production of NO and the expression of endogenous iNOS, wortmannin itself had no effect on CAT activity, but it induced the CAT activity in LPS- or IL-1β-treated C6 cells (Fig. 2D). Again, wortmannin was unable to induce the CAT activity in IFN-γ-treated cells. These results indicate that inhibition of PI 3-kinase by wortmannin is able to provide a necessary signal for the transcription of the iNOS gene in LPS- or IL-1β-stimulated C6cells.
      Table IEffect of inhibitors of PI 3-kinase phosphatases on LPS-induced production of NO in C6 glial cells
      StimuliNitrite
      nmol/mg/24 h
      Control3.4 ± 0.3
      LPS only3.7 ± 0.4
      Wort only3.5 ± 0.35
      LY29 only3.3 ± 0.25
      LPS + Wort32.6 ± 3.6
      LPS + LY2930.2 ± 2.8
      LPS + Wort + l-NMA5.1 ± 0.6
      LPS + LY29 + l-NMA4.9 ± 0.5
      LPS + Wort + arginase5.8 ± 0.4
      LPS + LY29 + arginase5.2 ± 0.7
      C6 glial cells preincubated in serum-free DMEM/F-12 for 30 min with l-NMA or arginase received LPS and/or wortmannin (Wort) and LY294002 (LY29). After a 24-h incubation, nitrite concentrations in the supernatants were measured as described under “Materials and Methods.” Data are expressed as the mean ± S.D. of three different experiments. The concentrations of different compounds were as follows: LPS, 0.5 μg/ml; wortmannin, 300 nm; LY294002, 30 μm; l-NMA, 0.1 mm; arginase, 100 units/ml.
      Figure thumbnail gr1
      Figure 1Wortmannin and LY294002 dose-dependently induce the expression of iNOS in LPS-stimulated C6 glial cells. Cells incubated in serum-free DMEM/F-12 received different concentrations of wortmannin (Wort) or LY294002 (LY) along with 0.5 μg/ml LPS. Panel A, after 24 h, the concentration of nitrite was measured in the supernatants as described under “Materials and Methods.” Data are the mean ± S.D. of three different experiments. Panel B, cell homogenates were electrophoresed, transferred onto nitrocellulose membrane, and immunoblotted with antibodies against mouse macrophage iNOS as described under “Materials and Methods.”
      Figure thumbnail gr2
      Figure 2Effect of wortmannin on the expression of iNOS in LPS- or cytokine-treated C6glial cells. Panel A, cells incubated in serum-free DMEM/F-12 received LPS and cytokines in the presence or absence of wortmannin. After a 24-h incubation, nitrite concentrations were measured in the supernatants. Data are the mean ± S.D. of three different experiments. Panel B, cell homogenates were electrophoresed, transferred onto nitrocellulose membrane, and immunoblotted with antibodies against mouse macrophage iNOS as described before. Panel C, after a 6-h incubation, cells were taken out directly by adding Ultraspec-II RNA reagent to the plates for isolation of total RNA, and Northern blot analysis for iNOS mRNA was carried out as described under “Materials and Methods.” GAPDH, glyceraldehyde-3-phosphate dehydrogenase.Panel D, cells were transfected with the construct containing the iNOS promoter fused to the CAT gene using Lipotaxi. 24 h after transfection, cells received LPS and cytokines in the presence or absence of wortmannin (Wort); after 14 h of stimulation, CAT activity was measured. Data are the mean ± S.D. of three different experiments. The concentrations of the different stimuli were as follows: LPS, 0.5 μg/ml; IL-1β, 50 ng/ml; IFN-γ, 50 units/ml; wortmannin, 300 nm.

      Expression of a Dominant-negative Mutant of p85α Induces the Expression of iNOS in LPS-stimulated C6 Glial Cells

      Induction of NOS by wortmannin or LY294002, inhibitors of PI 3-kinase, in LPS- or cytokine-stimulated C6 glial cells suggests that inhibition of PI 3-kinase activity may provide an essential signal for the expression of iNOS. To confirm this observation that inhibition of PI 3-kinase is able to induce the expression of iNOS in LPS-treated C6 glial cells, we transfected C6 glial cells with a dominant-negative mutant of p85α. PI 3-kinase is a heterodimer consisting of 85-kDa (p85) and 110-kDa (p110) subunits where p85 is the regulatory subunit that links PI 3-kinase activity in the catalytic subunit (p110) to the tyrosine-phosphorylated proteins. Expression of a dominant-negative mutant of p85α, in which the inter-SH2 region required for binding of the p110 subunit is disrupted, results in the inhibition of PI 3-kinase activity in different cell types including adipocytes and Chinese hamster ovary cells (
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jackson R.
      • Hawkin P.T.
      • Dhand R.
      • Clark A.E.
      • Holman G.D.
      • Waterfied M.D.
      • Kasuga M.
      ,
      • Teruel T.
      • Valverde A.M.
      • Navarro P.
      • Benito M.
      • Lorenzo M.
      ). We have also found that expression of the same dominant-negative mutant of p85α in C6 glial cells inhibited the lipid kinase activity of PI 3-kinase, but expression of the control empty vector had no effect (Fig. 3), indicating that the overexpressed dominant-negative mutant protein of p85α did not associate with the catalytic subunit of PI 3-kinase. In control C6 cells as well as in vector-transfected cells, LPS was unable to induce the production of NO and the expression of iNOS protein (Fig. 4). However, LPS induced the production of NO and the expression of iNOS protein in C6 cells transfected with the dominant-negative mutant of p85α (Fig. 4), suggesting that inhibition of PI 3-kinase activity is sufficient to induce the expression of iNOS in LPS-treated C6 glial cells.
      Figure thumbnail gr3
      Figure 3Expression of a dominant-negative mutant of p85α inhibits the lipid kinase activity of PI 3-kinase in C6 glial cells. Cells were transfected with various concentrations of a dominant-negative mutant of p85α and the control empty vector using Lipotaxi as described under “Materials and Methods.” After 24 h of transfection, cells were maintained in serum-free media for 24 h, and the lipid kinase activity of PI 3-kinase of was determined in cell lysates as described under “Materials and Methods.”
      Figure thumbnail gr4
      Figure 4Expression of a dominant-negative mutant of p85α induces the expression of iNOS in LPS-treated C6 glial cells. Cells were transfected with various concentrations of a dominant-negative mutant of p85α and the control empty vector using Lipotaxi as described. After 24 h of transfection, cells were stimulated with LPS (0.5 μg/ml) for 24 h, and nitrite concentrations (panel A) were measured in the supernatants as described. Data are the mean ± S.D. of three different experiments. Panel B, cell homogenates were electrophoresed, transferred onto nitrocellulose membrane, and immunoblotted with antibodies against mouse macrophage iNOS as described.

      Wortmannin Induces the Expression of iNOS in LPS- or IL-1β-treated C6 Glial Cells without Modulating the Activation of MAP Kinase and NF-κB

      Because the activation of NF-κB is necessary for the expression of iNOS (
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Xie Q.-W.
      • Kashiwabara Y.
      • Nathan C.
      ,
      • Kwon G.
      • Corbett J.A.
      • Rodi C.P.
      • Sullivan P.
      • McDaniel M.L.
      ,
      • Feinstein D.L.
      • Galea E.
      • Cermak J.
      • Chugh P.
      • Lyandvert L.
      • Reis D.J.
      ,
      • Nishiya T.
      • Uehara T.
      • Nomura Y.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Khan M.
      • Namboodiri A.M.S.
      • Singh I.
      ), and PD98059, an inhibitor of MEK, inhibits the LPS-induced expression of iNOS in astrocytes (
      • Pahan K.
      • Sheikh F.G.
      • Khan M.
      • Namboodiri A.M.S.
      • Singh I.
      ), to understand the basis of wortmannin-induced expression of iNOS in LPS- or IL-1β-treated C6 glial cells, we examined the effect of wortmannin on the activation of MAP kinase and NF-κB. Treatment of C6 glial cells with LPS alone resulted in the time-dependent activation of both Erk1 and Erk2 as evident from the Western blot analysis of stimulated C6 glial cells with antibodies against tyrosine-phosphorylated MAP kinase (Fig. 5). This activation was maximum after 10 min of treatment; however, with the increase in time of incubation phosphorylated Erk1 and Erk2 gradually decreased. Therefore, for subsequent experiments, cells were stimulated for 10 min, and activation of MAP kinase was monitored. Although LPS and IL-1β alone were ineffective in inducing the expression of iNOS, both of the stimuli, alone or together, induced the activation of MAP kinase in C6 glial cells (see Fig. 7A). Wortmannin, capable of inducing the expression of iNOS in LPS- and IL-1β-stimulated C6 cells, had no effect on LPS- and IL-1β-mediated phosphorylation of MAP kinase (see Fig. 7A), suggesting that wortmannin induced the expression of iNOS in LPS- or IL-1β-treated C6 cells without modulating the MAP kinase pathway. Next we examined the effect of wortmannin on the activation of NF-κB. Activation of NF-κB was monitored by both DNA binding as well as transcriptional activity of NF-κB. The DNA binding activity of NF-κB was evaluated by the formation of a distinct and specific complex in a gel shift DNA binding assay. Treatment of C6 glial cells with 0.5 μg/ml LPS resulted in the induction of DNA binding activity of NF-κB (Fig. 6). This gel shift assay detected a specific band in response to LPS which was competed off by an unlabeled probe (Fig. 6). In contrast to the inability of LPS or IL-1β to induce the expression of iNOS, both of these stimuli induced the DNA binding activity of NF-κB (Fig. 7B). Wortmannin alone neither induced the DNA binding activity of NF-κB nor modulated the LPS- and IL-1β-mediated DNA binding activity of NF-κB (Fig. 7B). We then tested the effect of wortmannin on NF-κB-dependent transcription of luciferase in C6 glial cells in the presence or absence of LPS and cytokines, using the expression of luciferase from a reporter construct, pNF-κB-Luc (Stratagene), as an assay. Consistent with the effect of wortmannin on DNA binding activity of NF-κB, wortmannin alone did not induce the NF-κB-dependent transcription of luciferase, and it also had no effect on the magnitude of LPS- and IL-1β-induced transcriptional activity of NF-κB (Fig. 7C). On the other hand, consistent with the inability of IFN-γ to induce the expression of iNOS in C6 glial cells, IFN-γ did not induce the DNA binding or transcriptional activity of NF-κB, whereas the combination of LPS and IFN-γ was able to induce the DNA binding as well as transcriptional activity of NF-κB (Fig. 7,B and C) and the induction of iNOS (Fig. 2). To examine whether wortmannin-induced expression of iNOS in LPS- or cytokine-treated C6 cells requires the activation of NF-κB, we studied the effect of pyrrolidine dithiocarbamate, an antioxidant inhibitor of NF-κB activation, on the induction of iNOS and the activation of NF-κB in cells treated with the combination of LPS and wortmannin. Pyrrolidine dithiocarbamate inhibited the activation of NF-κB and the induction of NO production in LPS- and wortmannin-treated C6 cells (Fig. 8). Taken together, these studies indicate that activation of NF-κB is necessary but not sufficient for the induction of iNOS, and the signal induced by inhibition of PI 3-kinase by wortmannin for the induction of iNOS is not mediated via activation of MAP kinase and NF-κB.
      Figure thumbnail gr5
      Figure 5Time course of LPS-induced activation of MAP kinase in C6 glial cells. Cells incubated in serum-free DMEM/F-12 received LPS (0.5 μg/ml). After different minute intervals, cells were lysed directly with SDS-sample buffer, boiled, electrophoresed in 4–20% gradient gels, transferred onto nitrocellulose membranes, and immunoblotted with antibodies against tyrosine-phosphorylated MAP kinase (New England Biolabs) as described under “Materials and Methods.” Phosphorylated bands were detected by exposure to film at −70 °C (upper panels) and quantitated by densitometry (lower panels). Data are from a single experiment representative of at least three others.
      Figure thumbnail gr7
      Figure 7Effect of wortmannin on the activation of MAP kinase and NF-κB in LPS- and cytokine-treated C6 glial cells. Panel A, cells incubated in serum-free DMEM/F-12 received LPS and cytokines in the presence or absence of wortmannin (Wort). After a 15-min incubation, cells were lysed and immunoblotted for MAP kinase as described. Panel B, cells incubated in serum-free DMEM/F-12 received LPS and cytokines in the presence or absence of wortmannin. After a 1-h incubation, cells were taken out to prepare nuclear extracts, and nuclear proteins were used for the electrophoretic mobility shift assay as described. Panel C, cells were transfected with pNF-κB-Luc using the Lipotaxi method. 24 h after transfection, the cells were stimulated with LPS and cytokines in the presence or absence of wortmannin for 4 h, and the expression of the luciferase reporter was quantitated as described under “Materials and Methods.” The concentrations of the different stimuli were as follows: LPS, 0.5 μg/ml; IL-1β, 50 ng/ml; IFN-γ, 50 units/ml; and wortmannin, 300 nm.
      Figure thumbnail gr6
      Figure 6LPS induces the DNA binding activity of NF-κB in C6 glial cells. Cells incubated in serum-free DMEM/F-12 were treated with LPS (0.5 μg/ml). After a 1-h incubation, cells were taken out to prepare nuclear extracts, and nuclear proteins were used for the electrophoretic mobility shift assay as described under “Materials and Methods.” Lanes 1–4 represent nuclear extract of control cells, nuclear extract of LPS-treated cells, nuclear extract of LPS-treated cells incubated with a 50-fold excess of unlabeled oligonucleotide, and nuclear extract of LPS-treated cells incubated with a 100-fold excess of unlabeled oligonucleotide. The upper arrow indicates the induced NF-κB band, and the lower arrow indicates the unbound probe.
      Figure thumbnail gr8
      Figure 8Pyrrolidine dithiocarbamate inhibits the induction of NO production and the activation of NF-κB in C6 glial cells treated with the combination of LPS and wortmannin. Cells preincubated with different concentrations of pyrrolidine dithiocarbamate (PDTC) for 1 h in serum-free media received the combination of LPS (0.5 μg/ml) and wortmannin (Wort; 300 μm). Panel A, after a 24-h incubation, nitrite concentrations were measured in the supernatants. Data are the mean ± S.D. of three different experiments. Panel B, after a 1-h incubation, cell were taken out to prepare nuclear extracts, and nuclear proteins were used for the electrophoretic mobility shift assay for the DNA binding activity of NF-κB as described under “Materials and Methods.”

      Inhibition of PI 3-Kinase Is Necessary for the Expression of iNOS in C6 Glial Cells

      Because inhibitors of PI 3-kinase induced the expression of iNOS in LPS- or IL-1β-treated C6 cells, we sought to examine whether inhibition of PI 3-kinase is necessary for the expression of iNOS in C6cells. Cells treated with LPS and IL-1β, alone or in combination, for different time intervals were assayed for the lipid kinase activity of PI 3-kinase. Although LPS or IL-1β alone had no effect on PI 3-kinase activity (Fig. 9, A and B), the combination of LPS and IL-1β inhibited the activity of PI 3-kinase within 5–10 min of incubation (Fig. 9C). Consistent with the inhibitory effect of wortmannin on PI 3-kinase activity in other cell types (
      • Okada T.
      • Kawano Y.
      • Sakakakibara T.
      • Hazeki M.
      • Ui M.
      ,
      • Powis G.
      • Bonjouklian R.
      • Berggren M.M.
      • Gallegos A.
      • Abraham R.
      • Ashendel C.
      • Zalkow L.
      • Matter W.F.
      • Dodge J.
      • Grindey G.
      • Vlahos C.J.
      ), wortmannin inhibited the lipid kinase activity of PI 3-kinase in LPS-treated C6cells (Fig. 9D). These results indicate that inhibition of PI 3-kinase activity may be necessary to induce the expression of the iNOS gene in C6 glial cells.
      Figure thumbnail gr9
      Figure 9Activity of PI 3-kinase in LPS- and cytokine-treated C6 glial cells. Cells treated with LPS (0.5 μg/ml) (panel A), IL-1β (50 ng/ml) (panel B), the combination of LPS and IL-1β (panel C), or the combination of LPS and wortmannin (panel D) in serum-free media were lysed, immunoprecipitated with monoclonal antibodies against p85α, and the lipid kinase activity of immunoprecipitated PI 3-kinase was assayed as described under “Materials and Methods.” Lipids were detected by exposure to film at −70 °C (upper panels) and quantitated by densitometry (lower panels). Data are from a single experiment representative of at least three others.

      Wortmannin Stimulates the LPS-induced Production of NO in Rat Primary Astrocytes without Modulating the Activation of NF-κB

      Because wortmannin induces the production of NO and the expression of iNOS in LPS- or cytokine-treated C6 glial cells without modulating the activation of NF-κB, we investigated the effect of wortmannin on LPS-induced production of NO and the activation of NF-κB in rat primary astrocytes. In sharp contrast to the inability of LPS to induce the expression of iNOS and the production of NO in C6 glial cells, LPS alone was able to induce the expression of iNOS and the production of NO in rat primary astrocytes as reported previously (
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Feinstein D.L.
      • Galea E.
      • Cermak J.
      • Chugh P.
      • Lyandvert L.
      • Reis D.J.
      ,
      • Feinstein D.L.
      • Galea E.
      • Roberts S.
      • Berquist H.
      • Wang H.
      • Reis D.J.
      ,
      • Dobashi K.
      • Pahan K.
      • Chahal A.
      • Singh I.
      ). Fig. 10 shows that LPS alone induced the production of NO, and the activation of NF-κB in rat primary astrocytes and wortmannin alone was unable to induce the production of NO and the activation of NF-κB. However, wortmannin markedly stimulated the LPS-induced production of NO (Fig. 10A) without modulating the degree of activation of NF-κB (Fig. 10B), suggesting that similar to C6 glial cells the wortmannin-induced stimulation of NO production in primary astrocytes is also not caused by the stimulation of NF-κB activation.
      Figure thumbnail gr10
      Figure 10Effect of wortmannin on the LPS-induced production of NO and activation of NF-κB in rat primary astrocytes. Cells incubated in serum-free DMEM/F-12 received different concentrations of wortmannin (Wort) in the presence or absence of 0.5 μg/ml of LPS. Panel A, after a 24-h incubation, nitrite concentrations were measured in supernatants. Data are the mean ± S.D. of three different experiments. Panel B, after a 1-h incubation, cells were taken out to prepare nuclear extracts, and nuclear proteins were used for the electrophoretic mobility shift assay as described under “Materials and Methods.”

      Effect of Inhibitors of PI 3-Kinase on Cell Viability

      C6 glial cells or astrocytes were incubated with different concentrations of wortmannin and LY294002 for 24 h, and their viability was determined as measured by the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. None of the inhibitors at the concentrations used in this study decreased or increased the viability of the cells (data not shown). Therefore, stimulation of the expression of iNOS in C6 and astrocytes by inhibitors of PI 3-kinase is not caused by any change in viability of the cells.

      DISCUSSION

      The signaling events transduced by proinflammatory cytokines and LPS for the induction of iNOS are poorly understood. A complete understanding of the cellular signaling mechanisms involved in the induction of iNOS should identify novel targets for the therapeutic intervention in NO-mediated neuroinflammatory diseases. Recently PI 3-kinase-associated signaling events have been shown to prevent apoptosis in a number of cell types including cerebellar granule neurons (
      • Okada T.
      • Kawano Y.
      • Sakakakibara T.
      • Hazeki M.
      • Ui M.
      ) and hematopoietic cells (
      • Powis G.
      • Bonjouklian R.
      • Berggren M.M.
      • Gallegos A.
      • Abraham R.
      • Ashendel C.
      • Zalkow L.
      • Matter W.F.
      • Dodge J.
      • Grindey G.
      • Vlahos C.J.
      ). Several lines of evidence presented in this study support the conclusion that the inhibition of PI 3-kinase activity, independent of the activation of MAP kinase and NF-κB, induces/stimulates the expression of iNOS in C6glial cells and astrocytes. Our conclusion is based on the following observations. First, LPS or IL-1β alone induced the activation of MAP kinase and NF-κB, but they were ineffective in the modulation of the activity of PI 3-kinase and in the induction of the expression of iNOS. However, the combinations of LPS and IL-1β or LPS and IFN-γ induced the activation of MAP kinase and NF-κB, caused a transient inhibition of PI 3-kinase, and induced the expression of iNOS and production of NO. Second, the compounds (wortmannin and LY294002) that inhibit PI 3-kinase had no effect on the degree of activation of MAP kinase and activation of NF-κB and the expression of iNOS in C6glial cells. However, these inhibitors induced the expression of iNOS and the production of NO in LPS- or IL-1β-treated C6glial cells. In addition, LPS was able to induce the expression of iNOS in C6 cells transfected with a dominant-negative mutant of p85α but not in cells transfected with the empty vector. Consistent with this observation the inhibitors of PI 3-kinase also induced iNOS promoter-derived expression of CAT in LPS- or IL-1β-treated C6 glial cells. On the other hand, these inhibitors of PI 3-kinase had no effect on LPS- or IL-1β-mediated activation of MAP kinase and NF-κB. These observations indicate that in addition to the activation of NF-κB, inhibition of PI 3-kinase is also necessary for the expression of iNOS in C6 glial cells. Our hypothetical model depicting the signals for the biosynthesis of iNOS in C6 glial cells is summarized in Fig. 11. Consistent with the apoptotic activity of NO (
      • Liu Q.
      • Schacher D.
      • Hurth C.
      • Freund G.G.
      • Dantzer R.
      • Kelly K.W.
      ,
      • Minshall C.
      • Arkins S.
      • Freund G.G.
      • Kelly K.W.
      ) and the antiapoptotic activity of activated PI 3-kinase (
      • Okada T.
      • Kawano Y.
      • Sakakakibara T.
      • Hazeki M.
      • Ui M.
      ,
      • Powis G.
      • Bonjouklian R.
      • Berggren M.M.
      • Gallegos A.
      • Abraham R.
      • Ashendel C.
      • Zalkow L.
      • Matter W.F.
      • Dodge J.
      • Grindey G.
      • Vlahos C.J.
      ), the observed up-regulation of LPS- or cytokine-induced expression of iNOS and production of NO in both C6 glial cells and rat primary astrocytes by inhibitors of PI 3-kinase indicates that PI 3-kinase may function as a negative regulator in the induction of iNOS and that this property of PI 3-kinase may contribute to its antiapoptotic activity.
      Figure thumbnail gr11
      Figure 11Hypothetical model describing the signaling pathways for the expression of iNOS in C6 glial cells. MAPK, MAP kinase.
      Proinflammatory cytokines (tumor necrosis factor-α, IL-1β, or IFN-γ) and LPS bind to their respective receptors and induce iNOS expression via activation of NF-κB (
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Xie Q.-W.
      • Kashiwabara Y.
      • Nathan C.
      ,
      • Kwon G.
      • Corbett J.A.
      • Rodi C.P.
      • Sullivan P.
      • McDaniel M.L.
      ,
      • Leist M.
      • Fava E.
      • Montecucco C.
      • Nicotera P.
      ,
      • Xie K.
      • Wang Y.
      • Huang S.
      • Xu L.
      • Bielenberg D.
      • Salas T.
      • McConkey D.J.
      • Jiang W.
      • Fidler I.J.
      ). The presence of a consensus sequence in the promoter region of iNOS for the binding of NF-κB (
      • Xie Q.-W.
      • Kashiwabara Y.
      • Nathan C.
      ) and the inhibition of iNOS expression with the inhibition of NF-κB activation establishes an essential role of NF-κB activation in the induction of iNOS (
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ,
      • Kwon G.
      • Corbett J.A.
      • Rodi C.P.
      • Sullivan P.
      • McDaniel M.L.
      ). Activation of NF-κB by various cellular stimuli involves the proteolytic degradation of IκBα and the concomitant nuclear translocation of the liberated NF-κB heterodimer (
      • Stefanova I.
      • Corcoran M.L.
      • Horak E.M.
      • Wahl L.M.
      • Bolen J.B.
      • Horak I.D.
      ,
      • Salkowski C.A.
      • Detore G.
      • McNally R.
      • van Rooijen N.
      • Vogel S.N.
      ). Although the biochemical mechanism underlying the degradation of IκBα remains unclear, it appears that degradation of IκBα induced by various mitogens and cytokines occurs in association with the transient phosphorylation of IκBα on serines 32 and 36 (
      • Beg A.A.
      • Ruben S.M.
      • Scheinman R.I.
      • Haskil S.
      • Rosen C.A.
      • Baldwin Jr., A.S.
      ). Upon phosphorylation, IκBα that is still bound to NF-κB apparently becomes a high affinity substrate for an ubiquitin-conjugating enzyme (
      • Sun S.-C.
      • Ganchi P.A.
      • Ballard D.W.
      • Greene W.C.
      ). After phosphorylation-controlled ubiquitination, the IκBα is rapidly and completely degraded by the 20 S or 26 S proteosome, and the NF-κB heterodimer is targeted to the nucleus (
      • Brown K.
      • Gerstberger S.
      • Carlson L.
      • Franzoso G.
      • Siebenlist U.
      ). Recently, it has been reported that 90-kDa ribosomal S6 kinase (p90 RSK), a downstream candidate of the well characterized Ras-Raf-MEK-MAP kinase pathway, phosphorylates the NH2-terminal regulatory domain of IκBα on serine 32 (
      • Ghoda L.
      • Lin X.
      • Greene W.C.
      ), suggesting the possible involvement of the MAP kinase pathway in the phosphorylation of IκBα and in the activation of NF-κB. Consistent with this observation, we also found that PD98059, an inhibitor of MEK, inhibited the LPS-induced activation of NF-κB in rat primary astrocytes (
      • Pahan K.
      • Sheikh F.G.
      • Khan M.
      • Namboodiri A.M.S.
      • Singh I.
      ).
      Earlier, we have also observed that cAMP derivatives that activate protein kinase A, mevalonate inhibitors that inhibit the p21ras, or antioxidants likeN-acetylcysteine inhibit the expression of iNOS by inhibiting the activation of NF-κB (
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Pahan K.
      • Namboodiri A.M.S.
      • Sheikh F.G.
      • Smith B.T.
      • Singh I.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ). On the other hand, cell-permeable ceramides analogs and inhibitors of protein phosphate 1/2A stimulate the expression of iNOS in rat primary astrocytes by stimulating the activation of NF-κB (
      • Pahan K.
      • Sheikh F.G.
      • Khan M.
      • Namboodiri A.M.S.
      • Singh I.
      ,
      • Pahan K.
      • Sheikh F.G.
      • Namboodiri A.M.S.
      • Singh I.
      ). Here also we have observed that activation of NF-κB is an essential, but not sufficient, signal for the induction of iNOS. First, IFN-γ did not induce the activation of NF-κB, therefore wortmannin was unable to induce the expression of iNOS and the production of NO in C6 cells. Second, pyrrolidine dithiocarbamate, an inhibitor of NF-κB activation, blocked the activation of NF-κB and thereby inhibited the expression of iNOS, induced by the combination of LPS and wortmannin, suggesting that LPS- and wortmannin-induced expression of iNOS is dependent on the activation of NF-κB. However, LPS- or IL-1β-induced activation of NF-κB was not sufficient to induce the expression of iNOS in the absence of inhibition of PI 3-kinase.
      In summary, studies reported in this manuscript underscore the necessity of inhibition of PI 3-kinase in the LPS- or cytokine-mediated induction of iNOS. Moreover, the signal induced by inhibition of PI 3-kinase for the induction of iNOS is not mediated by MAP kinase or NF-κB.

      Acknowledgments

      We thank Dr. Avtar K. Singh for a review of the manuscript and helpful suggestions and Jan Ashcraft for technical help.

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