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

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), 1 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 (1,2). 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 (3)(4)(5)(6)(7)(8). 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-1␤ (IL-1␤), tumor necrosis factor-␣, and interferon-␥ (IFN-␥). 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 (9 -13). NO derived from both astrocytes and macrophages is assumed to contribute to oligodendrocyte degeneration in demyelinating diseases and neuronal death during ischemia and trauma (3)(4)(5).
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)-B in the promoter region of iNOS (14) and inhibition of iNOS induction by inhibitors of NF-B activation have established an essential role of NF-B activation in the induction of iNOS (11)(12)(13)15). Suppression of NF-B and inhibition of iNOS expression (16,17) by inhibitors of tyrosine kinase in different cell types suggest the possible involvement of tyrosine phosphorylation in the activation of NF-B and the induction of iNOS. Inhibition of LPS-and cytokine-induced activation of NF-B 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-B and the induction of iNOS (11). Again, increasing cAMP and protein kinase A activity has been shown to inhibit the activation of NF-B and the induction of iNOS possibly because of the inhibition of Raf-1 (12). 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-B and the induction of iNOS in astrocytes, suggesting the possible involvement of the MAP kinase pathway in the LPS-and proinflammatory cytokinemediated induction of iNOS (18). Taken together, these studies suggest that any alteration of the Ras-Raf-MEK-MAP kinase signal transduction pathway alters the activation of NF-B and so the induction of iNOS in astrocytes and C 6 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 cytokinestimulated C 6 glial cells and rat primary astrocytes and that the signal is not mediated via MAP kinase and NF-B. Specific inhibitors of PI 3-kinase (wortmannin and LY294002) and expression of the dominant-negative mutant of p85␣, the regulatory subunit of PI 3-kinase, induced the expression of iNOS in LPS-or cytokine-stimulated C 6 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-B, suggesting that apart from the activation of NF-B 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 C 6 glial cells and astrocytes.

MATERIALS AND METHODS
Reagents-Recombinant rat IFN-␥, DMEM/F-12, fetal bovine serum, Hanks' balanced salt solution, and NF-B DNA-binding protein detection kit were from Life Technologies, Inc. Human IL-1␤ 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 (p85␣) were obtained from Calbiochem. Antibodies against mouse macrophage iNOS were obtained from Transduction Laboratories, [␥-32 P]ATP (3,000 Ci/ mmol) was from Amersham Pharmacia Biotech.
Induction of NO Production in Astrocytes and C 6 Glial Cells-Astrocytes were prepared from rat cerebral tissue as described by McCarthy and DeVellis (19). 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. C 6 glial 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 l of culture supernatant was allowed to react with 200 l of Griess reagent (20) 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 NaNO 2 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 mM Tris-HCl (pH 7.4) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 5 g/ml aprotinin, 5 g/ml antipain, 5 g/ml pepstatin A, and 5 g/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 125 I-labeled protein A (11)(12)(13).
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 g of total RNA was electrophoresed on 1.2% denaturing formaldehyde-agarose gels, elec-trotransferred to Hybond nylon membrane (Amersham Pharmacia Biotech), and hybridized at 68°C with 32 P-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: 5Ј-CTC CTT CAA AGA GGC AAA AAT A-3Ј; reverse primer: 5Ј-CAC TTC CTC CAG GAT GTT GT-3Ј) (11,12,21). After hybridization, the filters were washed two or three times in solution I (2 ϫ SSC, 0.05% SDS) for 1 h at room temperature followed by solution II (0.1 ϫ 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 (22) was amplified by using two primers (forward: 5Ј-GAG AGT GTG CAA GTA TTT GTA GGA G-3Ј and reverse: 5Ј-AAG GTG GCT GAG AAG TTT CA-3Ј) 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 g 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 p85␣ in C 6 Glial Cells-In the dominant-negative form of p85␣, 35 amino acids in the inter-SH2 region from residues 479 -513 of wild type p85␣, 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 (23). C 6 glial cells were transfected with either the dominant-negative form of p85␣ or an empty vector by Lipotaxi following manufacturer's protocol. 24 h after transfection, cells were treated with different stimuli.
Assay of MAP Kinase-Cells were lysed directly with 2 ϫ 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.
Assay of Transcriptional Activity of NF-B-To assay the transcriptional activity of NF-B, cells were transfected with pNF-B-Luc, an NF-B-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) (26,27) 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-(4,5dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay.

Inhibitors of PI 3-Kinase (Wortmannin and LY294002) Induce the Expression of iNOS and Production of NO in LPS-
stimulated Rat C 6 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 C 6 glial cells. C 6 glial cells were cultured in serum-free DMEM/F-12 in the presence of LPS and inhibitors of PI 3-kinase. Consistent with previous observations (12,13,16,20,28), the bacterial LPS or cytokines alone did not induce the production of NO in C 6 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 C 6 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 C 6 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 C 6 glial cells. The lowest dose of wortman-nin 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 C 6 glial cells, wortmannin also induced the production of NO in IL-1␤treated C 6 cells ( Fig. 2A). However, wortmannin was unable to induce the production of NO in IFN-␥-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-1␤-treated cells. Although LPS, IL-1␤, and IFN-␥ individually were unable to induce the expression of iNOS and the production of NO, different combinations of these stimuli (e.g. LPS ϩ IL-1␤; LPS ϩ IFN-␥) 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, C 6 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-1␤-treated C 6 cells (Fig. 2D). Again, wortmannin was unable  to induce the CAT activity in IFN-␥-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-1␤-stimulated C 6 cells.
Expression of a Dominant-negative Mutant of p85␣ Induces the Expression of iNOS in LPS-stimulated C 6 Glial Cells-Induction of NOS by wortmannin or LY294002, inhibitors of PI 3-kinase, in LPS-or cytokine-stimulated C 6 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 C 6 glial cells, we transfected C 6 glial cells with a dominant-negative mutant of p85␣. 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 tyrosinephosphorylated proteins. Expression of a dominant-negative mutant of p85␣, 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 (23,29). We have also found that expression of the same dominant-negative mutant of p85␣ in C 6 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 dominantnegative mutant protein of p85␣ did not associate with the catalytic subunit of PI 3-kinase. In control C 6 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 C 6 cells transfected with the dominant-negative mutant of p85␣ (Fig. 4), suggesting that inhibition of PI 3-kinase activity is sufficient to induce the expression of iNOS in LPS-treated C 6 glial cells.
Wortmannin Induces the Expression of iNOS in LPS-or IL-1␤-treated C 6 Glial Cells without Modulating the Activation of MAP Kinase and NF-B-Because the activation of NF-B is necessary for the expression of iNOS (11)(12)(13)(14)(15)(16)(17)(18), and PD98059, an inhibitor of MEK, inhibits the LPS-induced expression of iNOS in astrocytes (18), to understand the basis of wortmannininduced expression of iNOS in LPS-or IL-1␤-treated C 6 glial cells, we examined the effect of wortmannin on the activation of MAP kinase and NF-B. Treatment of C 6 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 C 6 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 mon-itored. Although LPS and IL-1␤ alone were ineffective in inducing the expression of iNOS, both of the stimuli, alone or together, induced the activation of MAP kinase in C 6 glial cells (see Fig. 7A). Wortmannin, capable of inducing the expression of iNOS in LPS-and IL-1␤-stimulated C 6 cells, had no effect on LPS-and IL-1␤-mediated phosphorylation of MAP kinase (see Fig. 7A), suggesting that wortmannin induced the expression of iNOS in LPS-or IL-1␤-treated C 6 cells without modulating the MAP kinase pathway. Next we examined the effect of wortmannin on the activation of NF-B. Activation of NF-B was monitored by both DNA binding as well as transcriptional activity of NF-B. The DNA binding activity of NF-B was evaluated by the formation of a distinct and specific complex in a gel shift DNA binding assay. Treatment of C 6 glial cells with 0.5 g/ml LPS resulted in the induction of DNA binding activity of NF-B (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-1␤ to induce the expression of iNOS, both of these stimuli induced the DNA binding activity of NF-B (Fig. 7B). Wortmannin alone neither induced the DNA binding activity of NF-B nor modulated the LPS-and IL-1␤-mediated DNA binding activity of NF-B (Fig. 7B). We then tested the effect of wortmannin on NF-B-dependent transcription of luciferase in C 6 glial cells in the presence or absence of LPS and cytokines, using the expression of luciferase from a reporter construct, pNF-B-Luc (Stratagene), as an assay. Consistent with the effect of wortmannin on DNA binding activity of NF-B, wortmannin alone did not induce the NF-B-dependent transcription of luciferase, and it also had no effect on the magnitude of LPS-and IL-1␤-induced transcriptional activity of NF-B (Fig. 7C). On the other hand, consistent with the inability of IFN-␥ to induce the expression of iNOS in C 6 glial cells, IFN-␥ did not induce the DNA binding or transcriptional activity of NF-B, whereas the combination of LPS and IFN-␥ was able to induce the DNA binding as well as transcriptional activity of NF-B (Fig. 7, B and C) and the induction of iNOS (Fig. 2). To examine whether wortmannin-induced expression of iNOS in LPS-or cytokinetreated C 6  the activation of NF-B and the induction of NO production in LPS-and wortmannin-treated C 6 cells (Fig. 8). Taken together, these studies indicate that activation of NF-B 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-B.
Inhibition of PI 3-Kinase Is Necessary for the Expression of iNOS in C 6 Glial Cells-Because inhibitors of PI 3-kinase induced the expression of iNOS in LPS-or IL-1␤-treated C 6 cells, we sought to examine whether inhibition of PI 3-kinase is necessary for the expression of iNOS in C 6 cells. Cells treated with LPS and IL-1␤, alone or in combination, for different time intervals were assayed for the lipid kinase activity of PI 3-kinase. Although LPS or IL-1␤ alone had no effect on PI 3-kinase activity (Fig. 9, A and B), the combination of LPS and IL-1␤ inhibited the activity of PI 3-kinase within 5-10 min of incu-bation (Fig. 9C). Consistent with the inhibitory effect of wortmannin on PI 3-kinase activity in other cell types (30,31), wortmannin inhibited the lipid kinase activity of PI 3-kinase in LPS-treated C 6 cells (Fig. 9D). These results indicate that inhibition of PI 3-kinase activity may be necessary to induce the expression of the iNOS gene in C 6 glial cells.
Wortmannin Stimulates the LPS-induced Production of NO in Rat Primary Astrocytes without Modulating the Activation of NF-B-Because wortmannin induces the production of NO and the expression of iNOS in LPS-or cytokine-treated C 6 glial cells without modulating the activation of NF-B, we investigated the effect of wortmannin on LPS-induced production of NO and the activation of NF-B in rat primary astrocytes. In sharp contrast to the inability of LPS to induce the expression of iNOS and the production of NO in C 6 glial cells, LPS alone was able to induce the expression of iNOS and the production of NO in rat primary astrocytes as reported previously (12,13,16,20,28). Fig. 10 shows that LPS alone induced the production of NO, and the activation of NF-B in rat primary astrocytes and wortmannin alone was unable to induce the production of NO and the activation of NF-B. However, wortmannin markedly stimulated the LPS-induced production of NO (Fig.  10A) without modulating the degree of activation of NF-B (Fig. 10B), suggesting that similar to C 6 glial cells the wortmannin-induced stimulation of NO production in primary astrocytes is also not caused by the stimulation of NF-B activation.
Effect of Inhibitors of PI 3-Kinase on Cell Viability-C 6 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 C 6 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 mecha- in serum-free media were lysed, immunoprecipitated with monoclonal antibodies against p85␣, 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 Ϫ70°C (upper panels) and quantitated by densitometry (lower panels). Data are from a single experiment representative of at least three others. nisms 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 (30) and hematopoietic cells (31). 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-B, induces/stimulates the expression of iNOS in C 6 glial cells and astrocytes. Our conclusion is based on the following observations. First, LPS or IL-1␤ alone induced the activation of MAP kinase and NF-B, 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-1␤ or LPS and IFN-␥ induced the activa-tion of MAP kinase and NF-B, 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-B and the expression of iNOS in C 6 glial cells. However, these inhibitors induced the expression of iNOS and the production of NO in LPS-or IL-1␤-treated C 6 glial cells. In addition, LPS was able to induce the expression of iNOS in C 6 cells transfected with a dominant-negative mutant of p85␣ 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-1␤-treated C 6 glial cells. On the other hand, these inhibitors of PI 3-kinase had no effect on LPS-or IL-1␤-mediated activation of MAP kinase and NF-B. These observations indicate that in addition to the activation of NF-B, inhibition of PI 3-kinase is also necessary for the expression of iNOS in C 6 glial cells. Our hypothetical model depicting the signals for the biosynthesis of iNOS in C 6 glial cells is summarized in Fig. 11. Consistent with the apoptotic activity of NO (32,33) and the antiapoptotic activity of activated PI 3-kinase (30,31), the observed up-regulation of LPSor cytokine-induced expression of iNOS and production of NO in both C 6 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.
Proinflammatory cytokines (tumor necrosis factor-␣, IL-1␤, or IFN-␥) and LPS bind to their respective receptors and induce iNOS expression via activation of NF-B (11-15, 34, 35). The presence of a consensus sequence in the promoter region of iNOS for the binding of NF-B (14) and the inhibition of iNOS expression with the inhibition of NF-B activation establishes an essential role of NF-B activation in the induction of iNOS (11,12,15). Activation of NF-B by various cellular stimuli involves the proteolytic degradation of IB␣ and the concomitant nuclear translocation of the liberated NF-B heterodimer (36,37). Although the biochemical mechanism underlying the degradation of IB␣ remains unclear, it appears that degradation of IB␣ induced by various mitogens and cytokines occurs in association with the transient phosphorylation of IB␣ on serines 32 and 36 (38). Upon phosphorylation, IB␣ that is still bound to NF-B apparently becomes a high affinity substrate for an ubiquitin-conjugating enzyme (39). After phosphorylation-controlled ubiquitination, the IB␣ is rapidly and completely degraded by the 20 S or 26 S proteosome, and the NF-B heterodimer is targeted to the nucleus (40). 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 NH 2 -terminal regulatory domain of IB␣ on serine 32 (41), suggesting the possible involvement of the MAP kinase pathway in the phosphoryla- tion of IB␣ and in the activation of NF-B. Consistent with this observation, we also found that PD98059, an inhibitor of MEK, inhibited the LPS-induced activation of NF-B in rat primary astrocytes (18).
Earlier, we have also observed that cAMP derivatives that activate protein kinase A, mevalonate inhibitors that inhibit the p21 ras , or antioxidants like N-acetylcysteine inhibit the expression of iNOS by inhibiting the activation of NF-B (11)(12)(13). 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-B (18,27). Here also we have observed that activation of NF-B is an essential, but not sufficient, signal for the induction of iNOS. First, IFN-␥ did not induce the activation of NF-B, therefore wortmannin was unable to induce the expression of iNOS and the production of NO in C 6 cells. Second, pyrrolidine dithiocarbamate, an inhibitor of NF-B activation, blocked the activation of NF-B 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-B. However, LPS-or IL-1␤-induced activation of NF-B 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-B.