From the Departments of Pediatrics and
§ Medicine, Medical University of South Carolina,
Charleston, South Carolina 29425
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ABSTRACT |
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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-1 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 (3-8). 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 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)- 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- Reagents--
Recombinant rat IFN- Induction of NO Production in Astrocytes and C6 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. C6
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 µl of culture supernatant
was allowed to react with 200 µl 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
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 mM
Tris-HCl (pH 7.4) containing protease inhibitors (1 mM
phenylmethylsulfonyl 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
(11-13).
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 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') (11, 12, 21). 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 (22) 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 p85 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 mM
phenylmethylsulfonyl 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 p85 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. (25) 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 M
NaCl, 1.5 mM MgCl2, 0.2 mM EDTA,
0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl 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 M
KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, and
0.5 mM phenylmethylsulfonyl fluoride) and stored at
Assay of Transcriptional Activity of NF- 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.
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. C6
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 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-1 Expression of a Dominant-negative Mutant of p85 Wortmannin Induces the Expression of iNOS in LPS- or
IL-1 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-1 Wortmannin Stimulates the LPS-induced Production of NO in Rat
Primary Astrocytes without Modulating the Activation of
NF- 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.
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 (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- (IL-1
) 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-1
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-1
-treated C6 glial cells. The expression of iNOS by
LPS in C6 glial cells expressing a dominant-negative mutant
of p85
, 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-
B in LPS- or IL-1
-stimulated C6 glial
cells. In contrast to the inability of LPS and IL-1
alone to induce
the expression of iNOS, both LPS and IL-1
individually stimulated
MAP kinase activity and induced DNA binding and transcriptional
activity of NF-
B. Wortmannin alone was unable to activate MAP kinase
and NF-
B. Moreover, wortmannin had no effect on LPS- or
IL-1
-mediated activation of MAP kinase and NF-
B, suggesting that
wortmannin induced the expression of iNOS in LPS- or IL-1
-stimulated
C6 glial cells without modulating the activation of MAP
kinase and NF-
B. 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-
B.
Taken together, the results from specific chemical inhibitors and
dominant-negative mutant expression studies demonstrate that apart from
the activation of NF-
B, inhibition of PI 3-kinase is also necessary
for the expression of iNOS and production of NO.
INTRODUCTION
Top
Abstract
Introduction
References
(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 (3-5).
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 (11-13, 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 cytokine-mediated
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 C6 glial cells.
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 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-
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
C6 glial cells and astrocytes.
MATERIALS AND METHODS
, 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,
[
-32P]ATP (3,000 Ci/mmol) was from Amersham Pharmacia Biotech.
in
C6 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). C6 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.
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 (24) 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 [
-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 1 N HCl and 200 µl of chloroform:methanol (1:1).
Phospholipids were separated by TLC and visualized by exposure to
iodine vapor and autoradiography (24).
70 °C until use. Nuclear extracts were used for the electrophoretic mobility shift assay using the NF-
B DNA-binding protein detection system kit (Life Technologies, Inc.) according to the
manufacturer's protocol.
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.
RESULTS
-treated C6 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 + IL-1
;
LPS + 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, 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-1
-treated C6 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 C6
cells.
Effect of inhibitors of PI 3-kinase phosphatases on LPS-induced
production of NO in C6 glial cells
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Fig. 1.
Wortmannin 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."
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Fig. 2.
Effect of wortmannin on the
expression of iNOS in LPS- or 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. 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-1 , 50 ng/ml; IFN-
, 50 units/ml; wortmannin, 300 nM.
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 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
tyrosine-phosphorylated 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 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 p85
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 p85
(Fig. 4),
suggesting that inhibition of PI 3-kinase activity is sufficient to
induce the expression of iNOS in LPS-treated C6 glial
cells.
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Fig. 3.
Expression of a dominant-negative mutant of
p85 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 p85
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."
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Fig. 4.
Expression of a dominant-negative mutant of
p85 induces the expression of iNOS in
LPS-treated C6 glial cells. Cells were
transfected with various concentrations of a dominant-negative mutant
of p85
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.
-treated C6 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 (11-18), and PD98059,
an inhibitor of MEK, inhibits the LPS-induced expression of iNOS in
astrocytes (18), to understand the basis of wortmannin-induced
expression of iNOS in LPS- or IL-1
-treated C6 glial
cells, we examined the effect of wortmannin on the activation of MAP
kinase and NF-
B. 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-1
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-1
-stimulated C6 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 C6 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 C6 glial cells with 0.5 µg/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 C6 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 C6 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
cytokine-treated C6 cells requires the activation of
NF-
B, we studied the effect of pyrrolidine dithiocarbamate, an
antioxidant inhibitor of NF-
B activation, on the induction of iNOS
and the activation of NF-
B in cells treated with the combination of
LPS and wortmannin. Pyrrolidine dithiocarbamate inhibited the
activation of NF-
B and the induction of NO production in LPS- and
wortmannin-treated C6 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.
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Fig. 5.
Time 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.
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Fig. 6.
LPS induces the DNA binding activity of
NF- B 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-
B band, and the lower
arrow indicates the unbound probe.
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Fig. 7.
Effect of wortmannin on the activation of MAP
kinase and NF- B 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-
B-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-1
, 50 ng/ml; IFN-
,
50 units/ml; and wortmannin, 300 nM.
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Fig. 8.
Pyrrolidine dithiocarbamate inhibits the
induction of NO production and the activation of
NF- B 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-
B as described under "Materials and
Methods."
-treated
C6 cells, we sought to examine whether inhibition of PI
3-kinase is necessary for the expression of iNOS in C6
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 incubation (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 C6
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 C6 glial cells.
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Fig. 9.
Activity of PI 3-kinase in LPS- and
cytokine-treated C6 glial cells. Cells
treated with LPS (0.5 µg/ml) (panel A), IL-1
(50 ng/ml) (panel B), the combination of LPS and
IL-1
(panel C), or the combination of LPS and
wortmannin (panel D) 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
70 °C (upper panels) and
quantitated by densitometry (lower panels). Data are from a
single experiment representative of at least three others.
B--
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-
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 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 (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 C6 glial cells
the wortmannin-induced stimulation of NO production in primary
astrocytes is also not caused by the stimulation of NF-
B
activation.
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Fig. 10.
Effect of wortmannin on the LPS-induced
production of NO and activation of NF- B 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."
DISCUSSION
B, induces/stimulates the expression of iNOS in C6
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 activation 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 C6
glial cells. However, these inhibitors induced the expression of iNOS
and the production of NO in LPS- or IL-1
-treated C6
glial cells. In addition, LPS was able to induce the expression of iNOS
in C6 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
C6 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 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 (32, 33) and the antiapoptotic activity of activated PI
3-kinase (30, 31), 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.
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Fig. 11.
Hypothetical model describing the signaling
pathways for the expression of iNOS in C6 glial
cells. MAPK, MAP kinase.
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
I
B
and the concomitant nuclear translocation of the liberated
NF-
B heterodimer (36, 37). Although the biochemical mechanism
underlying the degradation of I
B
remains unclear, it appears that
degradation of I
B
induced by various mitogens and cytokines
occurs in association with the transient phosphorylation of I
B
on
serines 32 and 36 (38). Upon phosphorylation, I
B
that is still
bound to NF-
B apparently becomes a high affinity substrate for an
ubiquitin-conjugating enzyme (39). After phosphorylation-controlled
ubiquitination, the I
B
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
NH2-terminal regulatory domain of I
B
on serine 32 (41), suggesting the possible involvement of the MAP kinase pathway in
the phosphorylation of I
B
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 p21ras, or antioxidants like
N-acetylcysteine inhibit the expression of iNOS by
inhibiting the activation of NF-B (11-13). 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
C6 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.
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ACKNOWLEDGEMENTS |
---|
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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FOOTNOTES |
---|
* This study was supported by National Institutes of Health Grants NS-22576, NS-34741, and NS-37766.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Pediatrics, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Tel.: 843-792-7542; Fax: 843-792-7130; E-mail: singhi{at}musc.edu.
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ABBREVIATIONS |
---|
The abbreviations used are:
NO, nitric oxide;
NOS, nitric-oxide synthase(s);
iNOS, inducible NOS;
LPS, lipopolysaccharide;
IL-1, interleukin-1
;
IFN-
, interferon-
;
NF-
B, nuclear factor
B;
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.
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REFERENCES |
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