From the Department of Pediatrics, Medical University of South
Carolina, Charleston, South Carolina 29425
Nitric oxide 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 stroke and other neurodegenerative diseases. The
present study underlines the importance of protein phosphatase (PP) 1 and 2A in the regulation of the differential expression of iNOS in rat
primary astrocytes and macrophages. Compounds (calyculin A,
microcystin, okadaic acid, and cantharidin) that inhibit PP 1 and 2A
were found to stimulate the lipopolysaccharide (LPS)- and
cytokine-mediated expression of iNOS and production of NO in rat
primary astrocytes and C6 glial cells. However, these
inhibitors inhibited the LPS- and cytokine-mediated expression of iNOS
and production of NO in rat resident macrophages and RAW 264.7 cells.
Similarly, okadaic acid, an inhibitor of PP 1/2A, stimulated the iNOS
promoter-derived chloramphenicol acetyltransferase activity in
astrocytes and inhibited the iNOS promoter-derived chloramphenicol
acetyltransferase activity in macrophages, indicating that okadaic acid
also differentially regulates the transcription of the iNOS gene in
astrocytes and macrophages. The observed stimulation of the expression
of iNOS in astrocytes and the inhibition of the expression of iNOS in macrophages with the inhibition of PP 1/2A activity clearly delineate a
novel role of PP 1/2A in the differential regulation of iNOS in rat
astrocytes and macrophages. Because the activation of NF-
B is
necessary for the induction of iNOS and the expression of tumor necrosis factor (TNF)-
also depends on the activation of NF-
B, we
examined the effect of okadaic acid on the LPS-mediated activation of
NF-
B and production of TNF-
in rat primary astrocytes and macrophages. Interestingly, in both cell types, okadaic acid stimulated the LPS-mediated DNA binding as well as transcriptional activity of
NF-
B and production of TNF-
. This study suggests that the stimulation of iNOS expression in astrocytes by inhibitors of PP 1/2A
is possibly due to the stimulation of NF-
B activation; however,
activation of NF-
B is not sufficient for the induction of iNOS in
macrophages and that apart from NF-
B some other signaling pathway(s)
sensitive to PP 1 and/or PP 2A is/are possibly involved in the
regulation of iNOS in macrophages. This differential induction of iNOS
as compared with similar activation of NF-
B by inhibitors of PP 1/2A
indicates the involvement of different intracellular signaling events
for the induction of iNOS in two cell types of the same animal
species.
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INTRODUCTION |
Nitric oxide (NO), a bioactive free radical, is enzymatically
formed from L-arginine by the enzyme nitric-oxide synthase
(NOS).1 The NOS are basically
divided into two forms. One constitutive form present in neurons and
endothelial cells is calcium-dependent enzymes, whereas the
inducible form present in macrophage and astrocytes is regulated at the
transcriptional level in response to stimuli (e.g.
cytokines/lipopolysaccharides) 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 pathophysiologies of inflammatory diseases
including demyelinating disorders (e.g. multiple sclerosis,
experimental allergic encephalopathy, and 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 induction of iNOS by bacterial
lipopolysaccharides (LPS) and a series of cytokines including
interleukin-1
(IL-1
), tumor necrosis factor-
(TNF-
) and
interferon-
(IFN-
). Astrocytes in the healthy brain do not
express iNOS but following ischemic, traumatic, neurotoxic, or
inflammatory damage the reactive astrocytes express iNOS in 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).
Characterization of intracellular pathways evoked to transduce the
signal from the cell surface to the nucleus for the induction of iNOS
in macrophages and astrocytes is an active area of investigation. Identification of the DNA-binding site for NF-
B in the promoter region of iNOS (14), and inhibition of iNOS induction by inhibitors of
NF-
B activation has 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 suggests the possible involvement of tyrosine phosphorylation in the activation of NF-
B and the induction of iNOS. Recently we have observed that PD 98059, an
inhibitor of 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). MAP kinases
exhibit dual specificity, regulating both Ser/Thr phosphorylation and
Tyr autophosphorylation (19-21). In addition, MAP kinases themselves require concurrent Thr and Tyr phosphorylation for activation and are
in turn substrates for MEK (19-21). MEK is also a dual specificity
kinase whose activation requires Ser/Thr phosphorylation (19-21).
These observations suggest that cellular regulation of this signaling
pathway may utilize Ser/Thr phosphatases to modulate the
phosphorylation state of critical phosphoproteins associated with the
activation of NF-
B and the induction of iNOS.
Because PP 1 and PP 2A are the two most abundant Ser/Thr phosphatases
in the cell, the present study was undertaken to investigate the
cellular regulation of the induction of iNOS by PP 1 and PP 2A in rat
primary astrocytes and macrophages. Our results clearly demonstrate
that calyculin A, microcystin, cantharidin, and okadaic acid,
inhibitors of PP 1 and PP 2A, stimulate the LPS- and cytokine-mediated expression of iNOS and production of NO in astrocytes and
C6 glial cells, whereas the same inhibitors inhibit the
LPS- and cytokine-mediated expression of iNOS and production of NO in
macrophages and RAW 264.7 cells. This differential regulation of the
induction of iNOS in astrocytes and macrophages by inhibitors of PP
1/2A suggests that different intracellular signaling events may be
involved for the induction of iNOS in astrocytes and macrophages.
However, despite this differential regulation of the induction of iNOS in astrocytes and macrophages, inhibitors of PP 1/2A stimulate the
activation of NF-
B and the production of TNF-
in both astrocytes and macrophages.
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MATERIALS AND METHODS |
Reagents--
Recombinant rat IFN-
, DMEM/F-12 medium, 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. Mouse recombinant TNF-
was obtained from
Boehringer Mannheim. LPS (Escherichia coli) was from Sigma.
NG-methyl-L-arginine
(L-NMA), okadaic acid, calyculin A, cantharidin, and
antibodies against mouse macrophage iNOS were obtained from Calbiochem.
Deltamethrin and fenvalerate were obtained from Biomol. [
-32P]ATP (3000 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 (22). 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 two more times after a gap of 1 or 2 weeks before subculturing to ensure the complete removal of all the oligodendrocytes and microglia. Cells were trypsinized, 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.
Isolation of Rat Macrophages and Induction of NO
Production--
Resident macrophages were obtained from rat by
peritoneal lavage with sterile RPMI 1640 medium containing 1% fetal
bovine serum and 100 µg/ml gentamicin as reported earlier (13). Cells were washed three times with RPMI 1640 at 4 °C. All cell cultures were maintained at 37 °C in a humified incubator containing 5% CO2 in air. Macrophages at a concentration of 2 × 106/ml in RPMI 1640 medium containing
L-glutamine and gentamicin were added in volumes of 800 µl to a 35-mm plate. After 1 h, nonadherent cells were removed
by washing, and 800 µl of serum-free RPMI 1640 medium with various
stimuli were added to the adherent cells. After incubation in 5%
CO2 in air at 37 °C, culture supernatants were
transferred to measure NO production.
Assay for NO Synthesis--
Synthesis of NO was determined by
assay of culture supernatants 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 (23) and incubated at
room temperature for 15 min. The optical density of the assay samples was measured spectrophotometrically at 570 nm. Fresh culture medium served as the blank in all experiments. Nitrite concentrations were
calculated from a standard curve derived from the reaction of
NaNO2 in the assay.
In Vitro PP 1/2A Assay--
The extraction and assay for PP 1/2A
were performed as described (24). Control and treated cells were
scraped off the dishes with phosphatase extraction buffer containing 20 mM imidazole-HCl, 2 mM EDTA, 2 mM
EGTA, pH 7.0, with protease inhibitors (1 mM PMSF, 5 µg/ml aprotinin, 5 µg/ml antipain, 5 µg/ml pepstatin A, and 5 µg/ml leupeptin). The cells were sonicated for 10 s and
centrifuged at 2000 × g for 5 min, and the
supernatants were used for the assay of phosphatase activities using
the protein phosphatase assay kit (Life Technologies Inc.) according to
the manufacturer's protocol.
Immunoblot Analysis for iNOS--
Following 24 h of
incubation in the presence or the absence of different stimuli, cells
were scraped off, washed with Hank's buffer, and homogenized in 50 mM Tris-HCl, pH 7.4, containing protease inhibitors (1 mM PMSF, 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 from 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), 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, 25). After hybridization, 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 amount of RNA was hybridized
with probe for glyceraldehyde-3-phosphate dehydrogenase. The relative
mRNA content for iNOS (iNOS/glyceraldehyde-3-phosphate dehydrogenase) 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 Activity--
The chloramphenicol
acetyltransferase (CAT) under the control of nitric-oxide synthase
promoter (iNOS) was created by subcloning a 1.5-kilobase promoter from
pGEM-NOS at SphI and SalI restriction site of
pCAT-basic vector (Promega). Full-length promoter (26) 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. Radioisotopic method was used to assay CAT
activity using a kit (Promega) as described by the manufacturer's
protocol.
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. (27) 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 PMSF, 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 PMSF, 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 PMSF) and stored at
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.
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) (28) using an assay kit from
Stratagene.
Cell Viability--
Cytotoxic effects of all 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 PP 1/2A Stimulate the LPS-induced Production of NO in
Rat Primary Astrocytes--
Rat primary astrocytes were cultured in
serum-free DMEM/F-12 in the presence of LPS and inhibitors of different
protein phosphatases. The concentration of NO as nitrite (a stable
reaction product of NO with molecular oxygen) was measured in culture
supernatants after 24 h. It is evident from Table
I that the bacterial LPS at a
concentration of 1.0 µg/ml induced the production of NO as nitrite by
about 9-fold. L-NMA, a competitive inhibitor of NOS activity, suppressed the LPS-mediated nitrite secretion, suggesting that LPS-induced nitrite release in rat primary astrocytes is dependent
on NOS-mediated arginine metabolism (Table I). Inhibitors of PP 1/2A
(calyculin A, microcystin, and okadaic acid) or PP 2B (cypermethrin,
deltamethrin, and fenvalerate) alone were neither stimulatory nor
inhibitory to nitrite production in control astrocytes (data not
shown). However, calyculin A, microcystin, and okadaic acid, when added
with LPS, stimulated the LPS-mediated induction of nitrite production
in astrocytes. In contrast, inhibitors of PP 2B (cypermethrin,
deltamethrin, and fenvalerate) had no effect on LPS-induced nitrite
production in astrocytes. These observations suggest that stimulation
of LPS-induced production of NO in astrocytes is specific for the
inhibitors of PP 1/2A. To understand the mechanism of stimulatory
effect of inhibitors of PP 1/2A on the LPS-mediated nitrite production
in astrocytes, we examined the effect of these inhibitors on the
protein and mRNA level of iNOS. Consistent with the production of
nitrite (Fig. 1A), Western
blot analysis with antibodies against murine macrophage iNOS and
Northern blot analysis for iNOS mRNA of LPS-stimulated astrocytes
clearly show that inhibitors of PP 1/2A (calyculin A, microcystin, and
cantharidin) enhanced the LPS-mediated induction of iNOS protein (Fig.
1B) and mRNA (Fig. 1C). Because the
inhibitors of PP 1/2A stimulated the LPS-mediated induction of iNOS, we
examined whether these inhibitors did in fact inhibit the activities of
PP 1/2A in LPS-treated astrocytes under these conditions. The
activities of PP 1/2A were measured in homogenates after 30 min of
incubation. Fig. 2 shows that okadaic acid inhibited the activities of PP 1/2A and stimulated the
LPS-mediated induction of iNOS protein and production of NO in
astrocytes in a dose-dependent manner.
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Table I
Effect of inhibitors of different protein phosphatases on
LPS-induced production of NO in rat primary astrocytes
Astrocytes preincubated in serum-free DMEM/F-12 for 30 min with
L-NMA and different inhibitors of protein phosphatases
received LPS (1.0 µg/ml). After 24 h of incubation, nitrite
concentration in the supernatants was measured as described under
"Materials and Methods." Data are expressed as the means ± S.D. of three different experiments.
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Fig. 1.
Inhibitors of PP 1 and PP 2A stimulate the
LPS-induced expression of iNOS in rat primary astrocytes. Cells
incubated in serum-free DMEM/F-12 received calyculin A (cal.
A), microcystin, or cantharidin along with 1.0 µg/ml of LPS.
A, after 24 h, concentration of nitrite was measured in
the supernatants as described under "Materials and Methods." Data
are the means ± S.D. of three different experiments.
B, cell homogenates were electrophoresed, transferred on
nitrocellulose membrane, and immunoblotted with antibodies against
mouse macrophage iNOS as described under "Materials and Methods."
C, after 6 h of incubation, cells were taken out
directly by adding ultraspec-II RNA reagent (Biotecx Laboratories Inc.)
to the plates for isolation of total RNA, and Northern blot analysis
for iNOS mRNA was carried out as described under "Materials and
Methods." Cal. A, calyculin A.
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Fig. 2.
Effect of okadaic acid on LPS-mediated
induction of iNOS in rat primary astrocytes. A, cells
incubated in serum-free DMEM/F-12 received different concentrations of
okadaic acid (OA) along with 1.0 µg/ml of LPS. After 30 min of incubation, protein phosphatase activity was measured. Data are
the means ± S.D. of three different experiments. B,
cells incubated in serum-free DMEM/F-12 received different
concentrations of okadaic acid in the presence or absence of 1.0 µg/ml of LPS. After 24 h of incubation, nitrite concentrations
were measured in supernatants. Data are the means ± S.D. of three
different experiments. C, cells incubated in serum-free
DMEM/F-12 received different concentrations of okadaic acid along with
1.0 µg/ml of LPS. After 24 h of incubation, cell homogenates
were electrophoresed, transferred on nitrocellulose membrane, and
immunoblotted with antibodies against mouse macrophage iNOS as
described before.
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Stimulation of LPS- and Cytokine-induced Production of NO by
Calyculin A in C6 Glial Cells--
Similar to primary
astrocytes, proinflammatory cytokines and the LPS induced the
production of nitrite as well as the expression of iNOS in rat
C6 glial cells (23, 29). Unlike astrocytes, LPS or
cytokines alone were not a sufficient inducer of NO production in rat
C6 glial cells. A combination of LPS and cytokines was required to induce the production of NO in C6 glial cells
(Refs. 13, 23, and 29 and Fig. 3).
Calyculin A (2 nM) stimulated the expression of iNOS
protein and the production of NO by more than 3-fold in LPS- and
cytokine-treated C6 cells (Fig. 3). The observed up-regulation of
cytokine-induced expression of iNOS and production of NO in both rat
primary astrocytes and C6 glial cells by inhibitors of PP
1/2A indicate that PP 1/2A may function as a negative regulator in
these intercellular signaling pathways.

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Fig. 3.
Stimulation of LPS- and cytokine-mediated
induction of iNOS by calyculin A in C6 glial cells.
Cells incubated in serum-free DMEM/F-12 received calyculin A along with
LPS and cytokines. A, after 24 h, concentration of
nitrite was measured in the supernatants as described earlier. Data are
the means ± S.D. of three different experiments. B,
cell homogenates were electrophoresed, transferred on nitrocellulose
membrane, and immunoblotted with antibodies against mouse macrophage
iNOS as described earlier. Concentration of different stimuli were as
follows: LPS, 0.5 µg/ml; TNF- , 20 ng/ml; IL-1 , 20 ng/ml;
IFN- , 20 units/ml.
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Inhibition of LPS- and Cytokine-induced NO Production by Inhibitors
of PP 1/2A in Rat Peritoneal Macrophages--
Because inhibitors of PP
1/2A stimulated the LPS- and cytokine-induced NO production in rat
primary astrocytes and C6 glial cells, we examined the
effect of these inhibitors on NO production and expression of iNOS in
rat resident macrophages. Similar to astrocytes, inhibitors of PP 1/2A
alone had no effect on the induction of NO production. However, in
contrast to the stimulation of NO production in astrocytes (Fig. 1 and
Table I), all of the three inhibitors of PP 1/2A (calyculin A,
microcystin, and cantharidin) inhibited the LPS-induced NO production
in macrophages (Fig. 4A). This
decrease in NO production was accompanied by a decrease in iNOS protein
(Fig. 4B) and iNOS mRNA (Fig. 4C). Okadaic
acid, another very specific and potent inhibitor of PP 1/2A, also
inhibited the LPS-mediated production of NO (Fig.
5A) and expression of iNOS
protein (Fig. 5B) in macrophages in a
dose-dependent manner. Similar to rat peritoneal
macrophages, calyculin A was also found to inhibit the LPS- and
cytokine-induced production of NO (Fig. 6A) and the expression of iNOS
protein (Fig. 6B) in the murine macrophage cell line RAW
264.7. Taken together, these results indicate that PP 1/2A activities
are required to induce iNOS gene expression in macrophages.

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Fig. 4.
Inhibition of LPS-induced expression of iNOS
by inhibitors of PP 1 and PP 2A in rat peritoneal macrophages.
Cells incubated in serum-free DMEM/F-12 received calyculin A
(Cal. A), microcystin, or cantharidin along with 1.0 µg/ml
of LPS. A, after 24 h, concentration of nitrite was
measured in the supernatants as described earlier. Data are the
means ± S.D. of three different experiments. B, cell
homogenates were electrophoresed, transferred on nitrocellulose
membrane, and immunoblotted with antibodies against mouse macrophage
iNOS as described earlier. C, after 6 h of incubation,
cells were analyzed for iNOS mRNA by Northern blotting technique as
described earlier.
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Fig. 5.
Effect of okadaic acid on LPS-mediated
induction of iNOS in rat primary astrocytes. A, cells
incubated in serum-free DMEM/F-12 received different concentrations of
okadaic acid (OA) in the presence or the absence of 1.0 µg/ml of LPS. After 24 h of incubation, nitrite concentrations
were measured in supernatants. Data are the means ± S.D. of three
different experiments. B, cells incubated in serum-free
DMEM/F-12 received different concentrations of okadaic acid along with
1.0 µg/ml of LPS. After 24 h of incubation, cell homogenates
were electrophoresed, transferred on nitrocellulose membrane, and
immunoblotted with antibodies against mouse macrophage iNOS as
described before.
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Fig. 6.
Calyculin A inhibits the LPS- and
cytokine-induced expression of iNOS in RAW 264.7 cells. Cells
incubated in serum-free DMEM/F-12 received calyculin A along with LPS
and cytokines. A, after 24 h, concentration of nitrite
was measured in the supernatants as described earlier. Data are the
means ± S.D. of three different experiments. B, cell
homogenates were electrophoresed, transferred on nitrocellulose
membrane, and immunoblotted with antibodies against mouse macrophage
iNOS as described earlier. Concentration of different stimuli were as
follows: LPS, 0.5 µg/ml; TNF- , 20 ng/ml; IL-1 , 20 ng/ml;
IFN- , 20 units/ml.
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Differential Effect of Okadaic Acid on iNOS Promoter-derived CAT
Activity in LPS-stimulated Rat Primary Astrocytes and
Macrophages--
Differential regulation of the induction of iNOS
mRNA and protein in astrocytes and macrophages by the inhibitors of
PP 1/2A suggests that these inhibitors may regulate the transcription of iNOS gene differentially in these two different cell lines. Therefore, to understand the effect of okadaic acid on the
transcription of iNOS gene, astrocytes and macrophages were transfected
with a construct containing the iNOS promoter fused to the CAT gene, and activation of this promoter was measured after stimulating the
cells with LPS in the presence or the absence of okadaic acid. Consistent with the effect of okadaic acid on the production of NO and
the expression of endogenous iNOS in these two different cell types,
okadaic acid stimulated the LPS-induced CAT activity in astrocytes but
inhibited the LPS-induced CAT activity in macrophages, supporting the
conclusion that okadaic acid differentially regulates the transcription
of iNOS gene in astrocytes and macrophages (Fig. 7).

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Fig. 7.
Effect of okadaic acid on iNOS
promoter-derived CAT activity in rat primary astrocytes and
macrophages. Astrocytes (A) and macrophages
(B) were transfected with the construct containing the iNOS
promoter fused to the CAT gene using lipofectamine. After 24 h of
transfection, cells received okadaic acid (OA) with or
without 1.0 µg/ml of LPS, and after 14 h of stimulation, CAT
activity was measured. Data are the means ± S.D. of three
different experiments.
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Effect of Okadaic Acid on the Activation of NF-
B in Rat Primary
Astrocytes and Macrophages--
Inhibitors of PP 1/2A stimulated the
induction of iNOS in astrocytes but inhibited the induction of iNOS in
macrophages, suggesting that PP 1/2A may transduce different signals in
two different cell types for the differential regulation of iNOS.
Because the activation of NF-
B is reported to be necessary for the
induction of iNOS, we examined the effect of okadaic acid on the
LPS-induced activation of NF-
B in astrocytes and macrophages to
understand the basis of this differential regulation of induction of
iNOS by inhibitors of PP 1/2A. Activation of NF-
B was monitored by both DNA binding and transcriptional activity of NF-
B. DNA binding activity of NF-
B was evaluated by the formation of a distinct and
specific complex in a gel shift DNA binding assay. Consistent with
previous reports (11, 12), treatment of astrocytes or macrophages with
1.0 µg/ml of LPS resulted in the induction of DNA binding activity of
NF-
B (Fig. 8). This gel shift assay
detected a specific band in response to LPS that was competed off by an unlabeled probe. Although okadaic acid alone at different
concentrations failed to induce the DNA binding activity of NF-
B in
astrocytes, okadaic acid alone induced the DNA binding activity of
NF-
B in macrophages. However, in both astrocytes and macrophages,
okadaic acid stimulated the LPS-induced DNA binding activity of NF-
B (Fig. 8). We then tested the effect of okadaic acid on
NF-
B-dependent transcription of luciferase in astrocytes
and macrophages in the presence or the absence of LPS, using the
expression of luciferase from a reporter construct, pNF-
B Luc
(Stratagene), as an assay. Consistent with the effect of okadaic acid
on DNA binding activity of NF-
B, the okadaic acid induced
NF-
B-dependent transcription in macrophages but not in
astrocytes and stimulated the LPS-induced NF-
B-dependent
transcription in both astrocytes and macrophages (Fig.
9) under the condition in which the
LPS-mediated expression of iNOS was stimulated in astrocytes and
inhibited in macrophages (Figs. 2 and 5).

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Fig. 8.
Effect of okadaic acid on LPS-mediated DNA
binding activity of NF- B in rat primary astrocytes and
macrophages. Astrocytes (A) and macrophages
(B) incubated in serum-free DMEM/F-12 were treated with
okadaic acid alone or together with LPS (1.0 µg/ml). After 1 h
of 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." Lane 1,
nuclear extract of control cells; lane 2, nuclear extract of
LPS-treated cells; lane 3, nuclear extract of LPS-treated
cells incubated with 100-fold excess of unlabeled oligonucleotide;
lane 4, nuclear extract of cells treated with okadaic acid
(5 nM) alone; lane 5, nuclear extract of cells
treated with okadaic acid (10 nM) alone; lane 6,
nuclear extract of LPS and okadaic acid (5 nM)-treated
cells; lane 7, nuclear extract of LPS and okadaic acid (10 nM)-treated cells. The upper arrow indicates the
induced NF- B band, whereas the lower arrow indicates the
unbound probe.
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Fig. 9.
Effect of okadaic acid on transcriptional
activity of NF- B in rat primary astrocytes and macrophages.
Astrocytes (A) or macrophages (B) were
transfected with pNF- B-Luc using the lipotaxi method (Stratagene).
After 24 h of transfection, the cells were stimulated with okadaic
acid (OA) in the presence or the absence of 1.0 µg/ml of
LPS for 4 h, and the expression of the luciferase reporter was
quantitated as described under "Materials and Methods."
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Inhibitors of PP 1/2A Stimulate the LPS-induced Production of
TNF-
in Rat Primary Astrocytes and Macrophages--
Okadaic acid
stimulated the transcription of iNOS in astrocytes and attenuated the
transcription of iNOS in macrophages. However, in contrast, okadaic
acid stimulated the activation of NF-
B in both astrocytes and
macrophages. Because the induction of TNF-
also depends on the
activation of NF-
B (30), we examined the effect of okadaic acid on
the LPS-induced production of TNF-
in astrocytes and macrophages.
Consistent with the stimulatory effect of okadaic acid on the
LPS-induced activation of NF-
B, okadaic acid stimulated the
LPS-induced production of TNF-
in both astrocytes and
macrophages (Table II).
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Table II
Effect of inhibitors of PP 1 and PP 2A on LPS-induced production of
TNF- in rat primary astrocytes and macrophages
Cells preincubated in serum-free DMEM/F-12 with different
concentrations of okadaic acid for 30 min were stimulated with 1.0 µg/ml of LPS. After 24 h of incubation, concentration of TNF-
was measured in supernatants as described under "Materials and
Methods." Data are expressed as the means ± S.D. of three
different experiments.
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Effect of Inhibitors of PP 1/2A on Cell Viability--
Astrocytes
or macrophages were incubated with different inhibitors of PP 1/2A 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 astrocytes and
inhibition of the expression of iNOS in macrophages by inhibitors of PP
1/2A are not due to any change in viability of either astrocytes or
macrophages.
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DISCUSSION |
Transient modulation of protein phosphorylation and
dephosphorylation is a major mechanism of intracellular signal
transduction pathways triggered by different cytokines (31, 32).
Therefore, it is reasonable to assume that inhibition of PP 1 and 2A
activities will influence cytokine-induced signal transduction pathways
for the induction of iNOS. The signaling events in cytokine-mediated induction of iNOS in astrocytes and macrophages are not well
understood. A complete understanding of the cellular mechanisms
involved in the induction of iNOS should identify novel targets for the
therapeutic intervention in NO-mediated neuroinflammatory diseases.
Several lines of evidence presented in this study support the
conclusion that inhibition of PP 1/2A activity differentially modulates
the LPS- and cytokine-induced expression of iNOS and production of NO
in rat primary astrocytes and macrophages. Our conclusion is based on
the following observations. First, compounds (calyculin A, microcystin,
okadaic acid, and cantharidin) that inhibit PP 1/2A stimulated the LPS-
and cytokine-mediated production of NO as well as expression of iNOS
protein and mRNA in astrocytes and C6 glial cells.
However, in contrast, these inhibitors inhibited the LPS- and
cytokine-mediated production of NO and expression of iNOS in rat
resident macrophages and RAW 264.7 cells. Second, the inhibitors of PP
1/2A stimulated iNOS promoter-derived CAT activity in LPS-treated
astrocytes but inhibited iNOS promoter-derived CAT activity in
LPS-treated macrophages. These observations suggest that the signaling
events (phosphorylation/dephosphorylation of serine and threonine
residues of proteins associated with transduction of these signals)
required for the induction of iNOS in astrocytes differ from those
required for the induction of iNOS in macrophages.
Cytokines (TNF-
, IL-1
, or IFN-
) and LPS bind to their
respective receptors and induce iNOS expression via activation of NF-
B (11-15, 33, 34). The nuclear expression and biological function of the NF-
B transcription factor are tightly regulated through its cytoplasmic retention by the inhibitor I
B
(35). 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). 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 (37). 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 but
not p70 S6 kinase or MAP kinase phosphorylates the N-terminal
regulatory domain of I
B
on serine 32 (38). In vivo,
only phorbol 12-myristate 13-acetate produced rapid activation of p90
RSK and that of NF-
B, whereas other potent NF-
B inducers
including TNF-
and the Tax transactivator of human T-cell
lymphotrophic virus type I failed to activate p90 RSK (38), suggesting
that more than a single I
B
kinase exists within the cell and that
these I
B
kinases may be differentially activated by different
NF-
B inducers. Upon phosphorylation, I
B
that is still bound to
NF-
B apparently becomes a high affinity substrate for an
ubiquitin-conjugating enzyme (39). Following phosphorylation-controlled
ubiquitination, the I
B
is rapidly and completely degraded by the
20 or 26 S proteosome, and NF-
B is targeted to the nucleus (40).
Okadaic acid and other inhibitors of PP 1/2A have also been shown to
induce the activation of NF-
B in monocytes, Jurkat T cells, and HeLa
cells (41, 42) as a result of phosphorylation of I
B
at protein
phosphatase 2A-sensitive phosphorylation sites, and these sites are
known to be different from cytokine-induced phosphorylation sites (43).
In contrast to the effect of okadaic acid on the activation of NF-
B
in other cell types (41, 42), okadaic acid by itself was unable to
induce the activation of NF-
B in rat primary astrocytes. However,
okadaic acid markedly stimulated the LPS- or cytokine-mediated
activation of NF-
B in astrocytes (Figs. 8 and 9). The increase in
the activation of NF-
B in LPS-stimulated astrocytes by okadaic acid
paralleled the increase in induction of iNOS, suggesting that
stimulation of iNOS expression in LPS-activated rat primary astrocytes
by inhibitors of PP 1/2A is probably mediated via enhanced activation of NF-
B. However, consistent with the effect of okadaic acid on the
activation of NF-
B in other cell types including monocytes, Jurkat T
cells, and HeLa cells (41, 42), okadaic acid by itself induced the
activation of NF-
B in macrophages. However, this activation of
NF-
B by okadaic acid in macrophages did not result in the induction
of iNOS, suggesting that activation of NF-
B by okadaic acid is not
sufficient for the induction of iNOS in macrophages. Moreover, similar
to astrocytes, the okadaic acid stimulated the LPS-mediated activation
of NF-
B in rat peritoneal macrophages, but in sharp contrast to the
effect of okadaic acid on the induction of iNOS in LPS-treated
astrocytes, the stimulation of NF-
B activation by okadaic acid in
LPS-treated macrophages did not parallel with the expression of iNOS.
Instead, consistent with a previous report (44), okadaic acid and other
inhibitors of PP 1/2A markedly inhibited the LPS- and cytokine-induced
expression of iNOS in macrophages. The basis for this differential
regulation of induction of iNOS in astrocytes and macrophages by
inhibitors of PP 1/2A is not understood at the present time.
Earlier, we have observed that cAMP-dependent protein
kinase A (PKA) also differentially modulates the induction of iNOS in astrocytes and macrophages (12). The inhibition of cytokine-induced activation of NF-
B and the induction of iNOS with the increase in
PKA activity in astrocytes (12) and the stimulation of the activation
of NF-
B and the induction of iNOS with the decrease in PP 1/2A
activities in astrocytes (Figs. 1 and 8) suggest that both PKA (a
serine-threonine protein kinase) and PP 1/2A (serine-threonine phosphoprotein phosphatases) may provide inhibitory signals to LPS- and
cytokine-induced signal transduction pathway for the induction of iNOS
in astrocytes. In contrast, in macrophages, inhibitors of PKA inhibited
the LPS-mediated activation of NF-
B and induction of iNOS (12), and
inhibitors of PP 1/2A stimulated the LPS-mediated activation of NF-
B
but inhibited the induction of iNOS, suggesting that both PKA and PP
1/2A are necessary components of the LPS-mediated signaling pathways
for the induction of iNOS. However, the molecular basis for the
differential regulation of activation of NF-
B and expression of iNOS
gene by inhibitors of PP 1/2A in rat peritoneal macrophages is not
known at the present time. In light of the fact that NF-
B is
necessary but not sufficient for the expression of iNOS gene (45,
46) and that many of the signal transduction events are cell type
specific (12, 47), the apparent stimulation of NF-
B and inhibition
of iNOS gene expression by inhibitors of PP 1/2A clearly delineate that
apart from the activation of NF-
B some other signaling pathway(s)
sensitive to PP 1/2A is/are responsible for the expression of iNOS gene in macrophages.
We thank Dr. Avtar K. Singh for review of the
manuscript and helpful suggestions and Jan Ashcraft for technical
help.