From the Department of Pediatrics, Medical University of South
Carolina, Charleston, South Carolina 29425
The sphingomyelin signal transduction
pathway is known to play a role in mediating the action of various
cytokines. Here we examined the possible role of the sphingomyelin
signaling pathway on lipopolysaccharide (LPS)- and cytokine-mediated
production of NO and the expression of inducible nitric-oxide synthase
(iNOS). Sphingomyelinase (SMase) treatment of astrocytes increased the cellular levels of ceramide without the induction of NO production. However, incubation of LPS or cytokine-stimulated astrocytes with SMase
or by increasing intracellular ceramide by cell-permeable ceramide
analogs (C2- or C6-ceramide) or inhibitor
of ceramidase (N-oleoyl ethanolamine) led to a time- and
dose-dependent increase in the production of NO. This
increase in NO production was accompanied by an increase in iNOS
activity, iNOS protein, and iNOS mRNA. Similar to astrocytes, SMase
or ceramide analogs also stimulated the LPS- and cytokine-mediated
expression of iNOS in the C6 glial cell line. Since
activation of NF-
B is necessary for the induction of iNOS, we
examined the effect of SMase and C2-ceramide on the activation of NF-
B. Although SMase or C2-ceramide alone
was ineffective in activating NF-
B, both stimulated the LPS-mediated
activation of NF-
B in LPS-activated astrocytes. Inhibition of
ceramide and LPS-mediated induction of iNOS by antioxidant inhibitors
of NF-
B (N-acetylcysteine and pyrrolidine
dithiocarbamate) suggest that the stimulatory effect of ceramide on the
induction of iNOS is due to the stimulation of NF-
B activation and
that cellular redox plays a role in the activation of NF-
B and
induction of iNOS. Inhibition of LPS-mediated as well as LPS and
ceramide-mediated induction of iNOS and activation of NF-
B by
PD98059, a specific inhibitor of activation of mitogen-activated
protein (MAP) kinase kinase (MEK), and FPT inhibitor II, a selective
inhibitor of Ras farnesyl protein transferase, indicate that the
Ras-MAP kinase pathway is involved in LPS-ceramide induced activation
of NF-
B and induction of iNOS, and that ceramide-mediated signaling
events probably converge into the LPS-modulated MAP kinase signaling pathway resulting in greater activation of NF-
B and iNOS induction. This study illustrates a novel role of the sphingomyelin-ceramide signaling pathway in stimulating the expression of iNOS via LPS- or
cytokine-mediated activation of NF-
B in astrocytes.
 |
INTRODUCTION |
The sphingomyelin pathway is a newly described signal transduction
pathway mediating the action of several extracellular stimuli and
leading to important biochemical and cellular responses (1, 2). This
pathway is initiated by the activation of neutral sphingomyelinase
(SMase)1 which hydrolyzes
membrane sphingomyelin to ceramide and phosphocholine. Ceramide, has
emerged as a second messenger molecule which is considered to mimic
most of the cellular effects of TNF-
, IL-1
, and LPS in terminal
differentiation, apoptosis, and cell cycle arrest (3, 4). These
conclusions are primarily based on the ability of exogenous,
cell-permeable, ceramide analogs and endogenous ceramide generated by
sphingomyelin-ceramide signaling events specifically to cause cell
differentiation and growth inhibition. Although the ceramide dependent
pathway for signal transduction is not very well established so far,
present knowledge indicates that a ceramide-mediated signal
transduction pathway includes activation of a specific proline-directed
Ser/Thr protein kinase (5), a specific protein phosphatase (1, 2) and
protein kinase C-
(6), and inhibition of phospholipase D
(7).
Since bacterial lipopolysaccharide (LPS) and cytokines (TNF-
,
IL-1
, and IFN-
) induce the production of ceramide and mediate the
induction of inducible nitric-oxide synthase (iNOS) (1, 2, 8-10) it
was of interest to examine the relationship between the sphingomyelin
signaling pathway and the expression of iNOS. Nitric oxide (NO), a
product of iNOS, is a diffusible gas that plays a part in many
physiological and diverse pathological conditions. At low concentration
NO has been shown to play a role in neurotransmission and vasodilation
while at higher concentration it is neurotoxic. This is of particular
importance in demyelinating conditions (e.g. multiple
sclerosis, experimental allergic encephalopathy, advanced HIV
encephalitis, X-adrenoleukodystrophy) and in ischemia and traumatic
injuries associated with infiltrating macrophages and the production of
proinflammatory cytokines (11-13), where astrocyte and
microglia-derived NO could contribute to oligodendrocyte degeneration and neuronal death.
In the present study we examined the possible involvement of the
sphingomyelin pathway in the induction or stimulation of iNOS in rat
primary astrocytes and C6 glial cells. We report that SMase
or cell-permeable ceramide analogs by themselves were unable to
activate NF-
B and induce iNOS, however, SMase and ceramide markedly
enhanced the LPS- and cytokine-mediated activation of NF-
B and
induction of iNOS. Antioxidant inhibitors of NF-
B activation (N-acetyl-L-cysteine and pyrrolidine
dithiocarbamate) inhibited the LPS and ceramide-mediated induction of
iNOS, indicating that ceramide stimulates the LPS-mediated induction of
iNOS by stimulating the activation of NF-
B and that cellular redox
plays a role in the activation of NF-
B and induction of iNOS.
Inhibition of LPS- and ceramide-mediated activation of NF-
B and
induction of iNOS by PD98059, an inhibitor of MAP kinase kinase, and
FPT inhibitor II, an inhibitor of Ras farnesyl protein transferase,
suggest that ceramide potentiates LPS-mediated MAP kinase signaling
pathways to stimulate activation of NF-
B and iNOS expression. The
ability of ceramides to potentiate the LPS- or cytokine-mediated
activation of NF-
B activation and expression of iNOS and production
of NO in astrocytes indicate that sphingomyelin-ceramide signaling
events may play a role in inflammatory neuropathies associated with the induction of iNOS and production of NO. To our knowledge, this is the
first example of a positive modulatory role in the regulation of iNOS
expression by ceramides in a cell.
 |
MATERIALS AND METHODS |
Reagents--
Recombinant rat IFN-
, DMEM/F-12, and fetal
bovine serum were from Life Technologies, Inc. Human IL-1
was from
Genzyme. Mouse recombinant TNF-
was obtained from Boehringer
Mannheim, Germany. Sphingomyelinase (Staphylococcus aureus),
LPS (Escherichia coli), NADPH, FAD, tetrahydrobiopterin,
N-acetylcysteine, pyrrolidine dithiocarbamate, and Dowex 50W
were from Sigma. C2- and C6-ceramides and
PD98059 were purchased from Biomol.
NG-Methyl-L-arginine, FPT
inhibitor II, and antibodies against mouse macrophage iNOS were
obtained from Calbiochem. [
-32P]ATP (3000 Ci/mmol) was
from NEN Life Science Products.
L-[2,3,4,5-H3]Arginine was from Amersham.
NF-
B DNA-binding protein detection kit was obtained from Life
Technologies, Inc.
Isolation and Maintenance of Astrocytes and C6 Glial
Cells--
Astrocytes were prepared from rat cerebral tissue as
described by McCarthy and DeVellis (14). Cells were maintained in
DMEM/F-12 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. To ensure the
complete removal of all the oligodendrocytes and microglia before
subculturing, the shaking was repeated twice after a gap of 1 or 2 days. 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.
Lipid Extraction--
Approximately 1.0 × 106
cells were exposed to 1.0 µg/ml LPS or 1.0 µg/ml LPS with 100 milliunits/ml of bacterial SMase for different periods and lipids were
extracted according to the method described by Welsh (15).
Quantification of Ceramide Levels by Diacylglycerol Kinase
Assay--
Ceramide content was quantified essentially according to
Priess et al. (16) using diacylglycerol kinase and
[
-32P]ATP. Briefly, dried lipids were solubilized in
20 µl of an octyl-
-D- glucoside/cardiolipin solution
(7.5% octyl-
-D-glucoside, 5 mM cardiolipin
in 1 mM DTPA) by sonication in a sonicator bath. The reaction was then carried out in a final volume of 100 µl containing the 20-µl sample solution, 50 mM imidazole HCl, pH 6.6, 50 mM NaCl, 12.5 mM MgCl2, 1 mM EGTA, 2 mM dithiothreitol, 6.6 µg of diacylglycerol kinase, and 1 mM [
-32P]ATP
(specific activity of 1-5 × 105 cpm/nmol) for 30 min
at room temperature. The labeled ceramide 1-phosphate was resolved with
a solvent system consisting of methyl acetate, n-propanol,
chloroform, methanol, 0.25% KCl in water:acetic acid
(100:100:100:40:36:2). A standard sample of ceramide was phosphorylated
under identical conditions and developed in parallel. Both standard and
samples had identical RF value (0.46). Quantification of ceramide 1-phosphate was carried out by
autoradiography and densitometric scanning using Imaging Densitometer
(Model GS-670; Bio-Rad) and software provided with the instrument by
the manufacturer. Values are expressed either as arbitrary units
(absorbance) or as percent change.
Assay for NO Synthesis--
Synthesis of NO was determined by
assaying 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 (17, 18) 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.
Assay for NOS Activity--
NOS activity was measured directly
by production of L-[2,3,4,5-3H]citrulline
from L-[2,3,4,5-3H]arginine (17, 18). In
these experiments, 50 µl of astrocyte homogenate was incubated at
37 °C in the presence of 50 mM Tris-HCl, pH 7.8, 0.5 mM NADPH, 5 µM FAD, 5 µM
tetrahydrobiopterin, and 12 µM
L-[2,3,4,5-3H]arginine (118 mCi/mmol) in a
total volume of 200 µl. The reaction was stopped by the addition of
800 µl of ice-cold 20 mM HEPES, pH 5.5, followed by the
addition of 2 ml of Dowex 50W equilibrated in the same buffer. The
samples were then centrifuged and the concentration of
L-[3H]citrulline was determined in the
supernatant by liquid scintillation counting. Protein was measured by
the procedure of Bradford (19).
Immunoblot Analysis for iNOS--
Following 24-h incubations in
the presence or absence of different stimuli, astrocytes were scraped
off, washed with Hank's buffer, and homogenized in 50 mM
Tris-HCl, pH 7.4, containing protease inhibitors. 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 as described earlier (18).
Northern Blot Analysis--
Stimulated primary astrocytes 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 ExpressHyb hybridization solution
(CLONTECH) as described by the manufacturer. The
cDNA probe was made by polymerase chain reaction amplification
using two primers (forward primer, 5
-CTCCTTCAAAGAGGCAAAAATA-3
;
reverse primer, 5
-CACTTCCTCCAGGATGTTGT-3
) (18, 20). After
hybridization, filters were washed two to 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 film (Kodak).
The same filters were stripped and rehybridized with probes for
glyceraldehyde-3-phosphate dehydrogenase. The relative mRNA content
for iNOS was measured after scanning the bands with a Bio-Rad (Model
GS-670) imaging densitometer.
Nuclear Run-on Assay--
For the measurement of gene
transcription, nuclei were prepared and in vitro
transcriptional activity was measured with nuclei (25 × 106 nuclei per assay) using 30 µCi of
[
-32P]UTP (400 Ci/mmol) as described by Caira et
al. (21). Briefly, the filters were prehybridized in 1 ml of
hybridization buffer (50% formamide, 5 × SSC, 1% SDS, 15%
dextran sulfate, 1 × Denhardt's solution, 50 µg/ml heparin).
Following 24 h of prehybridization in the above buffer,
hybridization was carried out with the labeled RNAs (1.3 × 105 cpm) at 42 °C for 60 h to 3 µg of the
immobilized plasmid pGEM-T as a control or to plasmids containing
inserts of rat glyceraldehyde-3-phosphate dehydrogenase, rat actin, and
human iNOS cDNAs. The filters were washed twice in 2 × SSC,
0.1% SDS for 15 min at 42 °C and twice in 0.5 × SSC, 0.1%
SDS for 15 min. Then the filters were treated with RNase buffer (300 mM NaCl, 10 mM Tris-HCl, pH 7.4, 40 mM EDTA, 10 µg/ml RNase A, and 350 units/ml RNase T1) at
37 °C for 30 min and then in the same buffer without RNase for
another 30 min and autoradiographed.
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. (22) 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
70 °C until further 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.
 |
RESULTS |
Bacterial SMase and LPS Are Capable of Inducing the Production of
Ceramide in Primary Rat Astrocytes--
Rat primary astrocytes were
cultured in serum-free media with LPS and SMase, alone or in
combination, for different times and the amount of ceramide was
quantitated by the diacylglycerol kinase assay (16). LPS alone induced
a significant increase in the ceramide content within 10-60 min of
exposure (Fig. 1). Almost 2.5-fold
increase in ceramide was observed after 30 min of exposure. Bacterial
neutral SMase, the enzyme capable of degrading sphingomyelin to
ceramide, alone also induced a marked generation of ceramide (8.0-fold
after 10 min) demonstrating that under these experimental conditions
SMase alone can degrade sphingomyelin and increase the level of
ceramide to the same level as the combined effect of LPS and SMase.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of LPS and SMase on ceramide content
of rat primary astrocytes. Astrocytes were exposed to either 1.0 µg/ml LPS or a combination of LPS (1.0 µg/ml) and SMase (100 milliunits/ml) for different time intervals. Lipids were extracted and
ceramide content was measured as described under "Materials and
Methods." Results are expressed as mean ± S.D. of three
different experiments.
|
|
Stimulation of LPS- and Cytokine-induced Expression of iNOS and
Production of Nitric Oxide by SMase in Rat Primary Astrocytes--
Rat
primary astrocytes were cultured in serum-free media in the presence of
LPS and SMase. The activity of iNOS was measured as production of
nitrite, a soluble product of NO in the culture medium (Table
I) and as conversion of arginine to
citrulline (Fig. 2B). It is
evident from Table I that in astrocytes, LPS (1 µg/ml) induced the
production of nitrite by more than 7-fold. Both
NG-methyl-L-arginine, a
competitive inhibitor of NOS, and arginase, an enzyme that degrades the
substrate (L-arginine) for NOS, suppressed the LPS-mediated
nitrite secretion suggesting that LPS-induced nitrite release in
primary astrocytes is dependent on NOS-mediated arginine metabolism.
SMase by itself was neither stimulatory nor inhibitory to nitrite
production in astrocytes. However, SMase, when added with LPS, potently
stimulated the LPS-mediated induction of nitrite production in
astrocytes. Almost 3-fold stimulation was observed when SMase was used
at a concentration of 100 or 200 milliunits/ml (Table I). Similar
effects of SMase were also observed on cytokine-induced nitrite
production in astrocytes (Fig.
3A). Addition of SMase
stimulated the TNF-
-, IL-1
-, or IFN-
-induced nitrite
production in astrocytes by 3-4-fold. Consistent with the production
of nitrite, the formation of L-citrulline from
L-arginine in an enzymatic assay with astrocyte homogenate was also stimulated approximately 3-fold by treatment of LPS- or
cytokine-stimulated astrocytes with SMase (100 milliunits/ml) (Figs.
2B and 3B). To understand the mechanism of the
stimulatory effect of SMase on LPS- and/or cytokine-induced activation
of iNOS in astrocytes, we examined the effect of SMase on the
expression of iNOS protein and iNOS mRNA. Immunoblot analysis with
antibodies against murine macrophage iNOS and Northern blot analysis
for iNOS mRNA of LPS-stimulated astrocytes clearly showed that
SMase enhanced the LPS-mediated induction of iNOS protein (Fig.
2C) and mRNA (Fig. 2D). To correlate the
expression of iNOS with ceramide, we measured ceramide levels under
these conditions. LPS, capable of inducing only modest generation of
ceramide (2.5-fold after 30 min of incubation) (Fig. 2A)
induced the expression of iNOS and production of NO. In contrast, SMase
(200 milliunits/ml) itself capable of inducing marked generation of
ceramide (7.5-fold after 30 min of incubation) (Fig. 2A) was
ineffective in inducing the expression of iNOS or production of NO.
However, increased ceramide level produced by SMase markedly stimulated
LPS-mediated induction of iNOS and production of NO (Fig. 2). Similar
stimulatory effects of SMase were also found on cytokine-induced
expression of iNOS (Fig. 3). In primary astrocytes, TNF-
, IL-1
,
or IFN-
alone were able to induce the expression of iNOS and the
addition of SMase along with cytokines stimulated the induction of iNOS
expression.
View this table:
[in this window]
[in a new window]
|
Table I
Effect of SMase on arginine-dependent nitrite
accumulation in LPS-stimulated rat primary astrocytes
Astrocytes were cultured for 24 h in serum-free DMEM/F-12 medium
with the listed reagents; and nitrite concentration in the supernatants
was measured as described under "Materials and Methods." Arginase
(100 units/ml), N-methyl-L-arginine
(L-NMA) (0.1 mM), and different concentrations
of SMase were added to the cells together with LPS (1.0 µg/ml). Data
are mean ± S.D. of three different experiments.
|
|

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 2.
Stimulation of LPS-induced expression of iNOS
by SMase in rat primary astrocytes. Cells incubated in serum-free
DMEM/F-12 received different concentrations of SMase along with 1.0 µg/ml LPS. A, after 30 min of incubation lipids were
extracted and ceramide content was measured as described under
"Materials and Methods." Results are expressed as mean ± S.D.
of three different experiments. B, after 24 h, activity
of iNOS was assayed in cell homogenates as mentioned in the methods
section. Data are mean ± S.D. of three different experiments.
C, cell homogenates were electrophoresed, transferred on
nitrocellulose membrane, and immunoblotted with antibodies against
mouse macrophage iNOS as mentioned in under "Materials and
Methods." D, 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."
|
|

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of SMase on cytokine-induced
production of NO and expression of iNOS in rat primary astrocytes.
Cells incubated in serum-free DMEM/F-12 received 100 milliunits/ml
SMase along with different cytokines (TNF- , 100 ng/ml; IL-1 , 100 ng/ml; IFN- , 200 units/ml). After 24 h of incubation,
concentration of nitrite was measured in supernatants (A)
and activity of iNOS was assayed in cell homogenates (B) as
mentioned under "Materials and Methods." Data are mean ± S.D.
of three different experiments. C, cell homogenates were
electrophoresed, transferred on nitrocellulose membrane, and
immunoblotted with antibodies against mouse macrophage iNOS as
mentioned under "Materials and Methods." D, after 6 h of incubation, cells were analyzed for iNOS mRNA using the
Northern blotting technique as described earlier. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
|
|
Exogenous Ceramide Stimulates the LPS- or Cytokine-induced iNOS
Expression in Rat Primary Astrocytes--
Ceramide, the breakdown
product of sphingomyelin, serves as the second messenger in the
sphingomyelin pathway. To evaluate its effect on the expression of
iNOS, the experiments described above for SMase studies were repeated
with the cell-permeable C2- and C6-ceramide
analogs. Similar to SMase, ceramide analogs alone did not induce the
production of nitrite but addition of C2- or
C6-ceramide to astrocytes along with LPS increased
expression of iNOS mRNA, iNOS protein as well as production of NO
(Fig. 4). This stimulatory effect peaked
at 10 µM for C2- or C6-ceramide. To examine the specificity of ceramide, we studied the effect of
C2-dihydroceramide, the inactive analog of
C2-ceramide, on LPS-mediated production of NO. In contrast
to C2-ceramide, C2-dihydroceramide was
ineffective in stimulating the LPS-induced production of nitrite (Fig.
5). A 10 µM concentration
of ceramide produced a maximal effect on LPS-induced production of NO,
however, a higher concentration of ceramide (25 µM) was
ineffective in inducing the production of NO.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 4.
Stimulation of LPS-induced expression
of iNOS in rat primary astrocytes by cell-permeable ceramide analogs
and by an inhibitor of ceramidase. Cells incubated in serum-free
DMEM/F-12 received C2- or C6-ceramide along
with LPS (1.0 µg/ml). However, N-oleoylethanolamine was
added to the cells 2 h prior to the addition of LPS. After 24 h of incubation, concentration of nitrite (A) was measured in the supernatants, and cell homogenates were electrophoresed, transferred on nitrocellulose membrane, and immunoblotted with antibodies against mouse macrophage iNOS (B) as mentioned
under "Materials and Methods." C, after 6 h of
incubation, cells were analyzed for iNOS mRNA by Northern blotting
technique as described earlier. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
|
|

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of C2-dihydroceramide on
the LPS-mediated production of NO in rat primary astrocytes. Cells
incubated in serum-free DMEM/F-12 received different concentrations of
C2-ceramide and C2-dihydroceramide in the
presence or absence of LPS (1.0 µg/ml). After 24 h of
incubation, concentration of nitrite was measured in the supernatants
as mentioned under "Materials and Methods."
|
|
Oleoylethanolamine (OE), an inhibitor of ceramidase, the enzyme
responsible for the catabolism of ceramide to sphingosine and fatty
acid, has been reported to induce apoptosis in neuroblastoma cells (23)
because of the increased concentration of ceramide due to the
inhibition of catabolism of ceramide (23). We examined the possible
role of increased intracellular ceramide by the inhibition of
ceramidase on the induction of iNOS mRNA, iNOS protein, and NO
production. Treatment of astrocytes with OE 2 h before the addition of LPS stimulated the expression of iNOS mRNA, iNOS
protein, and the production of NO (Fig. 4), supporting the conclusion
that the increase in concentration of intracellular ceramides
stimulates the expression of iNOS. The stimulatory effect of OE peaked
after 100 µM (data not shown). However, similar to
studies with SMase or exogenous ceramides, OE alone did not induce the
expression of iNOS in astrocytes.
SMase and Ceramide Stimulate the Transcription of the iNOS Gene in
Rat Primary Astrocytes--
To gain further insight into the mechanism
of the stimulatory effect of SMase and ceramide on LPS- or
cytokine-mediated expression of iNOS mRNA, we studied the influence
of SMase and ceramide on the rate of iNOS gene transcription, as
measured by nuclear run-on assays. Fig. 6
shows that LPS induced the transcription of the iNOS gene in
astrocytes, and addition of SMase (100 milliunits/ml) or
C2-ceramide (10 µM) along with LPS stimulated
LPS-induced transcription of the iNOS gene. These results clearly
suggest that SMase and ceramide stimulate LPS-induced expression of
iNOS mRNA, protein, and activity by stimulating transcription of
the iNOS gene.

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 6.
SMase and ceramide stimulate the relative
rate of nuclear transcription of the iNOS gene in LPS-stimulated rat
primary astrocytes. Cells incubated in serum-free DMEM/F-12
received 100 milliunits/ml SMase or 10 µM
C2-ceramide along with 1.0 µg/ml LPS. After 4 h
cells were taken out and nuclei were collected for nuclear run-on
assays. 32P-Labeled mRNA was transcribed in
vitro from isolated nuclei, and 1.3 × 105 cpm of
run-on products were hybridized to each blot as described under
"Materials and Methods." The plasmids used were pGEM-T without any
insert (negative control) or containing iNOS,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or actin
cDNA inserts.
|
|
SMase and Ceramide Stimulate the Expression of iNOS in Rat
C6 Glial Cells--
Similar to primary astrocytes, SMase
also stimulated LPS- and/or cytokine-induced production of nitrite as
well as the expression of iNOS in rat C6 glial cells (Fig.
7). Unlike astrocytes, neither LPS nor
cytokines alone was a sufficient inducer of NO production in rat
C6 glial cells (17, 18, 24). A combination of LPS and
cytokines was required to induce the production of NO in C6 glial cells (17, 18, 24) (Fig. 7). However, the addition of neutral
SMase along with LPS and cytokines to rat C6 glial cells
stimulated the expression of iNOS protein and the production of NO by
more than 2-fold. These observations suggest that both in primary
astrocytes and C6 glial cells the sphingomyelin-ceramide signaling events up-regulate the cytokine-induced expression of iNOS
and the production of NO.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 7.
SMase and LPS stimulate the production of NO
and the expression of iNOS in rat C6 glial cells. Cells incubated
in serum-free media received SMase (100 milliunits/ml) along with LPS
and cytokines. After 24 h of incubation, the production of nitrite
was measured in supernatants (A), and cell homogenates were
analyzed for iNOS protein by the immunoblotting technique
(B) as described before. Concentration of different stimuli
were: LPS, 0.5 µg/ml; TNF- , 25 ng/ml; IL-1 , 25 ng/ml; IFN- ,
50 units/ml.
|
|
SMase and Ceramide Stimulate LPS-mediated Activation of NF-
B in
Rat Primary Astrocytes--
Since the activation of NF-
B is
necessary for the induction of iNOS (8, 18), the observed enhancement
of iNOS expression by SMase or ceramides in rat primary astrocytes may
be due to the enhanced activation of NF-
B. To examine this
possibility, astrocytes were treated with either SMase or
C2-ceramide, alone or along with LPS, and activation of
NF-
B was examined as translocation of the transcription factor to
the nucleus. NF-
B activation was evaluated by the formation of a
distinct and specific complex in a gel-shift DNA-binding assay with
nuclear proteins. Treatment of astrocytes with 1.0 µg/ml LPS resulted
in the activation of NF-
B (Fig. 8).
This gel-shift assay detected a specific band in response to LPS that
was competed off by unlabeled probe. SMase or C2-ceramide
alone at different concentrations failed to induce NF-
B. However,
treatment of astrocytes with 1.0 µg/ml LPS in combination with
variable concentrations of C2-ceramide or SMase resulted in
enhancement of NF-
B activation, suggesting that the sphingomyelin
signaling pathway played a positive modulatory role in augmenting
NF-
B activation. Moreover, the inability of ceramide to activate
NF-
B and induce iNOS by itself and the augmentation of levels of
LPS-induced NF-
B activation and iNOS expression by ceramide suggest
that the sphingomyelin-ceramide signaling events instead of directly
activating the NF-
B converge into the LPS-mediated cascade resulting
in a greater signaling response. Consistent with this conclusion,
N-acetylcysteine and pyrrolidine dithiocarbamate,
antioxidant inhibitors of NF-
B activation (8, 25), inhibited the
LPS- and ceramide-mediated production of NO (Fig.
9A) and expression of iNOS
mRNA (Fig. 9B).

View larger version (72K):
[in this window]
[in a new window]
|
Fig. 8.
Enhancement of LPS-mediated activation of
NF- B by SMase and C2-ceramide in rat primary
astrocytes. Cells incubated in serum-free DMEM/F-12 were treated
with neutral SMase (100 milliunits/ml) or C2-ceramide (10 µM) alone or together with LPS (1.0 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 1-8
represent nuclear extract of control cells, nuclear extract of
LPS-treated cells, nuclear extract of LPS-treated cells incubated with
100-fold excess of unlabeled oligonucleotide, nuclear extract of cells
treated with LPS and C2-ceramide, nuclear extract of cells
treated with LPS and SMase, nuclear extract of LPS and SMase-treated
cells incubated with 100-fold excess of unlabeled oligonucleotide,
nuclear extract of C2-ceramide-treated cells, and nuclear
extract of SMase-treated cells. The upper arrow indicates the induced NF- B band whereas the lower arrow indicates
the unbound probe.
|
|

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 9.
N-Acetylcysteine and pyrrolidine
dithiocarbamate inhibit LPS and ceramide-induced expression of iNOS in
rat primary astrocytes. Cells preincubated in serum-free media
with different concentrations of N-acetylcysteine
(NAC) or pyrrolidine dithiocarbamate (PDTC) for
1 h received 1.0 µg/ml LPS and 10 µM
C2-ceramide. Concentration of nitrite (A) was
measured in the supernatants after 24 h of incubation, however,
after 6 h of incubation, cells were analyzed for iNOS mRNA
(B) by the Northern blotting technique as described earlier.
GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
|
|
Inhibition of the LPS- and Ceramide-mediated Expression of iNOS and
Activation of NF-
B by PD98059, an Inhibitor of MAP Kinase Kinase
(MEK), and FPT Inhibitor II, an Inhibitor of Ras Farnesyl Protein
Transferase--
To understand the possible role of Ras and the MAP
kinase pathway in LPS- and ceramide-mediated activation of NF-
B and
induction of iNOS in astrocytes, we examined the effect of PD98059 and
FPT inhibitor II on the LPS- as well as the LPS and ceramide-mediated activation of NF-
B and induction of iNOS. Preincubation of
astrocytes with either PD98059 or FPT inhibitor II blocked the
LPS-mediated as well as the LPS and ceramide-mediated production of NO
(Fig. 10A), the expression
of iNOS mRNA (Fig. 10B), and activation of NF-
B (Fig.
10C). These experiments suggest that the Ras-MAP kinase signaling pathway is involved in LPS-mediated induction of iNOS and
that ceramide potentiates these signaling pathways in LPS-stimulated astrocytes for the stimulation of NF-
B activation and iNOS
induction.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 10.
Effect of PD98059 and FPT inhibitor II on
LPS- and ceramide-mediated expression of iNOS and activation of NF- B
in rat primary astrocytes. Cells preincubated in serum-free media with 50 µM PD98059 or 200 µM FPT inhibitor
II for 1 h received 1.0 µg/ml LPS and 10 µM
C2-ceramide. Concentration of nitrite (A) was
measured in the supernatants after 24 h of incubation, however,
after 6 h of incubation, cells were analyzed for iNOS mRNA
(B) by the Northern blotting technique as described earlier. C, 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." GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
|
|
 |
DISCUSSION |
In the current work we provide evidence that the sphingomyelin
signal transduction pathway has a stimulatory effect on the LPS- and
cytokine-mediated expression of iNOS in astrocytes and C6
glial cells. This conclusion is based on the following observations. First, addition of SMase, the enzyme that hydrolyzes sphingomyelin in
the plasma membrane to ceramide and phosphocholine, by itself has no
effect on the production of NO both in astrocytes and C6 glial cells. Second, addition of SMase to LPS- or cytokine-stimulated astrocytes or C6 glial cells augments LPS- or
cytokine-induced production of NO and expression of iNOS. Third,
cell-permeable ceramide analogs (C2 and C6) as
well as OE, an inhibitor of ceramidase, led to an increase in the
production of NO and the expression of iNOS in LPS- or
cytokine-stimulated rat primary astrocytes and in C6 glial
cells.
The signaling events in cytokine-mediated induction of iNOS are not
completely established so far. LPS, TNF-
, IL-
, and IFN-
bind
to their respective receptors and induce iNOS expression via activation
of NF-
B (8, 18, 25, 26). NF-
B, a pleiotropic transcription
factor, is a heterodimer of 50- (p50, NF-
B 1) and 65-kDa (p65, rel
A) subunits located in the cytoplasm in an inactive form, bound to the
inhibitory protein I
B through the p65 molecule. Upon stimulation of
cells, I
B is phosphorylated, proteolytically degraded, and
dissociated from the complex (27). The dissociated NF-
B translocates
into the nucleus and transactivates
B-dependent promoters (28). Identification of the binding site of NF-
B in the
promoter region of the iNOS gene and the activation of NF-
B during
cytokine-induced iNOS expression establishes the role of NF-
B
activation in the induction of iNOS (8). In rat primary astrocytes,
although SMase or ceramide by itself was unable to induce NF-
B,
SMase, or ceramide markedly stimulated cytokine-mediated activation of
NF-
B. Increase in the activation of NF-
B in cytokine-stimulated astrocytes by ceramide paralleled the increase in induction of iNOS.
Moreover, inhibition of LPS- and ceramide-induced expression of iNOS by
antioxidant inhibitors of NF-
B activation (e.g.
N-acetylcysteine and pyrrolidine dithiocarbamate) in astrocytes
suggests a role for cellular redox in the ceramide-LPS or
proinflammatory cytokine induced activation of NF-
B and induction of
iNOS. These observations also indicate that stimulation of iNOS
expression in cytokine-activated rat primary astrocytes by
cell-permeable ceramide analogs or neutral SMase is probably mediated
via enhanced activation of NF-
B.
The potential immediate targets for ceramide signaling events
identified so far include ceramide-activated protein kinase (5),
protein kinase C-
(6), and ceramide-activated protein phosphatase
(1, 2). However, the role of ceramide in the activation of NF-
B is
not well understood thus far. Ceramide-mediated activation of NF-
B
was observed in permeabilized Jurkat T cells (29), however, no such
activation was observed in nonpermeabilized cells (30), suggesting that
permeabilization of Jurkat T cells may activate another factor
necessary for the activation of NF-
B in combination with ceramide. A
recent study reports that the IL-1
-stimulated formation of ceramide
and diacylglycerol in the insulin producing RINm5F cell line may
involve activation of c-Jun NH2-terminal kinase and
transcription factor ATF2 instead of activation of NF-
B and
production of NO (15). Studies described in this paper clearly show
that ceramide by itself has no effect on the activation of NF-
B and
induction of iNOS, however, it up-regulates the LPS or cytokine-induced
activation of NF-
B and induction of iNOS and that this up-regulation
is blocked by inhibitors of Ras farnesyl protein transferase and MEK
indicating that the Ras-MAP kinase pathway is involved in LPS- and
ceramide-mediated activation of NF-
B and induction of iNOS in
astrocytes. However, the precise mechanism of potentiation of a
cytokine-induced MAP kinase cascade by ceramide is not understood at
this time. The studies reported here suggest that
sphingomyelin-ceramide signaling events may converge into
cytokine-induced MAP kinase pathway leading to higher activation of
NF-
B and higher induction of iNOS.
Since the discovery of the sphingomyelin cycle, several inducers
(1
,25-dihydroxyvitamin D3, radiation, antibody
cross-linking, TNF-
, IFN-
, IL-1, nerve growth factor, and
brefeldin A) have been shown to be coupled to sphingomyelin-ceramide
signaling events (1, 2, 31, 32). In the case of TNF-
, the pathway is initiated by the action of TNF-
on its 55-kDa receptor leading to
phospholipase A2 activation, generation of arachidonic
acid, and subsequent activation of sphingomyelinase (33). Several studies support a critical role for sphingomyelin hydrolysis as a
stress-activated signaling mechanism with an important role for
ceramide in growth suppression and apoptosis in various cell types
including glial and neuronal cells (34, 35). Since NO can suppress
growth and is an important candidate to induce apoptosis (36), the
observed stimulation of NO production by ceramides in
cytokine-activated astrocytes may be an important factor in NO-mediated
cytotoxicity of neurons and oligodendrocytes in neurodegenerative diseases with neuroinflammatory conditions. The potentiation of LPS or
cytokine-mediated induction of iNOS and production of NO by ceramide as
compared with the inhibition of LPS- or cytokine-mediated induction of
iNOS and production of NO by compounds that activate protein kinase A
(18) and the compounds that inhibit the activation of Ras (37) suggest
a positive modulatory role for ceramide in the induction of iNOS in
astrocytes.
In summary, we have demonstrated that the sphingomyelin-ceramide
signaling events have a positive modulatory role on the expression of
iNOS in rat astrocytes which are probably mediated via activation of
NF-
B. The identification of a stimulatory effect of ceramides on
LPS- and cytokine-induced expression of iNOS defines a novel role for
the sphingomyelin signal transduction pathway in the regulation of
glial cell proliferation, programmed cell death, and cytotoxicity in
neurological disorders.
We thank Dr. Avtar K. Singh for review of the
manuscript and helpful suggestions.