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*
Department of Pharmacology, National Taiwan University College of Medicine, Taipei, Taiwan; and
Cancer Center, Veterans General Hospital, Taipei, Taiwan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B and AP-1 activation. The
immunocomplex kinase assay indicated that IB kinase activity
stimulated by LPS was inhibited by ceramide, which concomitantly
reduced the IB
degradation caused by LPS within 1–6 h. In
concert with the decreased cytosolic p65 protein level, LPS treatment
resulted in rapid nuclear accumulation of NF-B subunit p65 and its
association with the cAMP-responsive element binding protein. Ceramide
coaddition inhibited all the LPS responses. In addition, LPS-induced
PKC and p38 mitogen-activated protein kinase activation were overcome
by ceramide. In conclusion, we suggest that ceramide inhibition of
LPS-mediated induction of inducible NO synthase and cyclooxygenase-2 is
due to reduction of the activation of NF-B and AP-1, which might
result from ceramide’s inhibition of LPS-stimulated IB kinase, p38
mitogen-activated protein kinase, and protein kinase
C. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1, and IFN-
,
as well as apoptosis-inducing stimuli, such as UV light, anti-CD95,
anti-CD28, ionizing radiation, glucocorticoid, anti-cancer
drugs, and serum deprivation (3, 4, 5, 6). Accumulating evidence
has linked ceramide to cell growth, differentiation, apoptosis,
inflammation, immune responses, and many cellular signals in regulating
gene transcription (4). Viewing the crucial roles of
ceramide in cell responses, membrane-permeable ceramide analogs have
often been used to investigate the function of cellular ceramide.
Endotoxin LPS potently stimulates macrophages to up-regulate genes
whose products can enhance the ability of macrophages to invade tissue,
destroy bacteria, attract other immune system cells, and coordinate
their responses. Macrophages exposed to LPS undergo coinduction of
inducible NO synthase
(iNOS)3 and
cyclo-oxygenase-2 (COX-2) gene expression, leading to the formation of
two multifunctional inflammatory mediators, NO and
PGE2 (7, 8, 9). Evidence has indicated
that secondary to the stimulation by LPS transcription factors, NF-B
and AP-1 are critical and act in a coordinated manner for the induced
expression of iNOS and COX-2 (8, 10, 11, 12). One of the
earliest signaling events following LPS treatment in macrophages is
tyrosine phosphorylation and activation of some protein kinases, such
as protein kinase C (PKC), extracellular signal-regulated kinase (ERK),
p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal
kinase (JNK) (12, 13). Recently much progress has been
made with respect to the protein kinases required in the upstream
signaling for inflammatory gene expression. Activation of different
kinases ultimately results in either direct or indirect phosphorylation
and activation of various transcription factors. In this respect it has
been documented that PKC, ERK, p38 MAPK, and JNK are upstream signaling
kinases for the induction of AP-1 transcription (14, 15, 16, 17),
while their roles in NF-B transcription are still uncertain.
The most common transcriptionally competent form of NF-B is a
heterodimer, primarily composed of a 50-kDa DNA-binding subunit (p50)
and a 65-kDa trans-activator (p65 or Rel-A), that is
sequestered within the cytosol by association with the NF-B
inhibitors known as IBs (18, 19, 20). Both the p50 and p65
monomers contain Rel regions, 
300 aa in length, that bind to DNA,
interact with one another, and bind the IB inhibitors
(20). In nonstimulated cells, NF-B proteins are
retained in the cytoplasm, because IBs mask their nuclear
localization sequence. Upon exposure to proinflammatory stimuli,
phosphorylation targets IB for protein ubiquitination and subsequent
degradation through a proteasome-dependent pathway, and results in
dissociation of NF-B into the nucleus (21, 22). This
process leads to increased levels of NF-B at specific DNA enhancer
sequences (B binding sites) in the nucleus, resulting in the
activation of target gene transcription. A multisubunit protein kinase
complex, IB kinase (IKK), has recently been shown to phosphorylate
IB and IB
at the sites that mediate their ubiquitination
and degradation (21, 23, 24). Moreover, cointeraction of
p65 with the coactivator protein cAMP-responsive element binding
protein (CBP)/p300 can enhance its trans-activation
potential (25, 26, 27). Although upstream protein kinases
regulating this event have yet to be conclusively identified, recent
evidence observed in vivo suggests that p38 MAPK and ERK may play roles
in the enhanced trans-activation of p65 in response to
stimulation by IL-1 and TNF- (28, 29).
In macrophage cell lines, ceramide analogs have also been shown to partially mimic LPS-induced cytokine production (30, 31). Notably, because macrophages induce ceramide formation in response to a variety of environmental stimuli (13, 32), the effects of ceramide on LPS-induced gene regulation and its upstream signaling cascades have raised much interest.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phenol-extracted LPS (L8274) from E. coli was
obtained from Sigma (St. Louis, MO), and the protein content measured
by Bradford protein assay was 0.07% (w/w). Oligonucleotides
were synthesized on a PS 250 CRUACHEM DNA synthesizer (Glasgow, U.K.),
using the cyanoethyl phosphoroamidate method, and purified using gel
filtration. The sequences of the double-stranded oligonucleotides used
to detect the DNA-binding activities of NF-B and AP-1 are as follows
(the binding site is underlined): NF-B,
5'-GATCAGTTGAGGGGACTTTCCCAGGC-3'; and AP-1,
5'-GATCCGCTTGATGACTCAGCCGGAA-3'. DMEM, FBS, penicillin,
and streptomycin were obtained from Life Technologies (Grand Island,
NY). Rabbit polyclonal Abs against active (phosphorylated) ERK1/2,
JNK1/2, and p38 MAPK were purchased from New England Biolabs (Beverly,
MA). HRP-coupled anti-mouse and anti-rabbit Abs and the ECL
detection agent were purchased from Amersham Pharmacia Biotech
(Arlington Heights, IL). [-32P]ATP (5000
Ci/mmol) were obtained from NEN (Boston, MA). Rabbit polyclonal Abs
specific for p65 NF-B, IB, COX2, CBP, IKK, IKK, PKC,
and protein A/G agarose beads were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). The PGE2 assay
kit was obtained from Cayman Chemicals (Ann Arbor, MI). Plasmids
pGEX-IB5–55 and pGEX-c-Jun were provided
by Frank S. Lee (Pennsylvania Medical Center, Hershey, PA) and Min-L.
Kuo (National Taiwan University, Taipei, Taiwan), respectively. Rabbit
polyclonal iNOS and mouse mAbs specific for PKC
, -
, -
, and
-
were purchased from Transduction Laboratories (Lexington, KY).
Goat anti-rabbit IgG-fluorescein was obtained from Leinco
Technologies (St. Louis, MO). All materials for SDS-PAGE were obtained
from Bio-Rad (Hercules, CA). All other chemicals were obtained from
Sigma.
Cell culture
The RAW 264.7 macrophages, obtained from American Type Culture Collection (Manassas, VA), were cultured at 37°C in 5% CO2 using DMEM containing 10% FBS and antibiotics (100 U/ml of penicillin, and 100 µg/ml streptomycin). The medium was changed every 2 days. The peritoneal macrophages were prepared from BALB/c mice that have been i.p. injected with 1.5 ml of 3% thioglycolate 3 days before macrophage isolation. Mice were ether-anesthetized, and the peritoneal cavities were lavaged with ice-cold 0.9% NaCl to remove the elicited peritoneal macrophages and were cultured at 37°C in RPMI 1640, supplemented with 10% FBS, 100 U/ml of penicillin, and 100 µg/ml streptomycin. After 1 day of culture, peritoneal macrophages were treated with 3 µg/ml of LPS and C2-ceramide. Cells were seeded into 24-well plates for the nitrite and PGE2 assays, into 35-mm dishes for immunoblots, into 60-mm dishes for immunoprecipitation (IP) and kinase assays, and into 10-cm dishes for EMSAs.
Nitrite and PGE2 measurements
Nitrite and PGE2 production were measured in the RAW 264.7 macrophage supernatants. Briefly, the cells were cultured in 24-well plates in 500 µl of culture medium until confluence. The cells were treated with LPS and/or ceramide for 24 h, then the culture media were collected. Nitrite was measured by adding 100 µl of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamide in 5% phosphoric acid) to 100-µl samples of culture medium. The OD550 was measured using a microplate reader, and the nitrite concentration was calculated by comparison with the OD550 produced using standard solutions of sodium nitrite in the culture medium. PGE2 was measured with an ELISA kit following the manufacturer’s instructions.
Immunoblot analysis
To quantify various proteins, following 24 h (for iNOS and
COX-2) or various time (for JNK, ERK, p38 MAPK, and IB) of
incubation in the presence of various stimuli, cells were washed twice
in ice-cold PBS. Cells were then lysed in lysis buffer containing 20 mM
Tris (pH 7.5), 1 mM MgCl2, 125 mM NaCl, 1%
Triton X-100, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 25
mM -glycerophosphate, 50 mM NaF, and 100 µM sodium orthovanadate
and centrifuged. To assess PKC isoforms in cytosol and membrane
fractions, cells were lysed with buffer A containing 20 mM Tris-HCl,
0.5 mM EGTA, 2 mM EDTA, 2 mM DDT, 0.5 mM PMSF, and 10 µg/ml
leupeptin, pH 7.5, then sonicated and centrifuged. The supernatants and
pellets, respectively, represent the cytosolic and membrane fractions.
To assess the cellular localization of NF-B p65, cytosol and nuclear
extracts were prepared as described below. Samples of equal amounts of
protein (50–100 µg) were subjected to SDS-PAGE on 8–12%
polyacrylamide gels, then transferred onto a nitrocellulose membrane,
which was then incubated in buffer (150 mM NaCl, 20 mM Tris, and 0.02%
Tween, pH 7.4) containing 1% nonfat milk, and the protein band was
visualized by immunoblotting with specific Abs. Immunoreactivity was
detected by ECL following the manufacturer’s instructions.
Preparation of nuclear extracts and EMSAs
Nuclear extracts and EMSA were prepared according to the method described by Chen et al. (8). Briefly, nuclear extracts from stimulated or nonstimulated macrophages were prepared by cell lysis followed by nuclear lysis. Cells were then suspended in 30 µl of buffer (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.2 mM PMSF), vigorously vortexed for 15 s, left standing at 4°C for 10 min, and centrifuged at 2,000 rpm for 2 min. The pelleted nuclei were resuspended in buffer (20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM PMSF) for 20 min on ice, then the lysates were centrifuged at 15,000 rpm for 2 min. The supernatants containing the solubilized nuclear proteins were stored at -70°C until used for the EMSA. In EMSA, binding reaction mixtures (15 µl) contained 0.25 µg of poly(dI-dC) (Amersham Pharmacia Biotech) and 20,000 rpm of 32P-labeled DNA probe in binding buffer consisting of 10 mM Tris (pH 7.5), 1 mM EDTA, 4% Ficoll, 1 mM DTT, and 75 mM KCl. The binding reaction was started by the addition of cell extracts (10 µg) and was continued for 30 min at room temperature. The DNA-protein complex was resolved from free oligonucleotide by electrophoresis in a 5% polyacrylamide gel. The gels were dried and exposed to x-ray films.
IP and kinase assay
RAW 264.7 cells were cultured on 60-mm dishes. After various
stimulation time, cells were washed twice in ice-cold PBS, lysed in 1
ml of lysis buffer containing 20 mM Tris (pH 7.5), 1 mM
MgCl2, 125 mM NaCl, 1% Triton X-100, 1 mM PMSF,
10 µg/ml leupeptin, 10 µg/ml aprotinin, 25 mM -glycerophosphate,
50 mM NaF, and 100 µM sodium orthovanadate. After centrifugation, the
supernatant was collected, then anti-IKK or anti-JNK Ab (2
µg) was added with protein A/G-agarose beads (20 µl; Santa Cruz
Biotechnology) at 4°C overnight. The precipitates were washed three
times with lysis buffer and twice with kinase buffer (25 mM HEPES (pH
7.5), 20 mM MgCl2, 100 µM sodium orthovanadate,
and 2 mM DTT). The kinase reactions for the JNK and IKK complexes were
performed by incubating immunoprecipitated proteins in kinase mixture
(25 mM HEPES (pH 7.5), 20 mM MgCl2, 100 µM
sodium orthovanadate, 2 mM DTT, 10 µM ATP, and 5 µCi of
[-32P]ATP) respectively containing 1 µg of
GST-c-Jun and GST-IB as substrates at room temperature for 30
min. Laemmli’s loading buffer was added to stop the reaction, and
samples were resolved on SDS-PAGE followed by autoradiography.
IP and Western analysis
To determine the association between CBP and p65, total cell lysates prepared from lysis buffer as previous indicated were incubated with CBP Ab and 20 µl of protein A/G beads for 16 h at 4°C. Complex of proteins, p65, and CBP with beads were washed three times with lysis buffer before addition of SDS loading buffer. Samples were resolved on SDS-PAGE. Immunoreactivities of p65 and CBP were detected by ECL following the manufacturer’s instructions.
Immunocytochemistry
Murine RAW 264.7 cells were cultured in 12-mm coverslips. After
stimulation, cells were washed with cold PBS twice and added 10%
formaldehyde in PBS to fix the cells. Five minutes later, 1% Triton
X-100 was added to the cells followed by 5% nonfat milk for 15 min. To
detect the translocation of NF-B p65, the primary p65 rabbit Ab
diluted 1/100 in PBS was added to the cells and incubated for 1 h.
Then cells were washed with PBS twice, and fluorescein-conjugated goat
IgG fraction was added to rabbit IgG Ab diluted 1/100 in PBS. After 30
min, the cell fluorescein was detected using a Photomicrography
Digitalized Integrate System (MGDs, Taipei, Taiwan).
Statistical evaluation
Values are expressed as the mean ± SEM of at least three experiments. Student’s t test was used to assess the statistical significance of the differences; p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As in our previous reports (9, 12), stimulation of
murine macrophages with 0.1 µg/ml LPS for 24 h resulted in the
production of NO (from 5 ± 2 to 32 ± 4 µM) and
PGE2 (from 0.4 ± 0.1 to 6.7 ± 0.3
ng/ml). C2-ceramide at concentrations up to 50
µM by itself did not affect the release of NO or
PGE2 above levels seen in medium-treated
macrophages. Coaddition of C2-ceramide with LPS
inhibited the formation of NO and PGE2
accompanied by the induction of iNOS and COX-2 (Fig. 1
, A and B).
Dose-dependent NO production by LPS (0.03–0.1 µg/ml) was
significantly reduced by the presence of 10 and 50 µM
C2-ceramide. LPS-induced NO production was
30 ± 5% (n = 4) and 69 ± 10%
(n = 6) inhibited, and PGE2
production was 28 ± 7% (n = 5) and 54 ±
7% (n = 5) inhibited by 10 and 50 µM
C2-ceramide, respectively (Fig. 1A).
To confirm the specificity of C2-ceramide, we
found that the structural analog of C2-ceramide
that did not elicit functional activity,
C2-dihydroceramdie, did not affect the LPS
response at 50 µM (Fig. 1, A and C). To clarify
that the inhibitory action of C2-ceramide was not
due to cell toxicity, we examined cell viability. We found that
C2-ceramide at 50 µM had no significant effect
on cell viability with a 24-h incubation as evidenced from MTT,
crystal violet, and lactate dehydrogenase release assays (data not
shown). Even using the more sensitive annexin V and propidium
iodide staining to assess cell viability, no cell toxicity of
50 µM C2-ceramide was found in RAW
264.7 macrophages after a 24-h incubation (data not shown).
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C), which acts
as a macrophage priming signal through increasing the intracellular
calcium and by itself does not change the basal NO level. With regard
to C2-ceramide action, we intriguingly found that
in the absence of thapsigargin priming,
C2-ceramide (50 µM) had no significant effect
on LPS-induced NO production, whereas 56 ± 7% (n
= 3) inhibition was observed in priming cells. Furthermore,
C2-ceramide-specific inhibition in priming cells
was not mimicked by the same concentration of
C2-dihyroceramide. Following 50 µM
C2-ceramide and 3 µg/ml LPS incubation for
24 h, the cell viability did not change, as assayed by MTT and
lactate dehydrogenase release (data not shown). To explore the
mechanism responsible for the inhibitory action of
C2-ceramide, the following experiments were
performed in RAW 264.7 macrophages.
C2-ceramide inhibited LPS-induced NF-B and AP-1
activation
To determine whether decreases in iNOS and COX-2 levels were due
to inhibition of the essential nuclear translocation of the
transcription factors NF-B and AP-1 (8, 35) and, in
turn, their DNA binding, the nuclear extents of both transcription
factors were analyzed using EMSA. As shown in Fig. 2, cells treated with 0.1 µg/ml LPS for
1–3 h showed marked NF-B and AP-1 activation. Although
C2-ceramide (50 µM) alone had no effect on
either event, its coaddition resulted in a reduction of LPS responses
in a time-dependent manner (Fig. 2A). To elucidate whether
this inhibition resulted from the direct inhibition of DNA binding
rather than the alteration of nuclear translocation of both
transcription factors, C2-ceramide was directly
added to a nuclear extract prepared from LPS-stimulated cells. As shown
in Fig. 2B, ceramide at concentrations up to 50 µM had no
effect on NF-B and AP-1 binding to their DNA consensus
sequences.
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B
degradation
To understand whether the signal transduction leading to NF-B
activation occurred at the ceramide action site, we tested the effect
of ceramide on LPS-stimulated IKK activation. IP kinase assays using
GST-IB as a substrate indicated that LPS rapidly and in a
sustained manner stimulated IKK, with onset as early as 10 min and with
a duration of at least 5 h. Ceramide itself only slightly and
transiently stimulated IKK within 90 min. However, in the presence of
ceramide (50 µM), LPS-mediated IKK activity was transiently inhibited
at 10 min, but immediately recovered to the LPS control level until
2 h. Subsequent IKK activity after LPS treatment was significantly
reduced within 2–5 h by the presence of
C2-ceramide (Fig. 3A).
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B degradation
subsequent to IKK-dependent phosphorylation exhibited a rapid onset
(within 10 min) and a slower recovery after LPS treatment (Fig. 3B). C2-ceramide (50 µM) by itself
induced no detectable IB degradation within a 6-h incubation
(data not shown). Corresponding to the effect on IKK activity,
C2-ceramide inhibited the LPS response of
IB degradation, and the first signal of
C2-ceramide-induced inhibition was observed no
earlier than after 1 h (Fig. 3B). These results
indicate that C2-ceramide possesses inhibitory
action on LPS-induced IKK activation and IB degradation. Ceramide inhibited LPS-induced p65 nuclear translocation
Following inhibitor IB degradation, free NF-B can easily
gain access into the nucleus and elicit its trans-activation
ability. Next we asked whether C2-ceramide
affected nuclear translocation of NF-B subunit p65. Immunoblot
analysis revealed that C2-ceramide abrogated
LPS-stimulated p65 nuclear accumulation within 1–6 h, but could not
affect this response before 1 h. In parallel to the rapid movement
into the nuclear compartment in response to LPS, the initial decrease
in cytosolic p65 levels was already detectable after a 10-min
incubation. Consistently, C2-ceramide could
recover the sustained decrease in cytosolic p65 after LPS incubation
for 1–6 h (Fig. 4A).
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B, reveal that in the resting state the p65
signal was primarily limited to the cytosol (a), and that
ceramide itself did not affect the subcellular distribution of p65
(b–d). Upon LPS stimulation for as long as 6 h, p65
was localized in the nuclei as well as in the cytosol
(e–g). In the copresence of
C2-ceramide (50 µM), cells exhibited lower p65
nuclear immunointensity (h–j) compared with the
LPS-stimulated control (e–g). Ceramide reduced the interaction of CBP with p65
NF-B transcriptional competence requires interaction with the
transcription cofactor CBP. To assess the physical interactions of p65
with CBP, cells were subjected to coimmunoprecipitation analysis with
anti-CBP Ab, followed by Western blot analysis with anti-p65
Ab. Our results indicate that after LPS stimulation, p65 was able to
trap CBP from the total cell lysate, suggesting a strong recruitment of
cofactor CBP to the NF-B transcription factor. Upon
C2-ceramide (50 µM) addition, LPS-stimulated
p65/CBP interactions at 1, 2, 3, and 6 h were attenuated (Fig. 5).
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Ceramide has been shown to elicit JNK activation in several cell
types (13, 35, 36). Based on some reports indicating the
involvement of JNK activity in TNF- (37), iNOS
(38), and COX-2 (39) induction, we
investigated this possible mechanism. We found that, in contrast to
stimulation by anisomycin (0.03 or 0.1 µM), vinblastine (1 µg/ml),
UV irradiation (180 J/m2), or taxol (0.3 µM),
ceramide at 50 µM could not stimulate JNK within 30 min as assessed
by the IP kinase assay (Fig. 6A) as well as by
immunoblotting with phosphorylated JNK Ab (Fig. 6B). In
contrast, LPS at 0.1 µg/ml only slightly induced transient JNK
phosphorylation at 15 min, and this action was unaffected by
C2-ceramide (data not shown). These results rule
out the involvement of JNK in LPS-induced or ceramide-regulated iNOS
and COX-2 expression in RAW 264.7 cells.
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, LPS and
C2-ceramide stimulated p42/p44 ERK and p38 MAPK
phosphorylation. LPS and C2-ceramide acted in an
additive manner in ERK phosphorylation. On the contrary, their actions
on p38 MAPK phosphorylation were reciprocally inhibited.
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Because PKC is known as a key kinase for transducing iNOS and
COX-2 gene expressions (8, 42), we explored the possible
action of C2-ceramide in this respect. As shown
in Fig. 8, PKC, -, -, and -,
which initially existed more abundantly in the cytosol than in the
membrane fraction, showed marked translocation from cytosol to the
membrane fraction within 15–60 min of treatment with LPS.
C2-ceramide (50 µM) alone also caused the
membrane translocation of PKC, -, and -, with more obvious
stimulation seen for PKC and - than for PKC. Although the
membrane immunoreactivity of PKC was barely detected before or after
stimulation, decreased levels in the cytosol fraction were observed
after LPS treatment. On the contrary, C2-ceramide
caused increases in cytosol levels of PKC and -, both of which
increased at 15 min, and PKC decreased to near the control levels
after 30 min, while PKC maintained its cytosolic localization for at
least 60 min. Interestingly, we found that LPS-stimulated subcellular
distribution of all PKC isoforms except PKC was diminished by the
coaddition of C2-ceramide.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Fas ligand) or various stress insults
(e.g., irradiation, chemotherapy, and hypoxia/reperfusion), ceramide
might influence LPS-elicited signaling and relevant cellular responses
when a bacteria-infected host is subjected to inflammation,
chemotherapy, or other pathological conditions. To address this issue,
we herein examined the effects of the cell-permeable ceramide analog,
C2-ceramide, on LPS-initiated cell signaling and
responses in the murine RAW 264.7 macrophage cell line. In this study
we not only observed the inhibitory effects of
C2-ceramide on LPS-initiated NO and
PGE2 production, but also investigated its action
on the crucial signaling molecules that are required for iNOS and COX-2
expression. Unlike C2-ceramide, the inactive
analog C2-dihydroceramide did not alter LPS
responses at the same concentration. This suggests the specific action
of C2-ceramide as a lipid regulator.
NF-B is a prerequisite and ubiquitous transcription factor for the
expression of many inflammation-related genes, including iNOS, COX-2,
TNF-, IL-6, and GM-CSF. Although ceramide has been reported to
activate NF-B in some cell types (45, 46), this point
is still controversial (13, 47, 48). In this study we
found that C2-ceramide could not significantly
activate NF-B in RAW 264.7 cells. On the contrary, inhibition of
LPS-induced NF-B activation by ceramide was observed when ceramide
was present in RAW 264.7 cell cultures simultaneously with
LPS.
It is well established that the nuclear accumulation of NF-B relies
in large part upon IKK-dependent phosphorylation and subsequent
degradation of the cytosolic inhibitor, IB. In this context, LPS
stimulation in RAW 264.7 cells led to rapid IKK activation, IB
degradation, and p65 nuclear translocation. All these sequential
upstream signals responsible for NF-B activation lasted for up to
6 h after the addition of LPS. Moreover, our results show that
these sustained events maintained within 1–6 h of LPS incubation were
correspondingly reduced by the presence of
C2-ceramide. Following these inhibitory actions
of C2-ceramide, EMSA results consistently
showed the ability of C2-ceramide to inhibit
LPS-induced nuclear translocation and DNA binding of NF-B. This
delayed inhibitory action of C2-ceramide explains
the paradoxical results of MacKichn and DeFranco, who reported that
C2-ceramide failed to affect IB degradation
induced by LPS at 20 min (13). For
C2-ceramide action alone, weak IKK stimulation,
but not IB degradation or p65 translocation, was observed. We
suggest that the much less pronounced and transient IKK activation
compared with LPS can explain the noneffectiveness of
C2-ceramide on IB degradation and NF-B
activation. A further suggestion drawn from these results is that
C2-ceramide-targeted molecules, either through
direct or indirect action, are located in the upstream signaling
pathways involved in the sequential activation of IL-1R-associated
kinase, TNF receptor-associated factor-6, NF-B-inducing kinase, and
IKK after LPS binding to CD14/toll-like receptors (49, 50). Another intriguing finding is that
C2-ceramide treatment can transiently reduce IKK
activity at 10 min of LPS stimulation. Although the underlying
mechanism contributing to this rapid effect is presently unknown, the
instantly recovered IKK activation might be the reason why
C2-ceramide in RAW 264.7 cells did not
appreciably alter IB degradation or p65 nuclear translocation
respectively induced by LPS at 20 and 30 min.
In addition to NF-B-inducing kinase/IKK signaling transduction, LPS
has been reported to stimulate many serine/threonine protein kinases,
such as PKC, ERK, p38 MAPK, and JNK in macrophages. The physiological
relevance of these kinase signals to macrophage functions has been
proven by their crucial roles in iNOS and COX-2 induction (8, 38, 39, 40, 51). Recent studies further suggested that ERK
(28), p38 MAPK (28, 52), and PKC (53, 54) were responsible for the enhanced
trans-activation of NF-B via an IB
degradation-independent mechanism. In this study we found that the
stimulating effects of LPS on p38 MAPK and PKC, but not on ERK and JNK,
were also attenuated by C2-ceramide. Thus, we
suggest that C2-ceramdie inhibition of the LPS
response is primarily due to the interruption of LPS signals on IKK,
PKC, and p38 MAPK pathways, which play crucial roles in NF-B- and
AP-1-dependent gene expression. Our results do not address the
inhibitory mechanism of C2-ceramide on
LPS-activated PKC, p38 MAPK, and IKK. Whether any possible intermediate
signal transducers are involved in coordinating the signaling network
between ceramide and LPS needs further determination.
Relatively little is known about the way in which ceramide acts upon the signaling pathways. Although protein kinases and protein phosphatase are two principal targets in ceramide signaling pathway (55), the effects of ceramide on MAPKs and the mechanism involved are still unclear. The more well-established pathway is that ceramide-activated protein kinase can activate the Raf-MEK-ERK MAPK cascade (56). Consistent with this scenario, we detected increased ERK phosphorylation in C2-ceramide-treated RAW 264.7 cells. Similar findings were reported by Medvedev et al. (35) and Monick et al. (32) in mouse peritoneal and human alveolar macrophages, respectively, but not by MacKichan and DeFranco in the same macrophage cell line that we used (13). We currently can provide no evidence to explain this latter inconsistency.
With respect to the actions of ceramide on the other two MAPKs, our
results were supported by those of other studies with macrophages. The
ceramide-induced p38 MAPK activation shown in this study is consistent
with that observed in peritoneal macrophages (35). The
crucial kinase involved in the ceramide-induced apoptosis, JNK
(36), was not triggered by 50 µM
C2-ceramide during the period examined (i.e., 30
min). Instead, with increasing C2-ceramide
concentration to 100 µM, weak JNK activation was detected (data not
shown), and these results coincide with previous findings by MacKichan
and DeFranco in RAW 264.7 cells (13). In addition, this
JNK effect of ceramide might contribute to the weak cell apoptosis
(30% reduction in MTT assay) caused by 100 µM ceramide treatment
for 24 h in RAW 264.7 macrophages.
Besides MAPK, ceramide has been demonstrated to inactivate certain
members of the PKC family (57, 58), while activating
others (5, 54, 59). Consistent with a previous report
studying human leukemia cell lines (5),
C2-ceramide increased the cytosolic level of
PKC transiently and that of PKC in a more sustained manner in RAW
264.7 cells. Also supporting previous data indicating ceramide to be an
activator of PKC (54, 59), we observed the membrane
translocation of PKC after C2-ceramide
addition. Interestingly, four of the five PKC isoforms activated by LPS
in RAW 264.7 cells (, , , and , but not ) were overcome
by ceramide treatment. Certainly, the details and any intermediate
steps that contribute to the cross-talk between ceramide and LPS
signaling remain to be elucidated.
CBP was originally discovered based on its ability to interact with the
cAMP-responsive element binding protein and has been demonstrated to
interact with many proteins in a cell signal-regulated manner. It has
been shown that phosphorylation of p65 promotes the interaction with
CBP and results in the enhanced trans-activation potential of NF-B
based on at least three aspects (25, 26, 60). First, CBP
acts as a bridging factor between NF-B and DNA binding sites.
Second, CBP with intrinsic enzyme activity can acetylate histone, which
allows the unwinding or loosening of chromatin. Third, CBP can
acetylate p65 itself. Thus, it is apparent that CBP acetyltransferase
activity is required for the stimulation of NF-B. To date, upstream
kinases, at least protein kinase A, p38 MAPK, and ERK, have been
identified as regulating this event (28, 29, 61). In this
study we observed the inhibitory effect of
C2-ceramide on LPS-induced p65 interaction with
CBP and thus suggest that the attenuation of p38 MAPK activity by
ceramide can account for this event.
To understand whether the inhibitory action of
C2-ceramide is common to different types of
macrophages, we performed an NO assay in peritoneal macrophages
isolated from BALB/c mice. Intriguingly we found that
C2-ceramide failed to alter NO production caused
by LPS itself, while abolishing the priming actions of thapsigargin,
which transcriptionally up-regulates iNOS gene expression and
efficiently enhances NO production in BALB/c macrophages with low
sensitivity to LPS (33, 34, 62). These results suggest
that C2-ceramide inhibition of LPS-dependent
signaling cascades leading to iNOS gene induction is dependent on the
types of macrophages. Actually it is known that the iNOS gene is
regulated by several coordinated transcription factors (63, 64) and is distinctly induced in different cell types. For
example, priming factors, such as IFN-, IL-1, TNF-, and
Ca2+, are required for the maximal induction of
iNOS expression in peritoneal macrophages (33, 34, 65) and
C6-glioma cells (66). Viewing this
regulatory complexity, it is our future work to further investigate the
action of ceramide on LPS together with cytokine-induced NO formation
in other cell types, where the transmission of early signals into the
nucleus leading to iNOS expression might be varied. Moreover, the
noneffectiveness of ceramide on the LPS response in this study might
support the observation by Medvedev et al. that
C2-ceramide was unable to change LPS-induced
NF-B activation in peritoneal macrophages from C3H/OuJ mice
(35).
In conclusion, this study has identified the mechanisms through which
ceramide alters inducible iNOS and COX-2 gene expression in response to
LPS. Ceramide inhibition of LPS-mediated IKK, p38 MAPK, PKC, NF-B,
and AP-1 activation as well as the inhibition of p65/CBP interaction
may underlie the inhibitory nature of ceramide in macrophages. All
these results indicate that ceramide functions in a negative regulatory
mechanism to attenuate responses to LPS.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. W. W. Lin, Department of Pharmacology, National Taiwan University College of Medicine, Taipei, Taiwan.
3 Abbreviations used in this paper: iNOS, inducible NO synthase; COX-2, cyclo-oxygenase-2; MAPK, mitogen-activated protein kinase; IKK, IB kinase; PKC, protein kinase C; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; CBP, cAMP-responsive element-binding protein; IP, immunoprecipitation.
Received for publication August 7, 2000. Accepted for publication February 20, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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