From the Nutritional Immunology Laboratory, Jean
Mayer USDA Human Nutrition Research Center on Aging, the
¶ Department of Biochemistry, and the
Department of
Pathology, Sackler Graduate School of Biomedical Sciences, Tufts
University, Boston, Massachusetts 02111
Received for publication, July 25, 2002, and in revised form, December 13, 2002
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ABSTRACT |
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We have shown that the age-associated increase in
lipopolysaccharide (LPS)-stimulated macrophages (M It is well documented that T cell-mediated immune
function declines in old animals and elderly humans compared with their young counterparts (1, 2). The age-associated dysregulation in
macrophages (M Cyclooxygenase (COX) is the rate-limiting enzyme that catalyzes the
conversion of arachidonic acid (AA) to PG endoperoxide (PGH2), which is further converted to different PGs and
thromboxane. COX is hence a key factor in PG synthesis. Two isoforms of
COX have been identified: a constitutive form, COX-1 (9, 10), and the
inducible counterpart, COX-2 (11, 12). We have demonstrated that the
age-associated increase in M To determine the mechanism of the age-related and ceramide-induced
increase in COX-2 transcription, we investigated the role of various
nuclear transcription factors that are involved in COX-2 gene
expression. The binding sites for several nuclear
transcription factors, such as nuclear factor Along with NF- Our recent study showed that the intracellular concentration of
ceramide was higher in LPS-stimulated M Animals--
Specific pathogen-free male young (4-6 months) and
old (22-24 months) C57BL/6NIA mice were obtained from National
Institute on Aging colonies at Harlan Teklad (Madison, WI). Mice
exhibiting skin lesions, visible tumors, or splenomegaly were excluded
from the study. Mice were housed individually in microisolator cages at
a constant temperature (23 °C) with a 12-h light-dark cycle and were
fed autoclaved mouse chow Harlan 7012 (Harlan Teklad) and water
ad libitum. All conditions and handling of the animals was
approved by the Animal Care and Use Committee of the Jean Mayer Human
Nutrition Research Center on Aging, Tufts University, and were in
accordance with guidelines provided by the National Institutes of
Health Guide for the Care and Use of Laboratory Animals.
Peritoneal Macrophage Isolation--
Mice were injected per
peritoneum with 3 ml of 2.98% thioglycollate to elicit M Preparation of Cytosolic and Nuclear Extracts--
M Electrophoretic Mobility Shift Assay (EMSA)--
For the NF- Western Blot--
The cytosolic and nuclear samples were
prepared as described under "Preparation of Cytosolic and Nuclear
Extracts" and were used for I PGE2 Production and COX Enzyme
Activity--
Peritoneal M COX-2 mRNA Reverse Transcriptase-PCR--
M NF- Statistical Analysis--
Data were analyzed using SYSTAT
statistical software (SYSTAT 10, 2000; Evanston, IL). Paired Student's
t test was used to determine the effect of incubation time
and inhibitor. The difference between two age groups was assessed using
nonpaired Student's t test. Results are expressed as
mean ± S.E. Significance was set at p < 0.05.
NF- AP-1 and CREB Binding Activity in M Old M An I
To demonstrate that the effect of Bay 11-7082 is specific to NF Inhibition of NF-
To rule out the possibility that Bay 11-7082 may directly inactivate
the COX enzyme rather than inhibit transcriptional activation of COX-2
through reducing NF- Age-related Increase in PGE2 Production as Well as the
Involvement of NF- Inhibition of NF-
Nitric oxide and iNOS have been shown to be up-regulated with age (31,
38). The promoter region of the murine iNOS gene has a NF- Ceramide Increases LPS-induced Activation of NF-
To confirm that the altered NF- NF-
Furthermore, we examined whether this blocked NF- We previously showed that the higher PGE2 production
by M Binding of activated NF- To stimulate M If age-associated up-regulation of COX-2 is mediated through increased
NF- The prototypical and most abundant form of NF- Determining NF- The mechanism for the ceramide-induced increase in NF- In summary, our data demonstrated for the first time, that
NF-) prostaglandin
E2 (PGE2) production is because of
ceramide-induced up-regulation of cyclooxygenase (COX)-2 transcription
that leads to increased COX-2 expression and enzyme activity. To
determine the mechanism of the age-related and
ceramide-dependent increase in COX-2 transcription, we
investigated the role of various transcription factors involved in
COX-2 gene expression. The results showed that LPS-initiated
activations of both consensus and COX-2-specific NF-
B, but not AP-1
and CREB, were significantly higher in M
from old mice than those
from young mice. We further showed that the higher NF-
B activation in old M
was because of greater I
B degradation in the cytoplasm and p65 translocation to the nucleus. An I
B phosphorylation
inhibitor, Bay 11-7082, inhibited NF-
B activation, as well as
PGE2 production, COX activity, COX-2 protein, and mRNA
expression in both young and old M
. Similar results were obtained by
blocking NF-
B binding activity using a NF-
B decoy. Furthermore,
NF-
B inhibition resulted in significantly greater reduction in
PGE2 production and COX activity in old compared with young
M
. Addition of ceramide to the young M
, in the presence or
absence of LPS, increased NF-
B activation in parallel with
PGE2 production. Bay 11-7082 or NF-
B decoy prevented
this ceramide-induced increase in NF-
B binding activity and
PGE2 production. These findings strongly suggest that the
age-associated and ceramide-induced increase in COX-2 transcription is
mediated through higher NF-
B activation, which is, in turn, because
of a greater I
B degradation in old M
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)1
contributes to the impaired T cell function with aging. We, as well as
others, have demonstrated that immune cells, including M
, from old
animals and humans produced more PGE2 than those from their
young counterparts (3-7). We further showed that the increased
PGE2 production by M
contributes to the decline in T
cell-mediated function with aging (8).
PGE2 production is because of higher COX activity in M
from old mice compared with those from
young mice. This increased COX activity is, in turn, a result of
increased expression of COX-2 protein and mRNA (13). In a recent
study, we further demonstrated that the age-related increase in COX-2
mRNA was because of a higher level of ceramide in old M
compared
with those of young, which induced up-regulation of COX-2 transcription
(14). In addition, we showed that the effect of ceramide was not
mediated through components of the mitogen-activated protein
kinase pathway, c-Jun NH2-terminal kinase,
extracellular signal-regulated kinase, or p38 (14).
B (NF-
B),
nuclear factor interleukin-6, and cAMP-responsive element (CRE), have
been identified on the promoter region of the COX-2 gene (15-17). A
number of studies have suggested that NF-
B activation and binding to
its cognate site on the COX-2 promoter region are required to induce
COX-2 expression (16, 18, 19). Dysregulation of NF-
B activation has
been indicated in certain inflammatory diseases (20-22), in which
COX-2-catalyzed prostaglandin production may play an important role. It
is thus feasible that the age-associated increase in COX-2 expression
may be mediated through a corresponding change in the regulation of
NF-
B with aging. However, to date, age-related changes in M
NF-
B activity and the role of NF-
B in age-related up-regulation
of COX-2 have not been demonstrated.
B, another redox-sensitive transcription factor,
activator protein-1 (AP-1), has been shown to be involved in COX-2
transcriptional regulation (23, 24). Although an independent AP-1
binding site has not been recognized on the COX-2 promoter, it was
reported that the binding site for AP-1 in the COX-2 promoter is a CRE
binding site (15, 24, 25). A number of studies suggest that binding of
nuclear proteins, such as CRE-binding protein (CREB) and c-Jun, to CRE,
an element of COX-2 promoter, induces COX-2 transcription (26, 27).
Accordingly, we examined the roles of NF-
B, AP-1, and CREB in the
age-associated up-regulation of M
COX-2 transcription.
of old mice compared with
those of young mice and that this increased level of ceramide mediates
the age-associated up-regulation of COX-2 transcription (14). Whereas
the effect of ceramide on regulation of transcription factors has not
been well defined, previous studies showed that ceramide induced AP-1
(28) and NF
B activation (29). We hypothesize that up-regulated COX-2
transcription with aging is because of altered activation of
transcription factors involved in COX-2 expression by ceramide. We
demonstrate here that of the three transcription factors studied, only
NF-
B contributes to the age-associated up-regulation of COX-2. The
age-associated increase in NF-
B activation is because of enhanced
I
B degradation in the cytoplasm, resulting in increased nuclear
translocation of activated NF-
B. Ceramide induces increased COX-2
activation and the consequent PGE2 production through
up-regulating NF-
B activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Three
days later, the mice were euthanized via CO2 asphyxiation.
Peritoneal exudate cells were obtained by peritoneal lavage with cold
Ca2+- and Mg2+-free Hanks' balanced salt
solution (Sigma). Cells were centrifuged and resuspended in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10 mM HEPES (Sigma), 2 mM glutamine (Invitrogen), 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen), and
2% fetal bovine serum. Peritoneal exudates were enriched for M
by
their adherence to tissue culture-treated plastic dishes or plates for
2 h at 37 °C in 5% CO2. Nonadherent cells were
removed by vigorous washing and the remaining cells were at least 90% macrophages as assessed by the expression of cell surface markers Mac-1
and F4/80. Cells were monitored for their general condition and
viability throughout the study as assessed by morphology, adherence,
and trypan blue exclusion. No cytotoxicity was observed in treated
cells compared with untreated controls. We previously conducted a
number of studies using resident macrophages (8, 13, 30, 31). Because
of a large number of M
that were necessary for various experiments
and the limited number of resident M
obtainable from each mouse, we
used thioglycollate-elicited M
. Prior to use of thioglycollate in
our experiments, we compared the magnitude and pattern of
responsiveness between resident and thioglycollate-elicited M
.
Although there are differences between the two types of cells in their
ability to response to certain stimulation agents, the relative
response pattern, the age-related difference, as well as the response
to in vitro intervention are the same between these two cell
types. Particularly and of relevance to this study,
thioglycollate-elicited M
showed the age-associated difference in
COX-2 expression similar to that observed with resident M
(8,
13, 30).
(2 × 107) in a 10-cm dish were incubated overnight in
serum-free RPMI 1640 medium. The cells were washed and then stimulated by lipopolysaccharide (LPS, Escherichia coli serotype
0111:B4, Sigma) at 5 µg/ml for various lengths of time. This
concentration of LPS was used because our testing experiments indicated
it to be optimal for production of PGE2 and nitric oxide. A
parallel experiment was conducted using IL-1
(R & D Systems,
Minneapolis, MN) at 50 ng/ml as stimulator. For the NF-
B inhibition
study, the cells were preincubated with an inhibitor of I
B-
phosphorylation, Bay 11-7082 (Biomol Research Laboratories,
Plymouth Meeting, PA) for 30 min, or a NF-
B decoy (see
"NF-
B Decoy Approach" below) for 24 h before LPS
stimulation. To increase intracellular ceramide levels, cell-permeable
C2-ceramide (30 µM) (Matreya, Pleasant Gap, PA) was added
to the cell cultures with or without the presence of LPS and the cells
were incubated for different times. The concentration of 30 µM was chosen based on our previous study in which
different doses of ceramide were used and shown to induce an efficient
COX-2 expression and PGE2 production at this level (14). At
the end of the stimulation period, the cells were washed with cold PBS and then collected with a cell scraper. The cells were resuspended in a
hypotonic buffer (10 mM HEPES, pH 7.9, 2 mM
MgCl2, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethysulfonyl fluoride, and 0.5% Nonidet P-40) and incubated on ice for 10 min. After the cell lysates were centrifuged at 15,000 × g
for 1 min, the supernatants were collected as a cytosolic fraction and
stored at
70 °C. The remaining pellets were resuspended in a high
salt buffer (50 mM HEPES, pH 7.9, 300 mM NaCl,
50 mM KCl, 0.1 mM EDTA, 1 mM
dithiothreitol, 0.5 mM phenylmethysulfonyl fluoride, and 10% glycerol) and incubated in rotation at 4 °C for 30 min. The nuclear lysate was centrifuged at 15,000 × g at
4 °C for 30 min. The supernatant was collected as a nuclear fraction
and stored at
70 °C.
B
binding activity assay, both consensus and COX-2 promoter-specific
sequences were used. A double-stranded oligonucleotide containing an NF-
B consensus sequence (5'-AGTTGAGGGGACTTTCCCAGG C-3') was purchased from Promega (Madison, WI). A COX-2-specific NF-
B binding oligonucleotide (distal,
408/
388,
5'-GAGGTGAGGGGATTCCCTTAG-3') and its complementary sequence were
synthesized by the Tufts University Core Facility laboratory and were
annealed before labeling. For AP-1 and CREB binding assays, the
double-stranded consensus oligonucleotides (AP-1,
5'-CGCTTGATGAGTCAGCCGGAA-3' and CREB,
5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3') were purchased from Promega.
All the oligonucleotides were end labeled using
[
-32P]ATP (3000 Ci/mmol, PerkinElmer Life Sciences)
and T4 polynucleotide kinase (Promega). 32P-Labeled probes
were purified using MicroSpinTM G-25 columns (Amersham Biosciences).
For each reaction, nuclear extracts (2 µg of protein) were incubated
with labeled oligonucleotide in the presence of binding buffer
(Promega) at room temperature for 20 min. To ensure specificity of
probe binding, a 50-fold excess of unlabeled (cold) and mutant
oligonucleotides were added to the nuclear samples and incubated for 10 min before the labeled oligonucleotide was added. In supershift assays,
antibodies specific for the p65 or p50 subunit of NF-
B (Santa Cruz
Biotechnology, Santa Cruz, CA) were added to the binding reaction and
incubated for 30 min at room temperature before the labeled
oligonucleotide was added. Protein-DNA complexes were resolved at 350 V
for 1 h in 4% polyacrylamide gels and visualized using Kodak
x-ray film. Bands were quantified by ChemiImager (Alpha Innotech Corp.,
San Leandro, CA).
B-
and p65 detection,
respectively. For COX-2 and inducible nitric-oxide synthase (iNOS)
detection, M
were preincubated with or without Bay 11-7082 for 30 min and then stimulated by LPS (5 µg/ml) for 16 h. Total
cellular lysates were collected and 25 µg of protein from each sample
was electrophoresed in a 10% SDS-polyacrylamide gel and transferred to
nitrocellulose membranes. After blocking with 5% nonfat dry milk in
TBS containing 0.1% Tween 20 overnight, the membranes were incubated
with the respective antibodies (all from Santa Cruz Biotechnology) for
1 h. The membranes were rinsed and then incubated with the
corresponding secondary antibodies conjugated with alkaline phosphatase
(Tropix, Inc., Bedford, MA) for 1 h. After being rinsed, the
membrane was incubated in a Chemiluminescent Detection System (Tropix)
for 4 min and then exposed to film. The equal loading across the
samples was first estimated by staining the membranes with Ponceau S
(Sigma) and further confirmed by reprobing the stripped membranes with
-actin antibody (Sigma). All bands were quantified by ChemiImager (Alpha Innotech). The bands of interest molecules were normalized with
-actin bands and presented as relative density ratio.
(1 × 106 cells/well)
were plated to 24-well plates and isolated by adherence as described
above. The cells were preincubated with or without Bay 11-7082 for 30 min before being stimulated by 5 µg/ml LPS (Sigma), 30 µM ceramide (Matreya), or both for 12 to 24 h. After
the supernatants were removed and stored at
70 °C for analysis of
accumulated production of PGE2, the cells were layered with
1 ml of medium containing 30 µM AA and incubated at
37 °C for 10 min for determination of COX activity as described by
Fu et al. (32). Total cellular COX activity can be measured
by adding excess exogenous AA to M
, because the intracellular enzyme
pool is saturated with the substrate and is functioning at maximal
velocity. After 10 min, 2.1 mM aspirin was added to
inactivate the COX enzyme activity. Supernatants were immediately
removed and stored at
70 °C. Cells were then incubated with 1 M NaOH for 5 min, at which time the supernatant was removed
and stored at
20 °C for protein analysis by the bicinchoninic acid
protein assay kit (Pierce). PGE2 was measured by
radioimmunoassay (RIA) as previously described (4).
were
preincubated with or without Bay 11-7082 for 30 min and then stimulated
by LPS (5 µg/ml) for 4 h. Total RNA was isolated using the
Totally RNA Isolation kit (Ambion, Austin, TX). Two µg of total RNA
was reverse-transcribed to first-strand cDNA using random hexamer,
and amplified by PCR using the Superscript amplification kit
(Invitrogen). The PCR conditions for COX-2 mRNA were 1 cycle for 2 min at 94 °C, followed by 30 cycles of 1 min at 94 °C and 5 min
at 55 °C. Mouse exon 8 sense primer
(5'-ACTCACTCAGTTTGTTGAGTCATTC-3') and exon 10 antisense primer
(5'-GTAATTGGGATGTCATGATTAGTTT-3') were used to generate 583-bp
PCR products. To normalize the COX-2 mRNA reverse transcriptase-PCR
results, 18 S rRNA primers and the competitors at a ratio of 4:6
(Ambion) were used to generate 18 S rRNA PCR products from the same
cDNA samples used in COX-2 mRNA PCR assays. Our PCR conditions
for both murine COX-2 mRNA and 18 S rRNA were tested to be within
the linear range of PCR product formation (14). The PCR products were
resolved by electrophoresis in an ethidium bromide-stained 1.2%
agarose gel and the bands were visualized by ethidium bromide staining
and quantified using ChemiImager (Alpha Innotech).
B Decoy Approach--
The COX-2-specific NF-
B
binding oligonucleotide (distal,
5'-GAGGTGAGGGGATTCCCTTAG-3') and its
complementary sequence
(5'-CTAAGGGAATCCCCTCACCTC-3'), and their
mutated counterparts
(5'-GAGGTGAGGGCCTTCCCTTAG-3' and
5'-CTAAGGGAAGGCCCTCACCTC-3')
were custom synthesized by Qiagen Operon (Alameda, CA). The
underlined letters denote phosphorothioated bases and the bold
letters mark mutations. The complementary oligonucleotides were
annealed to double strands by heating at 90 °C for 5 min and then
cooling down to room temperature within 3 h. To be efficiently
delivered to the cells, the double-stranded oligonucleotides were mixed
with LipofectAMINE reagent (Invitrogen) and incubated at room
temperature for 40 min. The complex was then added to the cell cultures
at 0.5 to 10 nM for oligonucleotides and 5 µg/ml for
LipofectAMINE reagent. The cells were incubated in antibiotics and
serum-free RPMI 1640 medium for 24 h, after which the cells were
washed and then stimulated for varied times depending on the purpose.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B Binding Activity in Murine M
Increases with
Aging--
We previously showed that the age-associated increase in
PGE2 production is because of the ceramide-induced
up-regulation of COX-2 transcription with aging (14). Of the
transcription factors found in the promoter region of the COX-2 gene,
NF-
B is the most intensively studied and several investigations have linked its activity to the activation of the COX-2 gene (16, 18).
Although the age-related up-regulation of NF-
B activation has been
shown in rat gastric mucosa (33), kidney (34), liver, heart (35), and
brain (36), its binding activity in M
, a major source of
PGE2, has not been compared between young and old animals.
In this study, the peritoneal M
from young and old mice were
stimulated with LPS for different times as indicated in Fig.
1. NF-
B binding activity in nuclear
fractions was assessed by EMSA. Fig. 1A shows the results
obtained using an NF-
B consensus oligonucleotide as the binding
motif. Without stimulation, there was no detectable binding activity in
either young or old M
. At every time point following LPS
stimulation, the NF-
B binding activity was higher in old M
compared with that of young M
. To assure the specificity of the
binding, a 50-fold concentrated unlabeled (cold) NF-
B consensus
oligonucleotide was added to compete with the 32P-labeled
NF-
B oligonucleotide. The binding was competed out in both young and
old M
at peak time (2 h). When a mutated NF-
B oligonucleotide was
added, however, the binding was not affected, further indicating the
sequence specificity. To determine the composition of the NF-
B
proteins in the binding complex, we used antibody supershift in EMSA.
As seen in Fig. 1A, the binding complex was shifted by
antibodies to p65 and p50 units. Next, we used a COX-2-specific NF-
B
oligonucleotide in EMSA. Similar to the experiments in which the
consensus oligonucleotide was used, a consistently higher binding
activity was observed in old compared with young M
(Fig.
1B). The specificity was examined using the samples from old
M
at peak stimulation time (2 h) and 50-fold cold oligonucleotide
was shown to completely compete out the band. The supershift assay
yielded similar results to those observed in the assay using the
consensus oligonucleotide.
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Fig. 1.
NF- B binding
activity in murine M
increases with
aging. Peritoneal M
from young and old mice were stimulated
with LPS (5 µg/ml) for 0, 15 min, 30 min, 1, 2, or 4 h prior to
the preparation of nuclear extracts as described under "Experimental
Procedures." Nuclear extracts (2 µg) were incubated with
32P-labeled consensus (A) or COX-2-specific
NF-
B oligonucleotide (B) and subjected to EMSA. Cold
competition was conducted in the presence of 50-fold excess unlabeled
corresponding NF-
B oligonucleotide for 10 min before the
32P-labeled oligonucleotide was added. In supershift
analysis, antibody specific for p65 or p50 were added to the binding
reaction and incubated for 30 min before the 32P-labeled
oligonucleotide was added. The gel figures are representative samples
of six independent experiments. The bar figures are the
mean ± S.E., n = 6. Different from young at *,
p < 0.05, and #, p < 0.1.
Does Not Change with
Aging--
After establishing an age-related difference in NF-
B
binding activity, we examined whether two other transcription factors, AP-1 and CREB, which have been shown to be involved in COX-2
regulation, are affected by the aging process. Culture conditions and
LPS doses were the same as those used in the NF-
B gel shift assay. Both AP-1 and CREB were activated by LPS treatment, but the induction was less potent than that seen in NF-
B so that the autoradiography required 5-8-fold longer exposure times than that with the NF-
B probe. The binding activity for both AP-1 and CREB peaked at around 1 h poststimulation and no significant difference was detected between young and old mice in either AP-1 or CREB activation (data not shown).
Have Higher I
B Degradation and p65 Translocation
Than Young M
--
To determine the mechanism of the age-related
increase in NF-
B activation, we examined the two key steps preceding
the NF-
B binding activity: I
B degradation in the cytoplasm and
NF-
B translocation to the nucleus. The results indicated that there
was no difference between young and old M
in expression of I
B
under resting conditions. However, after LPS stimulation, there was
greater I
B-
degradation in old M
than that in young M
. The
degradation also occurred faster, as shown at 15 min after stimulation,
and appeared to recover more slowly when compared with young M
(Fig.
2A). We then examined the p65
appearance in nuclear extracts following its translocation from the
cytoplasm. As shown in Fig. 2B, p65, which was not detected
in the nucleus before stimulation, gradually increased during the time
from 15 min to 2 h after LPS stimulation, followed by a decline
between 2 and 4 h. These results indicate that the increased
NF-
B binding activity with age is because of different rates of
I
B degradation, and subsequent NF-
B translocation, two immediate
events prior to the binding of the NF
B to its target genes.
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Fig. 2.
Old M have higher
I
B degradation and p65 translocation than young
M
. Peritoneal M
from young and old mice were
stimulated with LPS (5 µg/ml) for 0, 15 min, 30 min, 1 h, 2 h, or 4 h. The cytosolic and nuclear samples were prepared as
described under "Experimental Procedures" and were used for
I
B-
and p65 detection, respectively. The corresponding samples
(25 µg of protein per lane) were used to determine I
B-
or p65
protein using Western blot analysis. After I
B-
or p65 bands were
visualized, the membranes were stripped and reprobed with the antibody
to
-actin to be used as normalization control. The results for
I
B-
and p65 are shown in A and B,
respectively. Each bar represents mean ± S.E. of three
separate experiments. * indicates a significant difference at
p < 0.05 or less.
B Inhibitor Prevents NF-
B Activation in Both Young and
Old Mouse M
--
As no age-related change was observed in AP-1 and
CREB activation, the relationship between NF-
B activation and
age-related increase in COX-2 expression was further investigated.
Because increased NF-
B activation in old M
was associated with a
higher I
B phosphorylation and the consequent degradation, to inhibit NF-
B activation, we used Bay 11-7082, an inhibitor of I
B
phosphorylation, and therefore NF-
B activation (37). This inhibitor
has been shown not to have as broad an effect as do other NF-
B
activation inhibitors such as aspirin, caffeic acid, and
N-acetylcysteine. Doses of Bay 11-7082 between 0.5 and 5 µM were used. These doses were chosen based on
preliminary experiments in which a range of 0.1 to 100 µM
Bay 11-7082 was tested. Below 0.5 µM, Bay 11-7082 did not
inhibit NF-
B binding and at 5 µM, it almost completely inhibited NF-
B binding. We stimulated M
from young and old mice with LPS for 2 h and used the COX-specific NF-
B binding
oligonucleotide to detect the formation of the DNA-protein complex in
the nuclear fractions. As shown in Fig.
3A, there were no detectable
levels of NF-
B binding activity in unstimulated samples. Consistent with the above mentioned experiments, LPS induced higher NF-
B activation in old rather than in young M
. Preincubation with unlabeled COX-2-specific NF-
B binding oligonucleotide (cold
competition) or mutant NF-
B binding oligonucleotide was conducted to
confirm the specificity. NF-
B activation was partially inhibited at
doses of 0.5-2 µM and almost completely inhibited at 5 µM by Bay 11-7082.
View larger version (52K):
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Fig. 3.
I B inhibitor
inhibits the NF-
B activation in mouse
M
. Peritoneal M
from young and old mice were
preincubated with the I
B-
phosphorylation inhibitor, Bay 11-7082, for 30 min and then stimulated with LPS (5 µg/ml) for 2 h prior
to the preparation of nuclear extracts as described under
"Experimental Procedures." Nuclear extracts (2 µg) were incubated
with 32P-labeled COX-2-specific NF-
B (A),
AP-1 (B), or CREB oligonucleotide (C) and
subjected to EMSA as described under "Experimental Procedures."
Cold competition was conducted in the presence of 50-fold excess
unlabeled corresponding oligonucleotides for 10 min before the
32P-labeled oligonucleotide was added. In NF-
B
EMSA (A), the specificity of the binding was determined only
in old M
by adding cold or mutated NF-
B prior to the
32P-labeled NF-
B. The results are representative
of three independent experiments.
B
activation, we also examined its effect on AP-1 and CREB activation. As
shown in Fig. 3B, neither AP-1 nor CREB binding activity was
affected by Bay 11-7082 under the same condition as that used for
determination of NF-
B activation. These results indicate that Bay
11-7082 can be used as a tool to determine the role of NF-
B
activation in age-related up-regulation of COX-2 expression.
B Preferentially Reduces PGE2
Production and COX Activity in Old M
--
Of the transcription
factors that control COX-2 transcriptional activation, NF-
B was the
only one that exhibited the age-related increase. Therefore, we next
examined whether changing NF-
B activation would alter the
LPS-stimulated production of PGE2, a representative COX
product in M
. After M
from either young or old mice were preincubated with I
B inhibitor Bay 11-7082 for 30 min, the cells were stimulated by LPS for 24 h and PGE2 production
was then determined. As shown in Fig.
4A, PGE2
production was low in unstimulated cells and greatly increased with LPS
stimulation. This LPS-induced PGE2 production was 5-fold
higher in old compared with young mice. LPS-induced PGE2
production was inhibited by Bay 11-7082 in a dose-dependent
manner between 0.5 and 2 µM, whereas at a dose of 5 µM, no further inhibition was observed. Because old M
have higher NF-
B activation and COX-2 expression than those of young M
, we next examined the effect of NF-
B inhibition on the ability of young and old M
to produce PGE2. Results indicated
that when NF-
B activation was reduced with Bay 11-7082, PGE2 production in old M
was inhibited more
significantly (p < 0.05) compared with that of young
M
(80 versus 58, 27 versus 12, and 39 versus 13% of their control levels were observed in young
and old mice in the presence of Bay 11-7082 at 1, 2, and 5 µM, respectively). COX is the rate-limiting enzyme in
prostaglandin biosynthesis and we have demonstrated that increased COX
activity is the major contributing factor to the age-related increase
in PGE2 production (13). We, therefore, examined COX
activity in cells treated under the same condition as in the test for
PGE2 production. As shown in Fig. 4B,
LPS-stimulated M
from old mice have significantly higher COX
activity than those from young mice. NF-
B inhibition by Bay 11-7082 reduced COX activity in a dose-dependent manner. Furthermore, the inhibition of COX activity in old M
was more significant (p < 0.05) compared with that of young
M
(75 versus 48, 36 versus 12, and 3 versus 0.2% of their control levels were observed in young
and old mice in the presence of Bay 11-7082 at 1, 2, and 5 µM, respectively).
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Fig. 4.
Inhibition of NF- B
activation dose dependently reduces M
PGE2 production and M
COX
activity. The peritoneal M
from young and old mice were
pretreated with increasing concentrations of I
B phosphorylation
inhibitor Bay 11-7082 for 30 min and then stimulated with LPS (5 µg/ml) for 24 h at 37 °C. The supernatant was collected and
analyzed for PGE2 production. After the supernatant was
collected for PGE2 production, the cells were washed and
then further incubated in the medium containing 30 µM AA
for 10 min at 37 °C. Aspirin (2.1 mM) was added at the
end of incubation to terminate the reaction. The supernatant was
collected and analyzed for the PGE2 synthesized utilizing
exogenous AA to assess the COX enzyme activity. PGE2
concentrations in the samples were determined using RIA and adjusted
for total cell protein. The results for PGE2 production and
COX enzyme activity are shown in A and B,
respectively. The data are mean ± S.E. of four independent
experiments in each of which a duplicate measurement was conducted. The
bars bearing different letters within the same case
(lower or upper) represent significant difference
with p < 0.05. The lowercase and
uppercase letters represent young and old mice,
respectively.
B activation, we also tested its direct effect
on COX activity by adding Bay 11-7082 to cultures after LPS
stimulation. After 24 h of LPS stimulation, COX-2 would be fully
activated. The presence of Bay 11-7082 for 30 min thereafter should not
change the levels of COX-2 enzyme, but would be adequate to affect
enzyme activity if it did have a direct effect on the enzyme. The
results showed that addition of Bay 11-7082 after LPS stimulation did
not change COX activity in either young or old M
(data not shown),
thus a direct effect on COX enzyme activity can be ruled out.
B Is Not Limited to LPS as
Stimulant--
Increased PGE2 production with age is not
limited to that stimulated by LPS. Previously we showed that in
addition to LPS, calcium ionophore or T cell mitogens also stimulated
more PGE2 production in splenocytes of old mice or
peripheral blood mononuclear cells of elderly humans compared with
their young counterparts (4-6). To further confirm this in the
elicited peritoneal M
, we conducted a dose-response experiment using
IL-1
, another common stimulant of COX-2. Fig.
5A shows that IL-1
dose
dependently induced PGE2 production in both young and old
M
, but old M
produced significantly more PGE2 than
young M
in response to IL-1
stimulation. Next, we stimulated the
cells with 50 ng/ml IL-1
in the presence of Bay 11-7082 and found
that IL-1
-stimulated PGE2 production was also inhibited
by Bay 11-7082 in a dose-dependent manner (Fig. 5B). The patterns of response in young and old M
were
similar to those when LPS was used as a stimulant. Similar results were obtained when COX activity was evaluated (data not shown).
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Fig. 5.
Age-associated difference is also present in
IL-1 -induced PGE2 production,
which is also inhibited by blocking NF-
B
activation. A, the peritoneal M
from young and old
mice were stimulated by IL-1
at different concentrations as
indicated for 24 h at 37 °C. The supernatant was collected and
analyzed for PGE2 production. PGE2
concentrations in the samples were determined using RIA and adjusted
for total cell protein. Data are mean ± S.E. of four individual
experiments. * indicates a significant difference between young and old
mice at p < 0.05, and # at p = 0.1. B, as mentioned above the peritoneal M
were pretreated
with increasing concentrations of I
B phosphorylation inhibitor Bay
11-7082 for 30 min and then stimulated with IL-1
(50 ng/ml) for
24 h at 37 °C. The supernatant was collected and analyzed for
PGE2 production. The data are mean ± S.E. of four
independent experiments. The bars bearing different letters
within the same case (lower or upper) represent
significant difference with p < 0.05. The
lowercase and uppercase letters represent young
and old mice, respectively.
B Reduces COX-2 mRNA and Protein
Levels--
Because our previous studies (13, 14) showed that the
age-related increase in COX activity is because of increased expression of the COX-2 mRNA and protein, we determined COX-2 mRNA and
protein levels in cells that were incubated with Bay 11-7082 prior to LPS stimulation. As shown in Fig.
6A, unstimulated M
had very low expression of COX-2. However, LPS significantly induced COX-2 expression and the LPS-stimulated COX-2 expression was higher in old
compared with young M
. Inhibition of NF-
B activation dose-dependently reduced the COX-2 mRNA expression in
both young and old M
. The change in COX-2 protein levels was
generally in accordance with the change in COX-2 mRNA levels,
although the dose response was not as pronounced as seen in mRNA
expression (Fig. 6B). These results demonstrate that there
is an age-dependent increase in activation of NF-
B,
which results in higher transcription of the COX-2 gene, increased
COX-2 mRNA, COX-2 protein, and greater PGE2
production.
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Fig. 6.
Inhibition of NF- B
dose dependently inhibits expression of COX-2 mRNA and protein, and
iNOS protein in young and old mouse M
. Peritoneal
M
from young and old mice were pretreated with increasing
concentrations of I
B phosphorylation inhibitor Bay 11-7082 for 30 min and then stimulated with LPS (5 µg/ml) at 37 °C for 4 and
16 h for mRNA and protein assays, respectively. A,
the isolated total RNA (2 µg) was used to generate first-strand
cDNA and then COX-2 mRNA level was determined using PCR as
described under "Experimental Procedures." To normalize COX-2
mRNA, the 18 S rRNA PCR products were generated from the same
cDNA samples used in COX-2 mRNA PCR assays. The results are
representative of three independent experiments. Total cell lysates (25 µg of protein per lane) were used to determine COX-2 (B)
or iNOS (C) protein using Western blot analysis. After COX-2
or iNOS bands were visualized, membranes were stripped and reprobed
with the antibody to
-actin to serve a loading control. Results are
representative of four independent experiments for COX-2 and two
independent experiments for iNOS.
B binding
site (39). Thus, to further prove the link between NF-
B activation
and its target genes in the context of age-related events, we chose to
measure iNOS protein expressed by young and old M
under the same
condition used for COX-2 determination. As demonstrated in Fig.
6C, LPS-induced iNOS expression was higher in old rather
than in young M
and was also inhibited by Bay 11-7082 in a
dose-dependent manner.
B in Young
M
--
Our recent work (14) showed that old M
produce higher
levels of intracellular ceramide, compared with those from young mice
after LPS stimulation. Furthermore, we showed that increasing ceramide
levels in young M
, by adding exogenous ceramide, significantly increased COX-2 expression. This effect was specific to ceramide and
did not depend on its downstream metabolite, sphingosine (14). Because
no age-related difference in mitogen-activated protein kinase activity
(14) or AP-1 and CREB activation was observed, we hypothesized that
NF-
B mediates ceramide-induced COX-2 up-regulation. In this study,
we added exogenous ceramide to the young M
and determined its effect
on NF-
B activation. First, we tested the effect of different
concentrations of ceramide on NF-
B activation. The results showed
that incubating cells in the presence of ceramide for 2 h induced,
although not as strongly as LPS, NF-
B activation in a
dose-dependent manner (Fig.
7A). We then tested the time course of ceramide-induced NF-
B activation in the absence or presence of Bay 11-7082. As shown in Fig. 7B, ceramide
induced NF-
B activation at all time points tested. This
ceramide-induced NF-
B activation was prevented by addition of the
I
B phosphorylation inhibitor Bay 11-7082. Furthermore, we determined
the effect of ceramide on LPS-stimulated NF-
B activation and
demonstrated that LPS induced higher NF-
B activation in the cells
supplemented with ceramide compared with those treated with vehicle
control (Fig. 7C). It should be mentioned that ceramide by
itself is a weak inducer of NF-
B activation and a much longer
exposure time was needed to obtain a comparable band density to that
seen with LPS. However, ceramide had an additive effect on LPS-induced
NF-
B activation.
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Fig. 7.
Ceramide induces by itself, and also enhances
LPS-stimulated NF- B binding activity, which is
inhibited by blocking NF-
B activation.
A, peritoneal M
from young mice were incubated in the
presence of ceramide at different concentrations as indicated for
2 h at 37 °C. B, peritoneal M
from young mice
were preincubated with or without Bay 11-7082 (5 µM) for
30 min. Ceramide (30 µM) was added to the cells and
incubation was continued at 37 °C for the additional times as
indicated. C, in a separate experiment, M
were stimulated
with LPS (5 µg/ml) during the same time course in the presence or
absence of ceramide (30 µM). The nuclear extracts were
prepared as described under "Experimental Procedures." Nuclear
extracts (2 µg) were incubated with 32P-labeled
COX-2-specific NF-
B oligonucleotide and subjected to an EMSA. The
experiments were repeated twice and similar results were
obtained.
B activation by ceramide or Bay
11-7082 is coupled to the changes in COX-2 activation, we measured
PGE2 production under the same condition. As shown in Fig.
8, addition of exogenous ceramide
increased PGE2 production and this ceramide-induced
increase was prevented by inhibiting NF-
B activation. Similar to
that seen in the NF-
B binding assay, addition of ceramide increased
LPS-stimulated PGE2 production, an effect that was also
prevented by inhibiting NF-
B activation.
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Fig. 8.
Addition of ceramide enhances LPS-induced
PGE2 production, which is reduced by inhibition of
NF- B activation. The peritoneal M
from
young mice were preincubated with or without I
B phosphorylation
inhibitor Bay 11-7082 (5 µM) for 30 min and then treated
with ceramide (30 µM), LPS (5 µg/ml), or ceramide plus
LPS for 12 h at 37 °C. The supernatants were collected and
analyzed for PGE2 production. PGE2
concentrations in samples were determined using RIA and adjusted for
total cell protein. Data are mean ± S.E. of four independent
experiments in each of which a duplicate measurement was conducted. The
bars bearing different letters represent significant
difference at p < 0.05.
B Decoy Blocks NF-
B Binding Activity and Inhibits
PGE2 Production and COX-2 Expression--
To further
confirm the role of NF-
B, we repeated some of the above experiments
by employing NF-
B decoy as an alternative and more specific approach
to block NF-
B binding to COX-2 promoter. Use of the NF-
B decoy
has been shown to successfully suppress COX-2 expression (40). The
COX-2-specific NF-
B decoys have the identical sequences to those
used for EMSA but modified on the 3 bases at each end by
phosphorothioation to prevent being digested in the cells. The NF-
B
decoy competes with the COX-2 promoter for binding to the activated
NF-
B dimers and thus, block COX-2 gene activation. As shown in Fig.
9A, NF-
B decoy
dose-dependently inhibited LPS-induced NF-
B binding
activity while its mutated form did not have any effect. The role of
NF-
B in the additive effect of ceramide on LPS-induced
PGE2 production was further confirmed by data shown in
Figs. 9 and 10. As seen in Fig. 9,
NF-
B decoy but not the mutant abrogated the ceramide and LPS-induced NF-
B binding activity (Fig. 9B).
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Fig. 9.
NF- B decoy blocks
LPS or LPS plus ceramide induced NF-
B binding
activity. A, NF-
B decoy or mutated NF-
B decoy at
indicated concentrations were prepared as described under
"Experimental Procedures." Peritoneal M
from young mice were
preincubated in the presence of the decoys for 24 h at 37 °C.
The culture medium was then replaced with new medium containing LPS (5 µg/ml) and incubated for 2 h. The nuclear extracts were prepared
as described under "Experimental Procedures." Nuclear extracts (2 µg) were incubated with 32P-labeled COX-2-specific
NF-
B oligonucleotide and subjected to an EMSA. B, the
cells were preincubated with only one high concentration (10 nM) of NF-
B decoy or its mutant. LPS (5 µg/ml) were
than added to stimulate the cells in the presence or absence of
ceramide (30 µM). The experimental procedures are the
same as described above in A.
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Fig. 10.
NF- B decoy inhibits
ceramide, LPS, or LPS plus ceramide-induced PGE2 production
through suppressing COX-2 expression. A, the peritoneal
M
from young mice were preincubated with NF-
B decoy or its mutant
for 24 h. LPS (5 µg/ml), ceramide (30 µM), or both
were then added to stimulate the cells for 12 h. The supernatants
were collected and analyzed for PGE2 production.
PGE2 concentrations in samples were determined using RIA
and adjusted for total cell protein. Data are mean ± S.E. of four
independent experiments. * indicates a significant difference at
p < 0.05. B, the cell treatments were
similar to those described above. Total cell lysates (25 µg of
protein per lane) were used to determine COX-2 protein levels using
Western blot analysis. After COX-2 bands were visualized, membranes
were stripped and reprobed with the antibody to
-actin to serve as a
loading control.
B binding would
similarly affect PGE2 production and COX-2 expression.
Mutated NF-
B decoy did not have a significant effect on
PGE2 production (data not shown). NF-
B decoy, however,
significantly inhibited LPS-stimulated PGE2 production both
in the presence and absence of ceramide (Fig. 10A). The
changes in PGE2 production was associated with similar
changes in COX-2 protein expression (Fig. 10B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
from old mice was because of their increased expression of
COX-2 mRNA (13). Furthermore, we showed that the increase in COX-2 mRNA was because of transcriptional up-regulation of COX-2 (14). Transcription factors NF-
B, AP-1, and CREB have been indicated in
regulation of COX-2 activation. In this study, we tested the involvement of these transcription factors in the age-associated and
ceramide-induced up-regulation of COX-2 activation. First we compared
the activation levels of NF-
B, AP-1, and CREB in peritoneal M
from young and old mice. We found that in response to LPS stimulation,
old M
had significantly higher consensus as well as COX-2-specific
NF-
B binding activity compared with young M
. However, neither
AP-1 nor CREB showed any significant change with age. The
age-associated change in transcription factor activation has not been
well studied and in particular, no information was available regarding
their changes with age in M
. Kim et al. (34) reported
that NF-
B binding activity in rat kidney increased with age.
Helenius et al. (35) showed an age-related increase in the
nuclear binding activity of NF-
B, but not those of Sp1 and AP-1, in
rat liver and heart. Increased NF-
B and AP-1 activation with age was
also observed in rat gastric mucosa cells (33). M
are the major
source of inflammatory mediators including PGE2. These
M
-originated mediators are involved in the pathogenesis of
inflammatory, cardiovascular, and neoplastic diseases (41-43). Most of
these diseases are more prevalent in the elderly population. Because
NF-
B has been indicated in regulation of various M
-originated mediators, our finding that consensus NF-
B activation in M
is up-regulated with aging might shed light on the mechanism of
age-related changes in other M
-originated mediators such as nitric
oxide and proinflammatory cytokines.
B to the COX-2 promoter region has been
suggested to be necessary for COX-2 transcriptional activation (16, 18,
19). To confirm this and also determine its role in the age-associated
increase of COX-2 activation, we inhibited NF-
B activation by
employing an I
B phosphorylation inhibitor, Bay 11-7082. In agreement
with the result reported by others (37), this inhibitor effectively
prevented NF-
B activation in this study. Furthermore, the NF-
B
inhibition dose dependently, up to 2 µM, reduced
LPS-induced PGE2 production in both young and old M
.
However, inhibition of NF-
B activation resulted in a significantly
larger reduction in PGE2 synthesis in old M
compared with that in young M
. Because old M
have higher NF-
B activity as well as PGE2 production compared with young M
, these
results further confirm the involvement of NF-
B in the age-related
up-regulation of COX-2. Because PGE2 synthesis is
determined by both substrate availability and COX activity, we measured
COX activity by providing excessive exogenous arachidonic acid so that
substrate availability would not be a limiting factor. The results
showed dose-dependent inhibition of COX activity by Bay
11-7082. The degree of inhibition was significantly higher in old M
compared with that in young M
.
for the activation of COX-2 and PGE2
production, we have mainly used LPS. It has been questioned whether the age-related difference in COX-2 expression is a phenomenon specific for
LPS, merely reflecting the difference in LPS signal transduction at its
receptor level. This is not likely because we previously observed an
age-related increase in PGE2 production when different immune cells from both mice and humans and several other stimulating agents were used (4, 6). In fact, M
Toll-like receptor 4 expression
decreases with aging (57). Thus, the age-related up-regulation of
LPS-stimulated COX-2 activation involves a post-receptor signal
transduction event, such as NF-
B, as suggested by this study. This
was further strengthened in this study by the observation that when
IL-1
was used in place of LPS to stimulate M
, those from old mice
have significantly higher PGE2 production compared with
those from young mice. Furthermore, the IL-1
-induced increase in
PGE2 production was abrogated by inhibiting NF-
B activation.
B activation, it will be predicted that other NF-
B target
genes may also demonstrate an up-regulation with age and their
expression could also be suppressed when NF-
B activation is blocked.
We chose iNOS as such a candidate to substantiate this speculation.
Increased NO production and iNOS expression have been shown in old
compared with young murine M
(31, 38). The murine iNOS gene has an
NF-
B binding site in its promoter region and pyrrolidine
dithiocarbamate, an NF-
B inhibitor, blocks both NF-
B activation
and NO production in LPS-stimulated M
(39). Increased iNOS
expression has also been shown in vascular smooth muscle cells from old
rats compared with those from young rats and this age-related
up-regulation is associated with NF-
B activation (44). In this
study, we confirmed the previous finding by showing higher iNOS
expression in old M
compared with young M
. More importantly, we
further demonstrated that blocking NF-
B activation reduced iNOS
expression. This observation added further support for the involvement
of NF-
B as a mechanism underlying the age-associated up-regulation
of certain genes and their products.
B complex is the p65
and p50 heterodimer (45). In most resting cells, NF-
B is sequestered
in the cytoplasm as an inactive precursor in complex with the
inhibitory protein I
B. By binding to the p65 component, I
B
inhibits transactivation of the p65 and p50 heterodimer, and thus
blocking the translocation of the dimer to the nucleus (46, 47). In
response to activation signals, I
B is phosphorylated and degraded,
allowing NF-
B release and further translocation to the nucleus where
it regulates gene expression. Thus, the degradation of I
B and
translocation of NF-
B are closely linked to the activity of NF-
B
binding to DNA. Although several members of the I
B family have been
identified, the best characterized I
B is I
B-
. In the current
study, we found that upon LPS stimulation, old M
had increased
degradation of I
B-
in the cytoplasm, which was accompanied by an
increased appearance of p65 in the nucleus. These results strongly
suggest that the increased degradation of I
B-
and the subsequent
p65 translocation to the nucleus are the main contributors to the
increased NF-
B binding activity seen in old M
compared with that
in young M
. An age-related decrease in cytoplasmic I
B-
and an
age-related increase in nuclear p65 was observed in unstimulated rat
kidney tissue by Kim et al. (34). However, another study
showed that NF-
B activation increased with age but I
B-
and p65
levels were unaffected in the rat gastric mucosa (33). I
B is
phosphorylated by the action of I
B kinase (IKK) (48). The activation
of IKK is in turn mediated by phosphorylation through NF
B-inducing
kinase (49, 50). The cause of increased I
B degradation was not
determined in this study and is the subject of our future
investigation. However, our recent work (14) suggested that ceramide
might be involved in the age-associated increase in I
B degradation.
We showed that, following LPS stimulation, M
from old mice
generated significantly more ceramide than those from young mice.
Furthermore, we showed that C2-ceramide significantly increased
LPS-induced COX activity and COX-2 expression in young M
; this
effect of ceramide was not mediated through mitogen-activated protein
kinase. In this study, we showed that ceramide dose dependently induced
NF-
B activation in young M
. This effect of ceramide was blocked
by the I
B phosphorylation inhibitor, Bay 11-7082. Addition of
ceramide increased LPS-stimulated NF-
B activation, which was also
inhibited by Bay 11-7082. These changes in NF-
B activation caused by
ceramide, in the presence or absence of LPS, were mirrored by
those in PGE2 production. Bay 11-7082 treatment prevented
the ceramide-induced effect on both NF-
B activation and
PGE2 production in the presence or absence of LPS. In this study, we also used a COX-2-specific NF-
B decoy as an alternative approach to block NF-
B activation. The decoy competes with the COX-2
promoter for binding to the activated NF-
B dimers and as a result,
blocks COX-2 gene activation. The results obtained using the NF-
B
decoy were similar to those obtained with Bay 11-7082. Taken together,
because old M
have higher levels of ceramide compared with young
M
and because increasing the level of ceramide in young M
enhances NF-
B activation, COX-2 expression, and PGE2 production, all of which are abrogated by Bay 11-7082 or an NF-
B decoy, these data strongly suggest that the age-associated increase in
COX-2 transcription is because of the ceramide-induced up-regulation of
NF-
B activation.
B activation after reducing ceramide
levels in old M
would have provided further support for our proposed mechanism, however, the approach is not feasible at the present time.
Although knockout mice for acidic sphingomyelinase are
available, these animals develop Neimann-Pick disease and die by the
age of 10 months, making them unsuitable to address the role of
ceramide in NF-
B up-regulation in aged mice (typically more than 20 months old). In addition, the neutral, but not the acidic
sphingomyelinase has been indicated to be responsible for the
age-related increase of ceramide levels in other tissues. However,
specific neutral sphingomyelinase inhibitors are not
commercially available.
B activation
has not been determined and will be the subject of our future study. It
has been suggested that ceramide may induce NF-
B activation through
its effect on the
isotype of protein kinase C (PKC-
). PKC-
was shown to activate NF-
B through phosphorylation of I
B-
in
NIH-3T3 fibroblasts (51). Furthermore, overexpression of PKC-
positively modulates IKK
activity, whereas the transfection of a
PKC-
dominant negative mutant severely impairs the activation of
IKK
(52). PKC-
-deficient mice have impaired NF-
B activation (53). It was reported that PKC-
can be activated in vitro
by ceramide and in vivo by sphingomyelinase, which
produces ceramide, in NIH-3T3 fibroblasts (54). Finally, ceramide was
shown to induce the translocation of PKC-
to both the nucleus and
membrane in rat astrocytes (55) and hepatocytes (56).
B activation is up-regulated with aging in murine M
, and that this age-associated up-regulation of NF-
B activation mediates the
higher age-associated expression of COX-2. Increased I
B degradation and p65 translocation with aging represent important determinants of
the increased NF-
B activation observed in old M
. Combined with
our previous study (14), in which ceramide was shown to mediate the
age-associated up-regulation of COX-2 transcription, the current study
suggests that increased ceramide levels in old M
induces higher
NF-
B activation, leading to increased COX-2 transcription. These
findings will help to further understand the mechanism of the
age-associated increase in COX-2 expression and associated diseases.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Sung Nim Han for technical assistance and Stephanie Marco for preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by NIA National Institutes of Health Grant RO1-AG09140-09, National Institutes of Health Grant ES11518, and United States Department of Agriculture agreement 58-1950-9-001.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: Food Science and Human Nutrition Department, Michigan State University, East Lansing, MI 48824.
** To whom correspondence should be addressed: Nutritional Immunology Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111. Tel.: 617-556-3129; Fax: 617-556-3224; E-mail: smeydani@hnrc.tufts.edu.
Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M207470200
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ABBREVIATIONS |
---|
The abbreviations used are:
M, macrophages;
LPS, lipopolysaccharide;
PGE2, prostaglandin
E2;
COX, cyclooxygenase;
NF-
B, nuclear factor
B;
AP-1, activator protein-1;
CREB, cAMP-responsive element-binding
protein;
EMSA, electrophoretic mobility shift assay;
AA, arachidonic
acid;
IL, interleukin;
iNOS, inducible nitric-oxide synthase;
RIA, radioimmunoassay;
IKK, I
B kinase;
CRE, cAMP-response
element.
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REFERENCES |
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1. | Miller, R. A. (1996) Science 273, 70-74[Abstract] |
2. | Chakravarti, B., and Abraham, G. N. (1999) Mech. Ageing Dev. 108, 183-206[CrossRef][Medline] [Order article via Infotrieve] |
3. | Bartocci, A., Maggi, F. M., Welker, R. D., and Veronese, F. (1982) in Prostaglandins and Cancer (Powles, T. J. , Backman, R. S. , Honn, K. V. , and Ramwell, P., eds) , pp. 725-730, Alan R. Liss, New York |
4. | Hayek, M. G., Meydani, S. N., Meydani, M., and Blumberg, J. B. (1994) J. Gerontol. 49, B197-B207[Medline] [Order article via Infotrieve] |
5. | Meydani, S. N., Meydani, M., Verdon, C. P., Shapiro, A. C., Blumberg, J. B., and Hayes, K. C. (1986) Mech. Ageing Dev. 34, 191-201[Medline] [Order article via Infotrieve] |
6. | Meydani, S. N., Barklund, M. P., Liu, S., Meydani, M., Miller, R. A., Cannon, J. G., Morrow, F. D., Rocklin, R., and Blumberg, J. B. (1990) Am. J. Clin. Nutr. 52, 557-563[Abstract] |
7. | Rosenstein, M. M., and Strausser, H. R. (1980) J. Reticuloendoth. Sci. 27, 159-166 |
8. | Beharka, A. A., Wu, D., Han, S. N., and Meydani, S. N. (1997) Mech. Ageing Dev. 93, 59-77[CrossRef][Medline] [Order article via Infotrieve] |
9. | DeWitt, D. L., and Smith, W. L. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1412-1416[Abstract] |
10. |
Merlie, J. P.,
Fagan, D.,
Mudd, J.,
and Needleman, P.
(1988)
J. Biol. Chem.
263,
3550-3553 |
11. |
Kujubu, D. A.,
Fletcher, B. S.,
Varnum, B. C.,
Lim, R. W.,
and Herschman, H. R.
(1991)
J. Biol. Chem.
266,
12866-12872 |
12. | Xie, W., Chipman, J. G., Robertson, D. L., Erickson, E. L., and Simmons, D. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2692-2696[Abstract] |
13. | Hayek, M. G., Mura, C. V., Wu, D., Beharka, A. A., Han, S. N., Paulson, K. E., Hwang, D., and Meydani, S. N. (1997) J. Immunol. 159, 2445-2451[Abstract] |
14. |
Claycombe, K. J.,
Wu, D.,
Nikolova-Karakashian, M.,
Palmer, H.,
Beharka, A.,
Paulson, E.,
and Meydani, S. N.
(2002)
J. Biol. Chem.
277,
30784-30791 |
15. | Xie, W., Fletcher, B. S., Andersen, R. D., and Herschman, H. R. (1994) Mol. Cell. Biol. 14, 6531-6539[Abstract] |
16. |
Yamamoto, K.,
Arakawa, T.,
Ueda, N.,
and Yamamoto, S.
(1995)
J. Biol. Chem.
270,
31315-31320 |
17. | Inoue, H., Nanayama, T., Hara, S., Yokoyama, C., and Tanabe, T. (1994) FEBS Lett. 350, 51-54[CrossRef][Medline] [Order article via Infotrieve] |
18. | Hwang, D., Jang, B. C., Yu, G., and Boudreau, M. (1997) Biochem. Pharmacol. 54, 87-96[CrossRef][Medline] [Order article via Infotrieve] |
19. | Newton, R., Kuitert, L. M. E., Bergmann, M., Adcock, I. M., and Barnes, P. J. (1997) Biochem. Biophys. Res. Commun. 237, 28-32[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Barnes, P. J.,
and Karin, M.
(1997)
N. Engl. J. Med.
336,
1066-1071 |
21. |
Neurath, M. F.,
Becker, C.,
and Barbulescu, K.
(1998)
Gut
43,
856-860 |
22. |
Miagkov, A. V.,
Kovalenko, D. V.,
Brown, C. E.,
Didsbury, J. R.,
Cogswell, J. P.,
Stimpson, S. A.,
Baldwin, A. S.,
and Makarov, S. S.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
13859-13864 |
23. |
Poligone, B.,
and Baldwin, A. S.
(2001)
J. Biol. Chem.
276,
38658-38664 |
24. |
Subbaramaiah, K.,
Lin, D. T.,
Hart, J. C.,
and Dannenberg, A. J.
(2001)
J. Biol. Chem.
276,
12440-12448 |
25. | von Knethen, A., and Brune, B. (2000) Biochemistry 39, 1532-1540[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Caivano, M.,
and Cohen, P.
(2000)
J. Immunol.
164,
3018-3025 |
27. | Mestre, J. R., Rivadeneira, D. E., Mackrell, P. J., Duff, M., Stapleton, P. P., Mack-Strong, V., Maddali, S., Smyth, G. P., Tanabe, T., and Daly, J. M. (2001) FEBS Lett. 496, 147-151[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Sawai, H.,
Okazaki, T.,
Yamamoto, H.,
Okano, H.,
Takeda, Y.,
Tashima, M.,
Sawada, H.,
Okuma, M.,
Ishikura, H.,
Umehara, H.,
and Domae, N.
(1995)
J. Biol. Chem.
270,
27326-27331 |
29. |
Majumdar, S.,
Lamothe, B.,
and Aggarwal, B. B.
(2002)
J. Immunol.
168,
2644-2651 |
30. | Wu, D., Mura, C., Beharka, A. A., Han, S. N., Paulson, K. E., Hwang, D., and Meydani, S. N. (1998) Am. J. Physiol. 275, C661-C668[Abstract] |
31. | Beharka, A. A., Wu, D., Serafini, M., and Meydani, S. N. (2002) Free Radical Biol. Med. 32, 503-511[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Fu, J.-Y.,
Masferrer, J. L.,
Seibert, K.,
Raz, A.,
and Needleman, P.
(1990)
J. Biol. Chem.
265,
16737-16740 |
33. | Xiao, Z. Q., and Majumdar, A. P. (2000) Am. J. Physiol. 278, G855-G865 |
34. | Kim, H. J., Kim, K. W., Yu, B. P., and Chung, H. Y. (2000) Free Radical Biol. Med. 28, 683-692[CrossRef][Medline] [Order article via Infotrieve] |
35. | Helenius, M., Hanninen, M., Lehtinen, S. K., and Salminen, A. (1996) Biochem. J. 318, 603-608[Medline] [Order article via Infotrieve] |
36. | Korhonen, P., Helenius, M., and Salminen, A. (1997) Neurosci. Lett. 225, 61-64[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Pierce, J. W.,
Schoenleber, R.,
Jesmok, G.,
Best, J.,
Moore, S. A.,
Collins, T.,
and Gerritsen, M. E.
(1997)
J. Biol. Chem.
272,
21096-21103 |
38. | Chen, L.-C., Pace, J. L., Russell, S. W., and Morrison, D. C. (1996) Infect. Immun. 64, 4288-4298[Abstract] |
39. |
Xie, Q. W.,
Kashiwabara, Y.,
and Nathan, C.
(1994)
J. Biol. Chem.
269,
4705-4708 |
40. |
von Knethen, A.,
Callsen, D.,
and Brune, B.
(1999)
Mol. Biol. Cell
10,
361-372 |
41. | O'Byrne, K. J., and Dalgleish, A. G. (2001) Br. J. Cancer 85, 473-483[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Yamamoto, Y.,
and Gaynor, R. B.
(2001)
J. Clin. Invest.
107,
135-142 |
43. |
Dubois, R. N.,
Abramson, S. B.,
Crofford, L.,
Gupta, R. A.,
Simon, L. S.,
Van De Putte, L. B.,
and Lipsky, P. E.
(1998)
FASEB J.
12,
1063-1073 |
44. |
Yan, Z. Q.,
Sirsjo, A.,
Bochaton-Piallat, M. L.,
Gabbiani, G.,
and Hansson, G. K.
(1999)
Arterioscler. Thromb. Vasc. Biol.
19,
2854-2862 |
45. | Verma, I. M., Stevenson, J. K., Schwarz, E. M., Van Antwerp, D., and Miyamoto, S. (1995) Genes Dev. 9, 2723-2735[CrossRef][Medline] [Order article via Infotrieve] |
46. | Baeuerle, P. A., and Baltimore, D. (1988) Cell 53, 211-217[Medline] [Order article via Infotrieve] |
47. | Beg, A. A., Ruben, S. M., Scheinman, R. I., Haskill, S., Rosen, C. A., and Baldwin, A. S. J. (1992) Genes Dev. 6, 1899-1913[Abstract] |
48. | May, M. J., and Ghosh, S. (1998) Immunol. Today 19, 80-88[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Delhase, M.,
Hayakawa, M.,
Chen, Y.,
and Karin, M.
(1999)
Science
284,
309-313 |
50. |
Nakano, H.,
Shindo, M.,
Sakon, S.,
Nishinaka, S.,
Mihara, M.,
Yagita, H.,
and Okumura, K.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
3537-3542 |
51. | Diaz-Meco, M. T., Dominguez, I., Sanz, L., Dent, P., Lozano, J., Municio, M. M., Berra, E., Hay, R. T., Sturgill, T. W., and Moscat, J. (1994) EMBO J. 13, 2842-2848[Abstract] |
52. |
Lallena, M. J.,
Diaz-Meco, M. T.,
Bren, G.,
Paya, C. V.,
and Moscat, J.
(1999)
Mol. Cell. Biol.
19,
2180-2188 |
53. | Leitges, M., Sanz, L., Martin, P., Duran, A., Braun, U., Garcia, J. F., Camacho, F., Diaz-Meco, M. T., Rennert, P. D., and Moscat, J. (2001) Mol. Cell 8, 771-780[Medline] [Order article via Infotrieve] |
54. |
Lozano, J.,
Berra, E.,
Municio, M. M.,
Diaz-Meco, M. T.,
Dominguez, I.,
Sanz, L.,
and Moscat, J.
(1994)
J. Biol. Chem.
269,
19200-19202 |
55. | Galve-Roperh, I., Haro, A., and Diaz-Laviada, I. (1997) FEBS Lett. 415, 271-274[CrossRef][Medline] [Order article via Infotrieve] |
56. | Calcerrada, M. C., Miguel, B. G., Martin, L., Catalan, R. E., and Martinez, A. M. (2002) FEBS Lett. 514, 361-365[CrossRef][Medline] [Order article via Infotrieve] |
57. |
Renshaw, M.,
Rockwell, J.,
Engleman, C.,
Gewirtz, A.,
Katz, J.,
and Sambhara, S.
(2002)
J. Immunol.
169,
4697-4701 |