From the Vascular Biology Research Center and Division of Hematology, University of Texas-Houston Medical School, Houston, Texas 77030
Received for publication, December 11, 2000, and in revised form, February 6, 2001
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ABSTRACT |
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The anti-inflammatory actions of
salicylates cannot be explained by inhibition of cyclooxygenase (COX)
activity. This study demonstrates that sodium salicylate at a
therapeutic concentration suppressed COX-2 gene transcription induced
by phorbol 12-myristate 13-acetate and interleukin 1 Salicylic acid is arguably the oldest anti-inflammatory drug
preparation, and its acetylated form, acetylsalicylic acid (aspirin), is the most commonly used non-steroidal anti-inflammatory drug. The
groundbreaking work of Vane (1) demonstrated that non-steroidal anti-inflammatory drugs, such as aspirin, owe their anti-inflammatory effects to the inhibition of prostaglandin synthesis. The last 30 years
has seen major developments in prostanoid biology, not least the
identification of cyclooxygenase-2
(COX-2),1 the inducible COX
isoform that is believed to play a major role in inflammation (2-4)
and tumorigenesis (5-8). Despite such advances, salicylate remains a
pharmacological enigma, as it inhibits prostanoid synthesis in intact
cells (9-11) but has little effect on purified COX-1 or COX-2 activity
(10). Recent studies have suggested that salicylate owes its
anti-inflammatory effects to the inhibition of nuclear factor Cell Culture--
HFF were obtained from ATCC and cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum (FBS), 100 µg/ml streptomycin, and 100 units/ml
penicillin at 37 °C in 5% CO2. In all protocols
90-95% confluent HFF were serum-deprived for 24 h to synchronize
the cells in the G0/G1 phase before treatment with serum or other stimuli.
Northern Analysis--
RNA isolation and Northern analysis was
performed as outlined previously (16). In brief, 25-30 µg of RNA
isolated from HFF was fractionated on 1% agarose and was transferred
to a positively charged nylon membrane. As a COX-2 probe, agarose
gel-purified, full-length, 1.9-kilobase COX-2 cDNA was used.
Hybridization and detection by autoradiography were performed according
to a procedure reported previously (16).
Western Blot Analysis--
Whole cell lysates were prepared by
lysing HFF with phosphate-buffered saline, pH 7.4, containing 0.1%
Triton X-100, 0.01% EDTA, 1 mM phenylmethylsulfonyl
fluoride, 1.5 mM pepstatin A, and 0.2 mM
leupeptin. Lysates were centrifuged at 13,000 rpm for 10 min. The
supernatants were boiled for 5 min with equal volumes of 2× gel
loading buffer (100 mM Tris, 10% Electrophoretic Mobility Shift Assay (EMSA)--
AP-2 and
NF- Transient Transfection--
A 5' flanking DNA fragment NaS Inhibited PMA- or IL-1 NaS Inhibited C/EBP but Not the NF- NaS Inhibited C/EBP Results from this study challenge the prevailing view that
salicylates, including aspirin, exert their therapeutic actions by
inhibiting NF- C/EBP It is intriguing that the inhibitory action of salicylate on COX-2
transcription stimulated by PMA and IL-1 by
inhibiting the binding of CCAAT/enhancer-binding protein
to its
promoter region of COX-2. By contrast, salicylate did not inhibit
nuclear factor
B-dependent COX-2 induction by tumor
necrosis factor
. The inhibitory effect of sodium salicylate was
restricted to serum-deprived quiescent cells. These findings indicate
that contrary to the current view that salicylate acts via inhibition
of nuclear factor
B the pharmacological actions of aspirin and
salicylates are mediated by inhibiting CCAAT/enhancer-binding protein
binding and transactivation. These findings have a major impact on
the conceptual understanding of the mechanism of action of salicylates
and on new drug discovery and design.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
(NF-
B)-mediated gene expression (12, 13). However, inhibition of
NF-
B is exerted only at suprapharmacological concentrations of
sodium salicylate (NaS) (>5 mM) with no effects at
pharmacological concentrations (14). Salicylate at such high
concentrations inhibits numerous cellular kinases nonspecifically (15).
Our group recently demonstrated that NaS and aspirin at
pharmacologically relevant concentrations equipotently inhibited COX-2
transcription (16) and that the maximal inhibitory effect occurs in
cells deprived of serum for 24 h. Because aspirin is rapidly
deacetylated and converted to salicylate in vivo its action
on COX-2 expression is attributed to salicylate. To elucidate the
mechanism by which salicylate suppresses COX-2 transcription we
evaluated the effects of NaS, at a representative therapeutic
concentration, on COX-2 expression in serum-deprived G0 and
serum-driven cycling human foreskin fibroblasts (HFF). The results show
that the inhibitory effect of salicylate on phorbol 12-myristate
13-acetate (PMA)- and interleukin 1
(IL-1
)-induced COX-2
transcription was confined to G0 cells. Salicylate
suppressed PMA- and IL-1
-induced COX-2 promoter activity by
inhibiting the binding of CCAAT/enhancer-binding protein (C/EBP)
to
its cognate site on the 5' promoter region. By contrast, salicylate did
not inhibit NF-
B binding to the COX-2 promoter or
NF-
B-dependent COX-2 induction by tumor necrosis factor
(TNF
).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 20%
glycerol, 4% SDS, 2 mg/ml bromophenyl blue). Nuclear extracts were prepared by a method described previously (16). 10-50 µg of
whole cell lysate or nuclear extract was applied to a 12% SDS polyacrylamide minigel using the Laemmli buffer system and transferred to a nitrocellulose membrane. Nonspecific IgGs were blocked with 5%
nonfat dried milk containing 1 mg/ml globulin-free bovine serum albumin
before being incubated with an antibody against COX-2 (Cayman Chemical)
or C/EBP isoforms (Santa Cruz Biotechnology). Protein bands were
detected using enhanced chemiluminescence.
B oligonucleotides were obtained from Promega. C/EBP
oligonucleotides were synthesized by Sigma-Genosys based on the
human COX-2 promoter (17). Wild type, 5'-GCTTACGCAATTTTTTTAAGG-3'; mutant, 5'-GCgactagAATTTTTTTAAGG-3'. The complimentary oligonucleotides were annealed and purified following the manufacturer's protocol. Each
probe was end-labeled with [
32P]ATP using T4 kinase
(Amersham Pharmacia Biotech). EMSA was perfomed by incubating 5 µg of
nuclear extract with a labeled probe (10,000 cpm; ~10fmol) in a
binding buffer containing 15 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM EDTA, 12% glycerol, 5 µg of bovine serum albumin, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM
dithiothreitol, and 1.5 µg of poly(dI-dC). To assess the specificity of DNA protein binding up to a 100-fold molar excess of unlabeled wild
type or mutant oligonucleotide was added. For supershift experiments
C/EBP isoform-specific antibodies (Santa Cruz Biotechnology) were
applied to the mixture for 30 min at room temperature. The mixture was
applied to a 4% polyacrylamide gel and electrophoresed at 150 V for 90 min, and the complex was detected by autoradiography.
891 to
+9 (
891/+9) and its deletion mutants of the human COX-2 gene were
constructed into a promoterless luciferase expression vector pGL3
(Promega) as described previously (16). Using the
891/+9 construct
NF-
B mutant 1 (
447 to
438), GGGGATTCCC to attcATTCCC; mutant 2 (
222 to
213), GGGACTACCC to aattCTACCC; and the C/EBP mutant (
132
to
124), TTACGCAAT to gcgatagcT were generated using
oligonucleotide-directed mutagenesis. For transfection experiments, 2 µg of reporter vector mixed with 10 µl of Lipofectin reagent (Life
Technologies, Inc.) was added to HFF. To optimize transfection
Dulbecco's modified Eagle's medium was replaced with OPTIMEM-1 (Life
Technologies, Inc.). After 6 h cells were washed twice with fresh
OPTIMEM-1 and refed with Dulbecco's modified Eagle's medium
supplemented as outlined above. After 16 h, cells were
serum-starved for a period of 24 h before treatment. The expressed
luciferase activity was determined in a luminometer (Bioscan Lumi/96;
Bioscan Inc., Washington D. C.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced COX-2 Expression Only in
Serum-starved Quiescent Fibroblasts--
We recently reported that NaS
and aspirin at therapeutic concentrations (10
7 to
10
3 M) equipotently suppress
IL-1
- and PMA-induced COX-2 transcription in serum-deprived human
endothelial cells and fibroblasts (16). To determine whether the
inhibitory effect of salicylate is cell cycle-dependent, we
performed experiments in HFF that were first serum-deprived for 24 h followed by the addition of 2.5% FBS to drive the cell cycle. This
fibroblast model has been extensively used to characterize
G0 cells, as well as cells in various phases of the cell
cycle (18). Our cell cycle analysis was consistent with previous
reports in that over 90% of HFF in medium deprived of FBS for 24 h were in G0, and after 2.5% FBS treatment cells entered
into S phase at 16 h, and over 50% of cells were in S phase at
24 h (19). At several time points, chosen by flow cytometry to
best represent the individual phases of the cell cycle, 0 h (G0), 4 h (G1), 24 h (S), and 42 h (G2/M), cells were pretreated with a representative
therapeutic concentration of NaS (10 µM) for 30 min
before adding PMA (100 nM), IL-1
(1 ng/ml), or TNF
(1 ng/ml). COX-2 mRNA and protein expression were determined 2 and
4 h post-treatment by Northern and Western blot analysis, respectively. NaS inhibited COX-2 mRNA (Fig.
1A) and protein levels (Fig.
1B) induced by PMA in serum-starved G0 cells,
but the inhibitory action was no longer apparent at 4, 24, and 42 h after 2.5% FBS treatment. Similarly, IL-1
-induced COX-2 protein
expression was inhibited by NaS in serum-deprived G0 cells
but not in cells treated with 2.5% FBS for 24 h (Fig.
1C). In contrast, TNF
-induced COX-2 protein expression
was not inhibited by NaS in G0 or S phase cells (Fig.
1C). In subsequent experiments, the inhibitory effects of NaS on PMA induced expression of COX-2 at earlier time points after
2.5% FBS addition was evaluated. NaS inhibited COX-2 mRNA and
protein by ~50% in G0 cells. However, after only 1 h of exposure to 2.5% FBS, NaS was no longer able to inhibit COX-2
mRNA and protein (Fig. 1, D and E). We have
previously shown that NaS and aspirin inhibited PMA- or IL-1
-induced
COX-2 promoter activity conferred by a core promoter fragment
891/+9
(16). Here we show that salicylate suppressed COX-2 promoter activity
in a G0-restricted manner (Fig.
2). As NaS is only effective in
G0 cells all subsequent experiments to determine its
mechanism of action were performed in the absence of serum unless
otherwise stated.
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Fig. 1.
Cell cycle-dependent effect of
NaS on COX-2 mRNA (A and D) and
COX-2 protein (B, C, and
E) induced by PMA, IL-1 , or
TNF
in HFF cells. Serum-starved HFF (0 h)
were treated with 2.5% FBS, which drives the cells into the cell
cycle. 30 min before time points determined by flow cytometry to best
represent each phase of the cell cycle, cells were washed and incubated
with fresh serum-free medium containing 10 µM NaS followed by addition of PMA
(100 nM), IL-1
(1 ng/ml), or TNF
(1 ng/ml) 30 min
later. COX-2 mRNA and protein expression were determined 2 h
and 4 h post-treatment by Northern and Western blot analysis,
respectively. A and D, the upper panel
shows a representative Northern analysis of the effect of NaS (10 µM) on COX-2 mRNA induced by PMA (100 nM). The lower panel shows the concurrent
expression of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) as the internal control. Densitometry results are
the ratio of COX-2/GAPDH mRNAs and are expressed as the mean ± S.E. of four separate experiments. B and E
show representative Western blot analysis (10 µg of total protein) of
the effect of NaS on PMA and (C) IL-1
(1 ng/ml)- or
TNF
(1 ng/ml)-induced COX-2 protein expression. Densitometry results
are expressed as the mean ± S.E. of three separate
experiments.
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Fig. 2.
Effect of NaS on PMA-induced luciferase
activity of a COX-2 promoter ( 891/+9) transiently expressed in
subconfluent HFF cells. The cell cycle was activated by the
addition of 2.5% FBS as for Fig. 1. NaS (10 µM) was
added 30 min prior to the addition of PMA (100 nM) at 0, 1, 2, 4, and 24 h post-serum. After 6 h of treatment, luciferase
activity was determined, and basal activity was subtracted. Each
bar represents mean ± S.E. of three separate
experiments. Black and white bars are without and
with NaS treatment, respectively. RLU, relative light
units.
B-mediated COX-2 Promoter
Activation--
The cis acting elements in the COX-2 promoter region
that are required for the transcriptional inhibition by salicylate were determined using fragment
891/+9 and its deletion or site-directed mutants (17) cloned into plasmid pGL3 and transiently expressed in
subconfluent HFF. The transfected cells were pretreated for 30 min with
NaS (10 µM) before the addition of PMA (100 nM), IL-1
(1 ng/ml), or TNF
(1 ng/ml) for 6 h at
which time cells were harvested and lysed, and luciferase activity was
determined. NaS retained its inhibitory action on PMA- and
IL-1
-induced promoter activity by 5' deletion mutants until the
96/+9 construct when neither PMA nor IL-1
stimulated the promoter
activity (Fig. 3, A and
B), whereas TNF
lost its stimulatory effect on the
362/+9 construct (Fig. 3C). Site-directed mutation of the
C/EBP site reduced PMA- and IL-1
- but not TNF
-induced promoter
activity. Neither the residual promoter activity seen with PMA or
IL-1
nor the promoter activity of the mutant induced by TNF
was
affected by salicylate (Fig. 3D). By contrast, site-directed
mutagenesis of NF-
B sites abolished the effect of TNF
but not PMA
or IL-1
(Fig. 3E). Mutation of NF-
B sites also had no
effect on salicylate inhibition of PMA- or IL-1
-induced COX-2
promoter activity (Fig. 3E). Taken together these results
suggest that the C/EBP site at
132/
124 is critically involved in
PMA- and IL-1
-induced COX-2 expression and salicylate suppression.
NF-
B sites at
447/
438 and
222/
213 are critical for
TNF
-induced COX-2 expression consistent with reported results (20).
However, neither NF-
B site is involved in promoter induction by PMA
and IL-1
or suppression by salicylate.
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Fig. 3.
Effect of NaS (10 µM) on PMA (100 nM)-,
IL-1 (1 ng/ml)-, or TNF
(1 ng/ml)-induced luciferase activity conferred by a 5' flanking
DNA fragment (
891/+9) and its 5' deletion mutants
(A-C), C/EBP site mutant (D), or
NF-
B site mutants (E).
Cells were serum-deprived for 24 h before treatment in serum-free
medium. After subtraction of the basal activity, the data are expressed
as the mean ± S.E. of three separate experiments.
White and black bars denote without and with
salicylate treatment, respectively. Dark ovals and
dark squares represent mutated C/EBP and NF-
B sites,
respectively.
Binding to the COX-2 Promoter--
The 5'
deletion mutation analysis suggested that the region
193 to
97,
containing an AP-2 and C/EBP/NF-IL6 site, is required for PMA-
or IL-1
-induced promoter activity, whereas TNF
requires the
region
447 to
438 containing a putative NF-
B site for optimal activity. EMSA were performed using an AP-2, C/EBP, or NF-
B
probe. AP-2 binding was unaffected by any of the stimuli (data
not shown). Concordant with the reporter assays, IL-1
and PMA but
not TNF
increased complex formation between C/EBP probe and nuclear
extract proteins (Fig. 4A,
left panel, and B). The specificity of binding is
demonstrated by the inhibition of the PMA-induced complex formation by
a 5- to 100-fold molar excess of unlabeled C/EBP wild type probe but
not by a 50-fold molar excess of unlabeled C/EBP mutant probe (Fig.
4A, right panel). NaS significantly reduced C/EBP binding activity induced by PMA and IL-1
(Fig. 4, A and
B) but not TNF
(Fig. 4B). A structural
analogue 3-OH benzoic acid failed to affect PMA-induced complex
formation (Fig. 4C). Hence, the inhibitory effect of
salicylate (2-OH benzoic acid) is selective and not common to all
substituted benzoic acids. The PMA-induced complex was only
supershifted in the presence of an anti-C/EBP
antibody but was
unaffected by antibodies against other C/EBP isoforms (Fig.
5). TNF
and IL-1
but not PMA
increased NF-
B binding that was insensitive to NaS inhibition (Fig.
6, left panel). The
specificity of binding is demonstrated by the inhibition of the
TNF
-induced complex formation by a 50-fold molar excess of unlabeled
NF-
B wild type probe (Fig. 6, right panel). C/EBP
full-length (46 kDa) and C/EBP
(40 kDa) proteins were expressed constitutively in nuclear extract and were not altered by PMA treatment
(Fig. 7). The truncated C/EBP
isoform
liver inhibitory protein was barely detectable and was not altered by
PMA treatment (data not shown). C/EBP
, C/EBP
, and C/EBP
were
undetectable (data not shown). To determine whether the increased
binding is because of phosphorylation of C/EBP
, control and
PMA-treated nuclear extracts were incubated with alkaline phosphatase
(10 units) for 30 min before being applied to EMSA. Alkaline
phosphatase treatment reduced the complex formation in both control and
PMA-treated HFF cells (Fig. 8).
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Fig. 4.
Effect of NaS (10 µM) or 3-OH benzoic acid (10 µM) on the complex formed by the
C/EBP/NF-IL6 DNA probe and nuclear extract proteins from serum-deprived
HFF cells treated with (A) PMA (100 nM)
(left panel), (B)
IL-1 (1 ng/ml) or TNF
(1 ng/ml), or (C) PMA (100 nM).
Specificity of binding was determined by the addition of a 5-100-fold
molar excess of wild type (WT) probe or a 50-fold molar
excess of mutant probe (A, right panel). The
densitometry shown in B was the mean ± S.E. of five
separate experiments and that shown in C was the mean ± S.E. of three separate experiments.
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Fig. 5.
Supershift assay shows complex of
C/EBP with DNA probe. Nuclear extracts
were prepared from serum-deprived HFF cells treated with PMA (100 nM). This figure is representative of five separate
experiments with similar results. Ab denotes specific
antibodies against isoforms of C/EBP.
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Fig. 6.
EMSA showing the effect of NaS (10 µM) on binding of nuclear extract
proteins of serum-deprived HFF treated with PMA (100 nM),
IL-1 (1 ng/ml), or TNF
(1 ng/ml) to a consensus NF-
B
oligonucleotide probe (left panel). Specificity
of binding was determined by the addition of a 50-fold molar excess of
wild type (WT) probe (right panel). Densitometry
is expressed as the mean ± S.E. of three separate
experiments.
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Fig. 7.
Western blot analysis of
C/EBP (left panel) and
C/EBP
(right panel) protein
expression in nuclear extracts of serum-deprived HFF treated without or
with PMA (100 nM) or PMA + NaS (10 µM). Only the full-length C/EBP
is shown. C/EBP
, -
, and -
isoforms were undetectable under
these conditions (50 µg of total protein). Densitometry is expressed
as the mean ± S.E. of three separate experiments.
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Fig. 8.
Alkaline phosphatase (Alk
Phos; 10 units) abolished binding of nuclear extract
proteins with C/EBP probe in vehicle or PMA (100 nM)-treated serum-deprived HFF. This EMSA figure is
representative of three experiments with similar results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-mediated transactivation of proinflammatory genes.
Our findings indicate that salicylate at a pharmacological concentration has no effect on NF-
B transactivation induced by TNF
, in contrast to the potent anti-NF-
B effects exerted by suprapharmacological concentrations (>5 mM).
Concentrations higher than 5 mM nonspecifically inhibit
many cellular kinases (15). This renders NF-
B as an unlikely target
for the pharmacological actions of salicylate. We propose that the
actions of salicylate are mediated instead by inhibition of C/EBP
binding and transactivation. Several pieces of evidence support this
proposal: 1) deletion or mutation of the C/EBP site abolishes the
stimulatory effect of PMA and IL-1
and concomitantly, the inhibitory
effect of salicylate; (2) salicylate inhibited the binding of C/EBP
to the C/EBP site; and (3) salicylate had no effect on TNF
-induced
NF-
B binding or COX-2 promoter activity conferred by wild type or
C/EBP mutant constructs. C/EBP
is a pleiotropic transactivator for myriad genes involved in inflammation, cell differentiation, and the
immune response (21-27). In this study, we have established the role
of C/EBP
in human COX-2 induction by PMA and IL-1
. C/EBP
has
been reported to be essential for COX-2 induction in murine cells in
response to a number of stimuli (28). Thus, salicylate blockade of
C/EBP
-mediated COX-2 expression provides a plausible explanation for
the pharmacological actions of aspirin and other salicylate compounds.
Furthermore, the action of salicylate may be extended to other
C/EBP
-mediated genes, and as such, our findings have a major impact
on the conceptual understanding of the actions of salicylate and on new
drug design and discovery.
is a member of the C/EBP family of basic leucine zipper
transcription factors (27). It forms homodimers and heterodimers with
other C/EBP isoforms to transactivate or repress promoter activity.
There are several C/EBP
isoforms comprising the full-length and
three truncated forms that include liver inhibitory protein, which is
considered as a dominant negative mutant of the full-length C/EBP
(29). In this study, the results indicate that quiescent HFF express
predominantly the full-length C/EBP
. C/EBP
was also detected, but
its role in regulating COX-2 expression is unclear. C/EBP
levels are
not altered by PMA, IL-1
, or salicylate. C/EBP
binding activity
has been previously shown to be enhanced by phosphorylation mediated by
several kinase pathways such as protein kinase C (30, 31),
mitogen-activated protein kinase (32), p90 ribosomal S6 kinase 2 (33),
and Ca2+-calmodulin-dependent protein kinase II
(34). These kinases directly or indirectly phosphorylate different
threonine or serine residues suggesting multiple mechanisms for
activating C/EBP
. Many of the activities of proinflammatory
mediators are mimicked by PMA that activates protein kinase C. We
therefore determined whether PMA-induced C/EBP
binding enhancement
was attributable to phosphorylation by treating nuclear extracts with a
general phosphatase (alkaline phosphatase), and the results confirm
that PMA-stimulated C/EBP
binding activity depends on
phosphorylation of this transactivator. Our results further suggest
that it is mediated via a protein kinase C-dependent
pathway.2 It is likely
that NaS blocks PMA-induced C/EBP
binding activity by targeting a
downstream kinase in the protein kinase C signaling pathway. Salicylate
has been shown to effect several protein kinases that include p90
ribosomal S6 kinase 2 (35), p38 mitogen-activated protein kinase (36),
p42/p44 mitogen-activated protein kinase (37), and c-Jun N-terminal
kinase (38). These studies were performed using suprapharmacological
concentrations of NaS, and as such, these kinases are not likely
involved in the specific effects demonstrated in this study. However, a
recent report demonstrated that NaS at pharmacological concentrations
inhibited PMA-induced phosphorylation of p70 S6 kinase (39). Hence, it
is possible that p70 S6 kinase may occupy an important position in
mediating C/EBP
phosphorylation and increasing its binding activity
and is the target of salicylate inhibition. This proposal is being investigated in our laboratory.
is confined to serum-deprived fibroblasts. Results from our recent work (19) show that
COX-2 is expressed more abundantly in serum-starved cells than
serum-treated cells suggesting that COX-2 expression is controlled by
an endogenous mechanism that is operative in proliferating cells.
Quiescent cells in vivo may be the major COX-2-expressing
cells and play a key role in the inflammatory response. That salicylate
suppresses COX-2 expression only in G0 cells is consistent
with its anti-inflammatory actions in vivo. However, the
mechanism that underlies the restricted efficacy of NaS is not
understood. We have excluded the possibility that the lack of effect,
after addition of 2.5% FBS, is because of sequestration of salicylate
by serum proteins as exposure of NaS to serum prior to its addition to
serum-deprived cells did not alter its inhibitory effect (data not
shown). However, it is possible that after addition of 2.5% FBS there
may be an alteration in the expression of C/EBP isoforms, and this
alteration abrogates the inhibitory action of salicylate. Switch of
C/EBP isoform expression has been shown to occur in liver cell
differentiation (40), but there is no published data on changes in
C/EBP isoforms after serum treatment. Certain isoforms of C/EBP, such
as liver inhibitory protein and C/EBP homologous protein, bind C/EBP
to form a heterodimer and inhibits C/EBP
binding to DNA and
transactivation of the promoter (41, 42). Whether these isoforms are
regulated in a cell cycle-dependent manner is unknown.
G0-restricted salicylate action may be because of an
alteration in C/EBP
phosphorylation in cycling cells. C/EBP
phosphorylation is probably regulated by coordinated activities of
kinases and phosphatases. This delicate balance may be altered by serum
or cell cycle progression. C/EBP
is located in cytosol and
translocated into the nucleus after stimulation with proinflammatory
cytokines (43, 44). It should be interesting to determine whether this
translocation process is altered by serum and cell cycle progression.
Understanding why NaS is only effective in G0 cells is
critical, as it will potentially provide a target for novel therapeutic
compounds with broad implications for the treatment of inflammation,
tissue injury, and tumor growth.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants NS-23327 and HL-50675 (to K. K. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Vascular Biology
Research Center and Division of Hematology, University of Texas-Houston Medical School, 6431 Fannin St., Houston, TX 77030. Tel.:
713-500-6801; Fax: 713-500-6812; E-mail:
Kenneth.K.Wu@uth.tmc.edu.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M011147200
2 M. A. Saunders and K. K. Wu, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
COX, cyclooxygenase;
C/EBP, CCAAT/enhancer-binding protein;
HFF, human
foreskin fibroblast;
NaS, sodium salicylate;
NF-B, nuclear factor
B;
PMA, phorbol 12-myristate 13-acetate;
IL, interleukin;
TNF
, tumor necrosis factor
;
FBS, fetal bovine serum;
EMSA, electrophoretic mobility shift assay.
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REFERENCES |
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