From the Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322
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ABSTRACT |
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Endothelial cell surface expression of VCAM-1 is
one of the initial steps in the pathogenesis of atherosclerosis.
The inflammatory response transcription factor nuclear factor
(NF)-B plays an important role in the regulation of
VCAM-1 expression by various stimuli including tumor
necrosis factor (TNF)-
. Other transcription factors may modulate
this response through interaction with NF-
B factors. Since
c-Fos/c-Jun (activating protein-1 (AP-1)) are expressed in vascular
endothelium during proinflammatory conditions, we investigated the role
of AP-1 proteins in the expression of VCAM-1 by TNF-
in
SV40 immortalized human microvascular endothelial cells (HMEC). TNF-
induced expression of both early protooncogenes, c-fos and
c-jun. The ability of TNF-
to activate the
B-motif (
L-
R)-dependent VCAM-1
promoter-chloramphenicol acetyltransferase (CAT) reporter gene
lacking a consensus AP-1 element was markedly inhibited by
co-transfection of the expression vector encoding c-fos
ribozyme, which decreases the level of c-fos by degrading c-fos mRNA, or c-fos or
c-jun oligonucleotides. Conversely, co-transfection of c-Fos and c-Jun encoding expression vectors potentiated the p65/NF-
B-mediated transactivation of the VCAM-1
promoter-CAT reporter gene. Furthermore the c-Fos encoding expression
vector potentiated by 2-fold the transactivation activity of a chimeric transcriptional factor Gal/p65 (containing the transactivation domain
of p65 and the DNA binding domain of the yeast transcriptional factor
Gal-4). Consistent with the promoter studies, curcumin and NDGA,
inhibitors of AP-1 activation, markedly inhibited the ability of
TNF-
to activate the expression of VCAM-1 mRNA
levels at concentrations that did not inhibit the activation of
NF-
B. In gel mobility supershift assays, the antibodies to c-Fos or c-Jun inhibited the binding of TNF-
-activated nuclear NF-
B to the
L-
R, suggesting that both c-Fos and c-Jun interacted with NF-
B. These results suggest that AP-1 proteins may mediate the effect of TNF-
in the regulation of VCAM-1 expression
through interaction with NF-
B factors in endothelial cells.
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INTRODUCTION |
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Several proinflammatory agents (e.g. tumor necrosis
factor- (TNF-
),1
interleukin-1
, and lipopolysaccharide) induce VCAM-1 gene
expression in endothelial cells through activating the redox-sensitive
transcription factor nuclear factor-
B (NF-
B) (1-3). Promoter
studies suggest that induction of the VCAM-1 gene by NF-
B
factors requires two NF-
B motifs (present at
63 and
77) (1, 2).
Since activated NF-
B is present in atherosclerotic lesions (4) and
is involved in the regulation of several proinflammatory responsive
genes in endothelial cells (5), it is considered to be one of the major
vascular inflammatory transcriptional factors.
Activation protein-1 (AP-1, c-Fos/c-Jun) designates another class of transcription factors that mediate inflammatory responses by various cytokines and growth factors in different cell types (6, 7). Inhibition of c-fos and/or c-jun by various agents (e.g. nordihydroguatiaretic acid (NDGA), curcumin, and N-acetylcysteine) has been shown to inhibit inflammatory responses in different tissues (6, 7). AP-1 proteins are dimers of the Fos (e.g. c-Fos, FosB, and Fra1) and the Jun (e.g. c-Jun, JunD, and JunB) families (8-15) generated through interaction between leucine zipper motifs (16-21). Members of the Fos family cannot make stable homodimers and therefore cannot act as transcriptional activators by themselves. AP-1 proteins are regulated by several growth factors, cytokines, and by various agents that induce oxidative stress (6, 7). Since several of these agents and the conditions that activate AP-1 proteins are present in atherogenic conditions, it is likely that AP-1 proteins play a role in the regulation of this inflammatory disease of the vasculature.
This study investigated the role of AP-1 proteins in the regulation of
the inflammatory response gene VCAM-1. We found that AP-1
proteins mediate some of the effects of TNF- in the regulation of
endothelial VCAM-1 expression. At least part of these
effects of AP-1 is independent of direct binding of AP-1 to its cognate enhancer element and is likely effected through direct interactions with NF-
B factors.
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MATERIALS AND METHODS |
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Cell Culture and Reagents--
Human dermal microvascular
endothelial cells (HMEC) were immortalized with simian virus 40 product, large T antigen (22). HMEC cells were generously provided by
E. W. Ades (Centers for Disease Control and Prevention, Atlanta,
GA) and maintained in EBM medium obtained from Clonetics (San Diego,
CA) supplemented with 15% fetal bovine serum (Atlanta Biological,
Norcross, GA), 10 ng/ml epidermal growth factor (Intergen, Purchase,
NY), and 1 µg/ml hydrocortisone (Sigma). Human recombinant TNF-
was obtained from Boehringer Mannheim. Curcumin and NDGA were purchased
from Sigma. All other reagents were of reagent grade.
CAT Assay--
One day prior to transfection, HMEC cells were
split at the ratio that would give 30-40% confluence. The
transfection was done by the calcium phosphate co-precipitation
technique. Overexpression studies were performed by co-transfection of
the expression vectors encoding the p65 subunit of NF-B and/or c-Fos
or c-Jun with the reporter gene p85VCAMCAT (5-10 µg) into HMEC cells
as described previously (23). The promoterless plasmid, poLUC (24), was used for adjusting the amount of transfected DNA. The cells were harvested and cell extracts were prepared by three cycles of rapid freeze-thaw in 0.25 M Tris, pH 8.0. Protein content was
determined using the Bradford (25) technique. The same amounts of
proteins were assayed for chloramphenicol acetyltransferase (CAT)
activity according to standard protocols (26). The CAT activity was
expressed as percent of chloramphenicol converted to acetyl
chloramphenicol. Acetylated and unacetylated forms of chloramphenicol
were separated on thin layer chromatography, and their amounts were
quantified by phosphorimaging. Each assay was performed in duplicate or
triplicate, and the results reported are representative of at least two
separate experiments.
Expression Vectors and CAT Reporter Genes--
The eucaryotic
expression vector, cytomegalovirus-p65, contains the p65 cDNA
cloned between a cytomegalovirus promoter, -globin intron, and
simian virus 40 poly(A) signal (27). Gal/p65 contains the DNA binding
domain of the yeast transcription vector Gal-4 and the transactivation
domain of p65. pGal-CAT is a reporter gene containing four tandem
elements of Gal-4 attached to the reporter gene CAT. These vectors were
generous gifts of Dr. C. Rosen (Human Genome Sciences, Rockville, MD).
The reporter gene, p85VCAMCAT, contains coordinates
85 to +12 of the
human VCAM-1 promoter (1). These reporter genes were
generous gifts of Dr. D. Dean (Washington University, St. Louis, MO).
The expression vectors encoding c-Fos and c-Jun protein were kindly
provided by Dr. Tjian, University of California, San Francisco. The
expression vector encoding c-fos ribozyme (28) was a
generous gift of Dr. Scanlon, National Medical Center, City of Hope,
California.
Nuclear Extract Preparation--
Confluent HMEC cells were
exposed to TNF- (100U/ml) for 1-2 h. Nuclear extracts were prepared
by a modification of the method of Dignam et al. (29).
Briefly, after washing with phosphate buffered saline, cells were
centrifuged and the cell pellet suspended in 500 µl buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, and 1.0 mM DTT). After
recentrifugation, the cells were resuspended by gentle pipetting up and
down in 80 µl of buffer A containing 0.1% Triton X-100. After
incubating for 10 min at 4 °C, the homogenate was centrifuged, and
the nuclear pellet was washed once with buffer A and resuspended in 70 µl of buffer C (20 mM HEPES, pH 7.9, 25% (v/v) glycerol,
0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT). This suspension was
incubated for 30 min at 4 °C followed by centrifugation at
20,000 × g for 10 min. The resulting supernatant
(nuclear extract) was stored at
70 °C. Protein concentrations were
determined by the Bradford (25) method. To minimize proteolysis, all
buffers contained 1.0 mM phenylmethylsulfonyl fluoride,
aprotinin (10 mg/ml), leupeptin (10 mg/ml), and antipain (10 mg/ml).
Gel Shift Assays--
The oligonucleotide containing L-
R
of the VCAM-1 promoter (VCAM-1 wild type oligo)
was synthesized. Its sequence is as follows: 5'-CTGCCCTGGGTTTCCCCTTGAAGGGATTTCCCTCCGCCTCTGCAACAAGCTCGAGATCCTATG-3'. The sequences of
L and
R are double underlined. The single
underlined sequences represent an unrelated tail sequence added to
serve as a template for synthesis of the double-stranded DNA. To
prepare double-stranded DNA, first an oligonucleotide
5'-CATAGGATCTCGAGC-3' (complementary to the 3' unrelated tail,
single underlined sequence) was annealed to VCAM-1 wild type
oligo. The second strand was extended with DNA polymerase (Klenow
fragment) in a reaction mixture containing 50 µCi of
[32P]dCTP and 0.5 mM of cold dATP, dGTP, and
dTTP. The reaction was followed by the addition of 0.5 mM
cold dCTP to ensure completion of the second strand. Unincorporated
nucleotides were removed by column chromatography over a Sephadex G-50
column. The DNA binding reaction was performed at 30 °C for 15 min
in a volume of 20 ml, which contained 225 µg/ml bovine serum albumin,
1.0 × 105 cpm 32P-labeled probe, 0.1 mg/ml poly(dI-dC), and 15 ml of binding buffer (12 mM
HEPES, pH 7.9, 4 mM Tris, 60 mM KCl, 1 mM EDTA, 12% glycerol, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride). After the binding reaction, the samples were subjected to electrophoresis in 1 × Tris-glycine buffer using 4% native polyacrylamide gels.
Northern Blot Analysis-- Total cellular RNA was isolated by a single extraction using an acid guanidium thiocyanate-phenol-chloroform (32). Total cellular RNA (10 µg) was size fractionated using 1% agarose formaldehyde gels in the presence of 1 µg/ml ethidium bromide. The RNA was transferred to a nitrocellulose filter and covalently linked by ultraviolet irradiation using a Stratlinker UV cross-linker (Stratagene, La Jolla, CA). Hybridizations were performed at 68 °C for 1 h in 5 × SSC (1 × = 150 mM NaCl, 15 mM sodium citrate), 1% sodium dodecyl sulfate, 5 × Denhardt's solution, 50% formamide, 10% dextran sulfate, and 100 µg/ml sheared denatured salmon sperm DNA. Approximately 1-2 × 106 cpm/ml of labeled probe (specific activity, >108 cpm/µg of DNA) was used per hybridization. Following hybridization, filters were washed with a final stringency of 0.2 × SSC at 65 °C. The nitrocellulose membrane was soaked for stripping the probe with boiled water prior to rehybridization. mRNA bands were quantified using PhosphorImager.
32P-Labeled DNA probes were prepared using the random primer oligonucleotide kit from Stratagene. The VCAM-1 probe was a HindIII-XhoI fragment of the cDNA consisting of nucleotide 132-1814 (33). The ICAM-1 probe was an EcoRI fragment of human cDNA (34). ![]() |
RESULTS |
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TNF- Induces c-fos and c-jun in HMEC Cells--
Because TNF-
is known to induce c-fos and c-jun in various
cell types (7, 35, 36), we tested whether similar responses occur in
HMEC. Confluent HMEC cells were treated with TNF-
(100 units/ml) for
15-120 min and RNA was prepared for analysis of c-fos and
c-jun expression. As shown in Fig.
1, TNF-
treatment produced a
severalfold induction of c-fos and c-jun
mRNA. c-fos mRNA was elevated 15 min after TNF-
treatment, peaked at nearly 30 min, and return to basal level at 2 h, kinetics similar to c-fos induction by serum and growth
factors (37-39). The kinetics of induction of c-jun by
TNF-
was similar to that observed for c-fos.
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Inhibition of c-fos or c-jun blocks TNF- Activation of the
VCAM-1 Promoter--
TNF-
induces p85VCAMCAT (a minimal
VCAM-1 promoter-coordinates
85 to +12, containing two
NF-
B motifs,
L-
R, but lacking a consensus AP-1 binding site,
attached to the reporter gene CAT) through activating NF-
B proteins
(1-3, 23). We investigated whether endogenous c-Fos contributes to the
TNF-
-mediated transactivation of p85VCAMCAT. The endogenous level of
c-fos was inhibited by co-transfection of an expression
vector encoding anti c-fos ribozyme that specifically
degrades c-fos mRNA (28). As expected, TNF-
(100 units/ml) activated p85VCAMCAT severalfold above the basal levels (Fig.
2, lanes 3 and 4)
in transient transfection assays in HMEC cells compared with untreated
cells (lanes 1 and 2). In cells co-transfected
with the expression vector encoding c-fos ribozyme, TNF-
was unable to transactivate p85VCAMCAT (lanes 5 and
6). Under similar conditions the expression vector
expressing c-fos ribozyme had no effect on the constitutive
activity of the SV40 promoter-driven reporter gene CAT, pSV2CAT
(compare lanes 7 and 8 with lanes 9 and 10). Under similar conditions antisense oligonucleotides
c-fos or c-jun (30, 31) inhibited the ability of
TNF-
to activate p85VCAMCAT nearly 80% (data not shown). These results suggest that endogenous c-Fos and c-Jun contribute to TNF-
-mediated transactivation of p85VCAMCAT.
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Co-expression of the Expression Vectors Encoding c-Fos or
c-Jun Potentiates the p65-mediated Transactivation of
p85VCAMCAT--
p65 is a member of the rel family and a
component of the NF-B heterodimers. p65 potently transactivates
p85VCAMCAT (23, 40). We investigated whether overexpression of c-Fos
and c-Jun potentiates the p65-mediated transactivation of p85VCAMCAT.
Eucaryotic expression vectors encoding c-Fos or c-Jun and/or p65 were
co-transfected with p85VCAMCAT into HMEC cells and the promoter
activity assessed. Consistent with previous observations, co-expressed
p65 transactivated p85VCAMCAT (Fig. 3)
(23, 40). Co-expression of the expression vectors encoding c-Fos and
c-Jun dose-dependently potentiated the p65-mediated
transactivation of p85VCAMCAT with maximum potentiation above 30-fold
at 5 µg of the each expression vector (Fig. 3A). When
co-transfected individually, both c-fos and c-jun
expression vectors also potentiated the p65-mediated transactivation of
p85VCAMCAT (Fig. 3B). These results suggest that both c-Fos
and c-Jun functionally interact with the p65 subunit of NF-
B to
potentiate p65-mediated transactivation of p85VCAMCAT.
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Co-expression of the Expression Vector Encoding c-Fos Potentiated
the Gal/p65-dependent Transactivation of
pGal-CAT--
p65 has a DNA binding domain and two
transactivation domains (41). The transactivation domains of p65
require co-factor proteins for transactivation activity (42). We
investigated whether c-Fos could interact with the transactivation
domains of p65. We utilized an expression vector encoding chimeric
transcriptional factor Gal/p65 and a Gal/p65 driven reporter gene
pGal-CAT. Gal/p65 contains a DNA binding domain of the yeast
transcriptional factor Gal-4 and transactivation domains of p65 subunit
of NF-B. pGal-CAT contains four multimerized DNA elements of the
transcriptional factor Gal-4 attached to a reporter gene, CAT.
Co-transfected Gal/p65 potently transactivated pGal-CAT in transient
transfection assays (42) (Fig. 4).
Co-transfection of the expression vector encoding c-Fos potentiated the
Gal/p65-mediated transactivation 2-fold. Co-transfection of the
expression vector encoding the p65 subunit of NF-
B completely
inhibited the Gal/p65-mediated transactivation of pGal-CAT. This was
likely through absorbing the co-factors interacting with the
transactivation domain of Gal/p65. These results suggest that c-Fos
interacts with transactivation domains of p65.
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Antibodies to c-Fos and c-Jun Inhibit the Binding of
TNF--activated NF-
B-like Proteins to the NF-
B Motifs
(
L-
R) of the VCAM-1 Promoter--
To determine whether c-Fos
and/or c-Jun were part of the NF-
B-like complex, electrophoretic
mobility shift assays were performed. Consistent with the previous
observations in human umbilical vein endothelial cells (23), TNF-
induces a major NF-
B-like complex A1 in the nucleus of HMEC cells
(Fig. 5, lanes 2 and
6) compared with the control (lanes 1 and
5). Preincubation of nuclear extracts with antibodies to
either c-Fos or c-Jun inhibited the binding of NF-
B-like proteins to
the
L-
R (lanes 3 and 7). To test the specificity of this interaction, the nuclear extracts were incubated with antibodies to either c-Fos or c-Jun in the presence of the peptides to which the antibodies were raised. In the presence of the
peptide to which the antibodies c-Fos were raised, the antibody c-Fos
was unable to inhibit the binding of NF-
B to the
L-
R motif
(lane 4). Similarly, in the presence of c-Jun peptide, the
antibody c-Jun was unable to inhibit the binding of the NF-
B-like proteins to the
L-
R motif (lane 8). These results
suggest that both c-Fos and c-Jun are part of the NF-
B proteins in
the nuclear extract of HMEC cells activated by TNF-
.
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Curcumin Inhibits the Ability of TNF- to Activate the Expression
of VCAM-1 without Affecting the Activation of NF-
B--
To explore
the role of c-Fos/c-Jun in the TNF-activation of endogenous
VCAM-1 expression, we investigated the effect of curcumin, an inhibitor of c-jun/c-fos expression (43). We
investigated the dose-dependent effect of curcumin on the
expression of the VCAM-1 gene by TNF-
. Pretreatment of
HMEC cells with 10 µM curcumin inhibited
TNF-
-activation of VCAM-1 but not ICAM-1 expression at
the mRNA levels (Fig. 6A).
At the same concentration, curcumin did not inhibit the
TNF-
-activation of NF-
B (Fig. 6B). These results
suggest that curcumin at 10 µM inhibits the TNF-
activation of VCAM-1 through a mechanism independent of
NF-
B activation.
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NDGA Inhibits the Expression of VCAM-1 by TNF- without
Inhibiting the Activation of NF-
B--
NDGA inhibits several of the
effects of TNF-
by inhibiting c-fos expression (7). We
investigated the effect of NDGA on the TNF-
-activation of
VCAM-1 expression in HMEC cells. Pretreatment of the cells
with NDGA at all concentrations tested (1-10 µM) inhibited the TNF-
-activation of VCAM-1 expression (Fig.
7A). Pretreatment of the cells
with NDGA at the same concentrations did not inhibit the TNF-
activation of NF-
B (Fig. 7B). These results suggest that
activation of the lipoxygenase pathway and c-Fos activation may
mediate, at least in part, the effect of TNF-
in regulating the
expression of VCAM-1 in HMEC cells.
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DISCUSSION |
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We demonstrate that AP-1 proteins c-Fos/c-Jun are involved in
TNF--activation of VCAM-1 expression. At least a part of
the effect of c-Fos/c-Jun is mediated through interaction with NF-
B, and is independent of the AP-1 element present on the VCAM-1
promoter. Furthermore, we demonstrate that anti-inflammatory agents and inhibitors of c-Fos/cJun expression inhibit the TNF-
-activation of
VCAM-1 in HMEC cells through a pathway that is independent of NF-
B activation (Fig. 8). These
studies suggest that in addition to NF-
B, c-Fos/c-Jun also mediate
the effect of TNF-
in the regulation of VCAM-1
expression.
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Inhibition of endogenous c-Fos/c-Jun by the expression vector encoding
ribozyme c-fos (28) or by phosphorothioate antisense oligonucleotides c-fos or c-jun (30, 31)
inhibited the TNF-activation of 85VCAMCAT, a minimal NF-B-driven
VCAM-1 promoter-reporter gene lacking the AP-1 element.
These studies suggest that the effect of c-fos and
c-jun is mediated, at least in part, through interaction
with NF-
B factors. While it is tempting to speculate that antisense
c-fos and c-jun, and ribozyme c-fos
functions by decreasing the endogenous concentration of c-Fos or c-Jun
in the
L-
R-bound NF-
B complex, we have not directly assessed
this in these studies and thus cannot rule out an indirect effect of the antisense oligonucleotides and ribozyme c-fos on the
expression of other transcriptional factors.
Co-transfection of the expression vectors encoding c-Fos and c-Jun
dose-dependently potentiated the expression of the
VCAM-1 promoter by co-expressed p65. Since low levels of
basal c-Fos/c-Jun are present in various cell types and TNF-
activates both c-Fos/c-Jun and NF-
B, it is likely that the level of
proinflammatory response is determined by the interaction of these two
class of transcriptional factors.
Co-transfection of the expression vector encoding the p65 subunit of
NF-B completely inhibited the Gal/p65-driven activity of pGal-CAT in
HMEC cells as observed earlier in COS cells (42). These studies
suggested that certain cofactors such as bridging proteins, mediators,
or intermediary proteins that associate or interact with the
transactivation domain of p65 are necessary for transactivation by p65.
We investigated whether c-Fos or c-Jun could act as a co-factor in the
transactivation by p65. Consistent with the 85VCAMCAT studies,
co-transfection of the expression vector encoding c-fos potentiated the
Gal-p65 driven transactivation of pGal-CAT. Under similar conditions,
co-transfection of the expression vector encoding c-Jun did not
potentiate the transactivation activity of Gal/p65 suggesting that the
interaction of c-Jun with p65 may require its DNA binding domain. These
studies at least suggest that c-Fos could act as a cofactor through
interaction with the transactivation domain of p65 in HMEC cells.
AP-1 proteins can regulate the expression of several genes in various
ways. 1) Through binding to the AP-1 element, AP-1 proteins can
activate the transcription of various genes containing the AP-1
element; 2) after binding to their DNA element, AP-1 proteins can
synergize with other transcriptional factors and modulate the
transactivation mediated by them; and 3) independent of their DNA
binding element, AP-1 proteins can interact with other transcriptional factors and modulate the transactivation mediated by them (44-46). Since deletion or mutation of the AP-1 element present in the VCAM-1 promoter had no effect on activation of the
VCAM-1 promoter by TNF- (1, 2), the AP-1 element does not
appear to play a role in TNF-
-activation of the VCAM-1
promoter. This would suggest that the regulation by AP-1 proteins of
VCAM-1 is mainly through interaction with NF-
B factors
and is independent of the AP-1 element.
Curcumin inhibits transcriptional activation of the c-jun
gene by PMA and TNF- and blocks the increase in TRE binding activity of c-Jun/AP-1 protein (43, 47). Curcumin decreases TPA-induced nuclear
abundance of c-Fos protein through the induction of a hyperphosphorylated unstable form of c-Fos (48). Curcumin has also been
shown to inhibit the activation of NF-
B (49). In our experimental
conditions, curcumin (10 µM) blocked the
TNF-
-activation of VCAM-1 expression in HMEC cells
without inhibiting NF-
B activation. These studies suggest that
transcriptional factors other than NF-
B, such as c-Fos/c-Jun/AP-1,
may also play an important role in the regulation of VCAM-1
gene expression by TNF-
.
NDGA, an inhibitor of fos expression (7), inhibits the TNF-
activation of VCAM-1 expression without blocking NF-
B
activation. These results suggest that NDGA inhibited the
transactivation activity of NF-
B either by directly changing the
phosphorylation of nuclear NF-
B or by inhibiting the activity of
other factors that interact with NF-
B. In recent studies, the
phosphorylation of nuclear NF-
B was analyzed under conditions that
inhibited TNF-
activation of VCAM-1 expression but did
not inhibit nuclear translocation of NF-
B (50). These studies
suggest that factors other than phosphorylation of NF-
B are involved
in inhibiting the transactivation activity of NF-
B. Consistent with
these observation, our studies suggest that AP-1 proteins may play an
important role in regulating the activity of NF-
B.
The antibodies to c-Fos and c-Jun inhibited the binding of NF-B to
the
L-
R motif, as observed earlier with Ig/HIV
B motif (44).
Assuming that c-Fos and c-Jun are part of the
L-
R-bound NF-
B
complex, the antibodies to c-Fos or c-Jun could inhibit the binding of
NF-
B to
L-
R either by changing the conformation of the
DNA-binding domain of NF-
B or directly interfering with the binding
of NF-
B to
L-
R. The other possibility is that c-Fos and c-Jun
are not part of the NF-
B complex but that their presence in the
nucleus facilitates the binding of NF-
B to the NF-
B motif. These
results suggest that the TNF-
-activated signal transduction pathways
leading to activation of NF-
B and AP-1 may interact in the nucleus
to regulate VCAM-1 gene expression.
In conclusion, our studies have demonstrated that TNF- induces
85VCAMCAT (a minimal VCAM-1 promoter-CAT reporter gene) and expression of VCAM-1 gene through interaction of c-Fos and
c-Jun with NF-
B proteins, suggesting that the AP-1/NF-
B protein
complex may be one of the transactivating complexes induced by TNF-
in HMEC cells. Furthermore, c-Fos/c-Jun may play a role in the
regulation of VCAM-1 gene expression under conditions such
as growth state of the cell and shear stress that affect the levels of
c-Fos/c-Jun in endothelial cells.
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ACKNOWLEDGEMENTS |
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We are thankful to Kate W. Harris for editing the manuscript, and Lynn Olliff for technical support in preparing plasmids.
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FOOTNOTES |
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* This work was supported in part by an American Heart Association national grant-in-aid, an American Heart Association Georgia Affiliate grant-in-aid, and an Emory University research grant (to M. A.), and National Institutes of Health Grant PO1-HL48667 (to R. M. M. and R. Wayne Alexander).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: Division of
Cardiology, Dept. of Medicine, 1639 Pierce Drive, WMB#319, Emory
University, Atlanta, GA 30322. Tel.: 404-727-3415; Fax: 404-727-3330;
E-mail: mahmad{at}emory.edu.
§ This work was performed during tenure as an Established Investigator of the American Heart Association.
1
The abbreviations used are: TNF-, tumor
necrosis factor-
; CAT, chloramphenicol acetyltransferase; VCAM,
vascular cell adhesion molecule; DTT, dithiothreitol; HMEC,
SV40-transformed human microvascular endothelial cells; AP-1,
activating protein-1; NDGA, nordihydroguatiaretic acid; NF, nuclear
factor; ICAM, intercellular adhesion molecule.
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REFERENCES |
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