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and GR Differentially Down-Regulate the Expression of Nuclear Factor-
B-Responsive Genes in Vascular Endothelial Cells
Department of Molecular Medicine, Osaka University Graduate School of Medicine (C-4), Suita, Osaka 565-0871, Japan
Address all correspondence and requests for reprints to: Dr. Soji Kasayama, Department of Molecular Medicine, Osaka University Graduate School of Medicine (C-4), 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail: kasayama{at}imed3.med.osaka-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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activator fenofibrate and
the GR activator dexamethasone on TNF-stimulated expression of IL-6
and vascular cell adhesion molecule-1 in vascular endothelial cells.
Both fenofibrate and dexamethasone reduced TNF-induced IL-6
production in human vascular endothelial cells, but only fenofibrate
reduced TNF-stimulated vascular cell adhesion molecule-1 expression
in these cells. Transient transfection of bovine aortic endothelial
cells with an IL-6 promoter construct or a vascular cell adhesion
molecule-1 promoter construct revealed that fenofibrate inhibited
TNF-induced IL-6 promoter as well as vascular cell adhesion
molecule-1 promoter activities, whereas dexamethasone inhibited only
the former. EMSA demonstrated that both fenofibrate and dexamethasone
reduced nuclear factor-
B binding to its recognition site on the IL-6
promoter, but only fenofibrate reduced such binding to the vascular
cell adhesion molecule-1 promoter. Thus, down-regulation of nuclear
factor-B activity by PPAR occurs in both the IL-6 and vascular
cell adhesion molecule-1 genes, whereas that by GR occurs only in the
IL-6 gene in vascular endothelial cells. These results strongly suggest
the existence of a target gene-specific mechanism for the nuclear
receptor-mediated down-regulation of nuclear factor-B
activity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1. This up-regulation represents part of the inflammatory
process affecting vascular walls (1, 2, 3). To inhibit the
expression of these inflammatory molecules is therefore one of the
targets of antiinflammatory drugs. Glucocorticoids are thought to exert
their antiinflammatory effects at least partly through inhibiting the
expression of many cytokines, including TNF (4), IL-2
(5), IL-3 (6), IL-5 (7), IL-6
(8), and IL-8 (9). Furthermore, in the case
of vascular ECs glucocorticoids reportedly have a direct inhibitory
effect on the expression of adhesion molecules such as intercellular
adhesion molecule-1 (10), E-selectin (10, 11), and vascular cell adhesion molecule-1 (VCAM-1)
(12). Recent observations indicate that the GR can repress
the gene transcription of these inflammatory molecules by interaction
with transcription factors such as activating protein-1
(13, 14, 15) and nuclear factor-B (NF-B) (11, 16, 17, 18, 19).
Recently, other nuclear receptor activators, such as estrogens
(20, 21), progestins (22), retinoic acids
(23), and fibric acid derivatives (24), have
also been shown to inhibit the expression of several adhesion molecules
in cultured vascular ECs. These results suggest the potential basis
that these nuclear receptor activators use to exert their biological
effects at inflammatory sites on vascular walls in atherosclerosis and
cutaneous vascular diseases. There is little information, however,
about the precise molecular mechanisms by which these nuclear receptor
activators inhibit the expression of adhesion molecules in vascular
ECs. Several studies have indicated that the transcriptional inhibition
of the adhesion molecules is the result of the inhibition of NF-B
activity by the nuclear receptor activators (22, 23, 24).
NF-B is a widely expressed transcription factor that has a positive
regulatory effect on the expression of genes for several cytokines and
cell adhesion molecules (25). It is a dimer that typically
comprises p65 (RelA) and p50 subunit (26). In its
unactivated form, NF-B is retained in the cytoplasm through
interaction with the inhibitory protein IB (26). When
cells are stimulated with extracellular signals such as a
proinflammatory cytokine and an oxidative stressor, NF-B is
activated through phosphorylation and ubiquitination of IB
(inhibitor of NF-B), leading to proteolytic degradation
(26). Subsequently, NF-B is released from the
inhibitory protein and migrates to the nucleus, where it activates
transcription of target genes.
The promoters of both the IL-6 gene and the VCAM-1 gene contain binding
sites for NF-B, although the nucleotide sequences of these promoters
are not exactly the same (27, 28). In the study presented
here, we examined the effects of the PPAR activator and the GR
activator on the expression of IL-6 and VCAM-1 in human vascular
ECs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was
obtained from Dainippon Pharmaceutical (Osaka, Japan). Fenofibrate was
obtained from Kaken Pharmaceutical. Pioglitazone was
purchased from Takeda Pharmaceutical (Osaka, Japan). Dexamethasone was
obtained from Sigma (St. Louis, MO). RU486 was purchased
from BIOMOL Research Laboratories, Inc. (Plymouth Meeting,
PA).
Immunocytochemistry
After treatment with acetone/methanol (1:1, vol/vol), HUVECs
were incubated with rabbit antibody against GR or goat antibody against
PPAR (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
diluted 1:100, followed by incubation with FITC-conjugated second
antibody (Jackson ImmunoResearch Laboratories, Inc., West
Grove, PA) or Cy3-conjugated second antibody (Jackson ImmunoResearch Laboratories, Inc.), respectively. The specimens
were examined with an Axiovert 135 fluorescent microscope equipped with
LSM410 confocal microscopy (Carl Zeiss, Jena,
Germany).
For immunostaining of NF-B p65 protein, 2% paraformaldehyde-treated
HUVECs were incubated with 5% goat serum in PBS for 1 h and then
with antibody against p65 (Santa Cruz Biotechnology, Inc.)
diluted 1:200, followed by incubation with biotinylated antirabbit IgG
antibody. The next steps were performed with the use of
Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA).
Determination of secreted IL-6
HUVECs were plated onto 96-well collagen-coated dishes (2
x 104 cells/well) in the growth medium. The next
day, the culture medium was replaced by MCDB131–10% FBS without bFGF.
The cells were treated with test compounds or vehicle (0.1%
dimethylsulfoxide) for 24 h and thereafter were stimulated with 20
ng/ml TNF for 24 h. Culture supernatants were collected and
centrifuged at 1500 rpm for 5 min to remove any particulate material.
Human IL-6 concentrations in the culture supernatants were determined
by ELISA (kit from R & D Systems, Minneapolis, MN).
Determination of cell surface adhesion molecules
HUVECs were plated onto 96-well collagen-coated dishes (2
x 104 cells/well) in the growth medium. The next
day, the culture medium was replaced by MCDB131–10% FBS without bFGF.
Then the cells were treated with test compounds or vehicle (0.1%
dimethylsulfoxide) for 24 h and thereafter were stimulated with 20
ng/ml TNF for 4 h. ELISA for cell surface VCAM-1 was performed
as described previously (22).
Transient transfection
To investigate the effect of test compounds on IL-6 and VCAM-1
promoter activities, we transiently transfected BAECs with an IL-6 or a
VCAM-1 reporter construct. The IL-6 reporter, provided by Dr. S. Akira
(Osaka University, Osaka, Japan), consists of 5'-flanking region
(-840/+12) of the human IL-6 gene and firefly luciferase. The VCAM-1
reporter, provided by Dr. M. Kurabayashi (Gunma University, Maebashi,
Japan), consists of 5'-flanking region (-258/+40) of the human VCAM-1
gene and firefly luciferase. BAECs on 24-well plates were transfected
with each of the reporter plasmids (1 µg) together with seapansy
luciferase control plasmid (0.1 µg; Toyo Beanet, Tokyo, Japan) using
SuperFect Transfection Reagent (QIAGEN, Valencia, CA). Two
hours after the transfection, the cells were treated for 24 h with
test compounds and subsequently with 20 ng/ml TNF. The cell lysates
were assayed for each luciferase activity in Lumat LB9501 luminometer
(Berthold Systems, Aliquippa, PA).
In some experiments to investigate the effects of dexamethasone on
these promoter activities, 4 ng of the human GR expression vector
pRShGR (provided by Dr. K. Umesono) (29) together with
an IL-6- or a VCAM-1-reporter construct were transfected into BAECs.
Four hours after the transfection, the cells were treated with test
compounds as described above.
Nuclear extraction and EMSA
HUVECs were treated with test compounds, and thereafter were
stimulated with TNF as described above. The nuclear extracts were
prepared by the method of Schreiber et al.
(30). EMSA for NF-B was performed as we described
previously (31). The sequence of double stranded
oligonucleotides used was 5'-CATGGGAAAATCCCACATTT-3' for
the human IL-6 gene promoter (27) and
5'-CTGCCCTGGGTTTCCCCTTGAAGGGATTTCCCT-CCGCC-3'
for the human VCAM-1 gene promoter (28). The NF-B
binding consensus sequence is underlined.
Statistics
The data were analyzed by ANOVA, and the Bonferroni method was
used to estimate the level of significance of differences between
means. P < 0.05 was considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and GR in human vascular ECs was localized
predominantly to the cytoplasm in HUVECs (Fig. 1
). When the cells were treated with
fenofibrate, PPAR staining was observed in the nuclei (Fig. 1). In
contrast, GR was localized in the nuclei of the cells regardless of
whether the cells were treated with dexamethasone (Fig. 1). Staining
was specific for each of the nuclear receptors, as the staining was
blocked by each of their antibodies subjected to preabsorption with
excess antigen (data not shown).
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-induced IL-6
production in human vascular ECs, the IL-6 concentration in
the culture supernatants was increased to about 3-fold. TNF-induced
IL-6 production was inhibited by treatment with fenofibrate in a
concentration-dependent manner (Fig. 2A):
At 5 x 10-5
M or more, fenofibrate showed significant inhibition of
IL-6 production. Treatment with fenofibrate alone
(10-4 M) had
no significant effect on IL-6 production, whereas the PPAR
activator
pioglitazone
(10-4 M)
showed no apparent inhibitory effect on TNF-induced IL-6 production
(Fig. 2A). Dexamethasone at
10-9 M or more
showed significant inhibition of TNF-induced IL-6 production in a
concentration-dependent manner, but it did not affect basal IL-6 levels
(Fig. 2B). The GR antagonist RU486
(10-5 M)
prevented the dexamethasone
(10-7
M)-induced inhibition of TNF-stimulated IL-6 production
(Fig. 2B).
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-induced VCAM-1
expression in human vascular ECs-induced cell surface VCAM-1 expression in HUVECs. As shown in
Fig. 3A, fenofibrate inhibited
TNF-induced VCAM-1 expression in a concentration-dependent
manner, with its inhibitory effect becoming noticeable at
10-4 M or
more. Pioglitazone
(10-4 M) had
no effect on the TNF-induced VCAM-1 levels. Dexamethasone also had
no effect on the TNF-induced VCAM-1 levels even when the cells were
treated with 10-5
M dexamethasone (Fig. 3B).
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increased IL-6
promoter activity by 16-fold compared with that in unstimulated cells
(Fig. 4A). Treatments with fenofibrate
concentrations of 5 x
10-5 M or more
significantly reduced the TNF-induced IL-6 promoter activity,
whereas fenofibrate alone had no significant effect on basal IL-6
promoter activity. As shown in Fig. 4B, TNF also enhanced VCAM-1
promoter activity by 20-fold in BAECs. Fenofibrate at concentrations of
5 x 10-5
M or more clearly inhibited VCAM-1 promoter activity.
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-stimulated promoter activity of either gene. As Inoue et
al. (32) demonstrated that these cells express GR at
a low levels, we introduced a GR expression vector into these cells. In
BAECs transfected with this vector, dexamethasone
(10-7
M) significantly inhibited the TNF-induced
IL-6 promoter activity (Fig. 5A), but
failed to inhibit the TNF-induced VCAM-1 promoter activity (Fig. 5B).
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B binding to
its recognition sites in IL-6 and VCAM-1 promoter regionsB binding to its recognition sites in IL-6 as well as VCAM-1
promoters, EMSAs were performed using nuclear extracts from HUVECs and
radiolabeled oligonucleotide probes corresponding to the NF-B
binding sites in the promoters of the two genes. The EMSA using
oligonucleotides for the IL-6 gene demonstrated that fenofibrate
(10-4 M) as
well as dexamethasone
(10-7 M)
reduced the amount of gel-retarded complexes induced by TNF (Fig. 6A). The EMSA using oligonucleotides for
VCAM-1 gene, however, demonstrated that fenofibrate, but not
dexamethasone, reduced the amount of gel-retarded complexes induced by
TNF (Fig. 6B). Pioglitazone elicited no change in the
gel-retarded complexes in either EMSA (Fig. 6, A and B). The
TNF-induced NF-B complexes consisted mainly of p65 (RelA) and
p50, as evidenced by the supershifted bands resulting from the addition
of p65 antibody and/or p50 antibody (Fig. 6, A and B).
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B in human vascular ECsB dimer from cytoplasms is a
prerequisite for activation of NF-B (26). Thus, we
investigated the effects of fenofibrate and dexamethasone on the
cellular localization of the NF-B p65 protein. As shown in Fig. 7, TNF stimulated translocation of the
p65 protein from cytoplasms to nuclei in HUVECs, whereas neither
fenofibrate (10-4
M) nor dexamethasone
(10-7 M) could
prevent this translocation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in human vascular ECs was previously
demonstrated by RT-PCR and Western blot analyses (33, 34).
In this study we showed for the first time that PPAR is localized
predominantly to cytoplasms in HUVECs and is translocated into nuclei
upon stimulation with the PPAR activator fenofibrate. PPAR1 mRNA
is also expressed in human vascular ECs (35, 36). We
therefore compared the effects of the pharmacological activators of
PPAR, PPAR, and GR on the expression of the known key molecules
in vascular inflammation, IL-6 and VCAM-1, in HUVECs. Our results
showed that the PPAR activator fenofibrate clearly inhibits
TNF-stimulated IL-6 production, but the PPAR activator
pioglitazone does not. In this connection, Staels et
al. (37) have recently shown that PPAR activators,
but not PPAR activators, inhibit IL-1-mediated IL-6 production in
vascular smooth muscle cells. Then two sets of results mean that
PPAR acts as a down-regulator of IL-6 production in both vascular
endothelial cells and vascular smooth muscle cells. In our experiments
the GR activator dexamethasone also inhibited TNF-induced IL-6
production in HUVECs, and this inhibition was completely restored by
the GR antagonist RU486, indicating that it is mediated by GR. IL-6 is
known to control macrophage and T cell activation as well as vascular
smooth muscle cell proliferation (38, 39) and has been
detected in human and rabbit atherosclerotic regions (40, 41). Therefore, the inhibition of IL-6 production in vascular
ECs may contribute to the antiinflammatory effects of PPAR and GR on
vascular walls.
VCAM-1 has been shown to play an important role in mediating
mononuclear leukocyte-selective adhesion to vascular endothelium
(3, 42). This is expressed in vascular inflammatory
disorders such as atherosclerosis, vasculitis, renal diseases,
arthritis, and graft resection (3, 43), and its soluble
form is elevated in sera from patients with such diseases
(44, 45, 46), indicating that VCAM-1 is one of the key
molecules involved in vascular inflammatory disorders. This means that
this adhesion molecule also is a molecular target of antiinflammatory
drugs in vascular walls. Furthermore, our study showed that fenofibrate
inhibits TNF-induced VCAM-1 expression in HUVECs, but that
pioglitazone does not. These results support the findings
obtained by using human saphenous vein ECs (24). Very
interestingly, however, dexamethasone failed to inhibit the
TNF-induced VCAM-1 expression in HUVECs. Thus, PPAR proved to
have inhibitory effects on both IL-6 and VCAM-1 expression in the human
vascular ECs, but GR inhibited only IL-6 expression.
As suggested by the experiments involving transient transfection of an
IL-6 or a VCAM-1 promoter construct, the inhibitory effects of
fenofibrate on both IL-6 and VCAM-1 expression may be exerted at least
partly as a result of transcriptional inhibition of these genes. In
experiments using BAECs transfected with human GR expression vector, on
the other hand, dexamethasone only reduced IL-6 promoter activity, not
VCAM-1 promoter activity. Thus, PPAR and GR had similar effects on
the inhibition of the IL-6 gene promoter, but different effects on the
VCAM-1 gene promoter in vascular ECs.
Both of the genes encoding IL-6 and VCAM-1 are known to have
NF-B-binding sites in their promoter regions (27, 28).
In our EMSA using oligonucleotide probes derived from the human IL-6
gene promoter, fenofibrate as well as dexamethasone clearly suppressed
TNF-activated NF-B binding. By contrast, in the EMSA using
oligonucleotide probes from the human VCAM-1 gene promoter, only
fenofibrate inhibited such binding. Thus, the differences in the
effects of fenofibrate and dexamethasone on NF-B binding to the two
promoters correlate to those on protein expression and promoter
activity. The totality of these findings suggests that the inhibition
of both IL-6 and VCAM-1 expression by fenofibrate as well as the
inhibition of IL-6 expression by dexamethasone are mediated at least in
part through interference with NF-B activity. The TNF-stimulated
NF-B proteins consist mainly of p65 (RelA) and p50 (NF-B1)
in HUVECs. Immunocytochemical analysis demonstrated that neither
fenofibrate nor dexamethasone prevented nuclear translocation of the
p65 protein. Thus, the inhibitory effects of fenofibrate and
dexamethasone on NF-B binding must be exerted chiefly during stages
after the nuclear translocation of NF-B. In contrast to our
findings, Simoncini et al. (47) have shown that
nuclear translocation of both p50 and p65 after LPS treatment was
inhibited by dexamethasone in human saphenous vein ECs. Although the
reason for this discrepancy is not clear, differences in the sources of
vascular ECs or in the stimulators for NF-B may be responsible. In
this connection, Brostjan et al. (18) have
shown that although IB-dependent down-regulation of NF-B
activity by glucocorticoids has been postulated (48, 49),
such a mechanism is not involved in vascular endothelial cells.
The molecular mechanisms of interference of NF-B activity by PPAR
and GR remain unidentified. Studies using overexpression systems have
shown that GR can be physically associated with p65 protein, thereby
antagonizing it (16, 17). In similar experimental systems,
both ER (50) and PR (51) have been shown to
be capable of physical association with the p65 protein. Delerive
et al. (52) have recently shown that in
glutathione-S-transferase pull-down experiments PPAR also
physically interacts with p65. In view of these findings in
overexpression systems, physical interaction between these nuclear
receptors and NF-B proteins might also occur physiologically in
vascular ECs. We therefore propose a hypothesis of down-regulation of
NF-B activity by PPAR and GR via their interaction with NF-B
in vascular ECs.
As one of the reasons for target gene-specific
down-regulation of NF-B activity by PPAR and GR, it has been
suggested that repression of gene transcription by nuclear receptors
can result from competition for limited amounts of coactivators
(53, 54). If such a competition model is responsible for
the target gene-specific down-regulation of NF-B, the
predominant coactivator(s) involved in PPAR- and GR-mediated gene
repression may be different. Alternatively, coactivators may not be
involved in the mechanisms by which PPAR and GR exert their specific
repression of NF-B-driven genes. In this connection, it has been
shown that overexpression of the coactivator cAMP-responsive
element-binding protein-binding protein does not relieve
PPAR-mediated transcriptional repression of p65 (52).
Furthermore, a recent study (55) has shown that
cAMP-responsive element-binding protein-binding protein functions as an
integrator of GR/p65 physical interaction, rather than as a limiting
cofactor for which GR and p65 compete. On the other hand, De Bosscher
et al. (56) recently proposed a model in which
glucocorticoids repress NF-B-driven genes by interfering with the
interaction of p65 with the basal transcription machinery. The sum
total of these finding suggests that the repression of NF-B-driven
genes by PPAR or GR in vascular ECs may be evoked in a specific
interaction of the nuclear receptor, NF-B, coactivator(s), and/or
some components of the basal transcription machinery, which may, in
turn, be responsible for the target gene-specific down-modulation
of NF-B activity by the nuclear receptor.
Down-regulation of IL-6 and VCAM-1 gene expression on vascular walls
may lead to important clinical consequences, as these molecules are
involved in the pathogenesis of various inflammatory disorders. In the
study presented here we showed that the PPAR activator fenofibrate
has a more beneficial effect than the GR activator dexamethasone on the
expression of IL-6 and VCAM-1 in vascular ECs. Thus, this PPAR
activator may have the potential to relieve vascular inflammation,
sometimes even more effectively than glucocorticoids.
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Acknowledgments |
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Footnotes |
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Abbreviations: BAEC, Bovine aortic endothelial cell; bFGF,
basic fibroblast growth factor; EC, endothelial cell; HUVEC, human
umbilical vein endothelial cell; IB, inhibitor of NF-B; NF-B,
nuclear factor-B; VCAM-1, vascular cell adhesion molecule-1.
Received March 7, 2001.
Accepted for publication April 25, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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transcription in macrophages: involvement
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Acad Sci USA 97:3919–3924This article has been cited by other articles:
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) or with
(
) 20 ng/ml TNF
). Values are the mean ± SD in triplicate
analyses in a typical experiment of four separate experiments.
Statistical analyses were made for cells treated with TNF
