© 1995 American Heart Association, Inc.
Articles |
Oxidized LDL Induces Transcription Factor Activator Protein–1 but Inhibits Activation of Nuclear Factor–
B in Human Vascular Smooth Muscle Cells
From the Atherosclerosis Research Unit, King Gustaf Vth Research Institute, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden.
Correspondence to Dr Mikko Ares, King Gustaf Vth Research Institute, Karolinska Hospital, S-171 76 Stockholm, Sweden.
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Abstract |
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Abstract Oxidized LDL (Ox-LDL) has been implicated in the development of atherosclerotic lesions, mainly due to its enhanced uptake by macrophages and its ability to alter gene expression in arterial cells. In the present study we demonstrated that Ox-LDL activates activator protein–1 (AP-1), a transcription factor generally induced by mitogenic substances. Lysophosphatidylcholine, which is generated during oxidation of LDL, stimulated AP-1 in a dose-dependent manner. In contrast, the radical-dependent transcription factor nuclear factor–
B (NF-B) was not activated by Ox-LDL, and at a
concentration of 50 µg/mL, Ox-LDL inhibited
lipopolysaccharide-induced activation of NF-B.
Oxysterols but not lysophosphatidylcholine inhibited
lipopolysaccharide-induced NF-B activation,
suggesting that they may be responsible for the inhibitory
effect of Ox-LDL. In conclusion, Ox-LDL has opposing effects on the
activities of NF-B and AP-1, suggesting involvement of mechanisms
for transcriptional regulation that are strongly affected by lipid
oxidation products.
Key Words: oxidized LDL • transcription factor activator protein–1 • lysophosphatidylcholine • nuclear factor–B • oxysterol
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Oxidative modification of LDL particles in the arterial intima is believed to be an early key event in the development of atherosclerotic lesions.1 This modification leads to the formation of intimal macrophage foam cells by rendering LDL susceptible to uptake by the scavenger receptor pathway. Ox-LDL also influences the secretory activities and functional properties of endothelial cells, SMCs, and macrophages, suggesting a complex role for lipoprotein oxidation in atherosclerosis. The biological actions of Ox-LDL include both stimulatory and inhibitory effects on gene transcription as well as general cytotoxicity and have been attributed to the formation of reactive molecules such as lipid peroxides, lysoPC, aldehydes, and oxysterols.2 3 However, the interactions between products generated by lipid peroxidation and intracellular signaling remain poorly understood.
Transcription factor NF-B is present in most cell types as a
cytosolic heterodimer composed of NFKB1 (p50) and RelA (p65) subunits
bound to an inhibitor protein, I-B. NF-B regulates
the inducible expression of a variety of genes involved in inflammatory
and immune responses.4 Following activation, NF-B
dissociates from I-B and is translocated to the nucleus. A wide
variety of agents, including cytokines, UV light, antibodies to
cell-surface proteins, LPS, and H2O2,
activate NF-B. The finding that substances that generate
radicals activate NF-B and the observation that several
antioxidants inhibit the stimulatory effect of almost all known NF-B
activators have led to the conclusion that intracellular
radicals have an integrative role in NF-B activation.5
Oxidation of LDL is associated with the generation of substances with
radical properties such as lipid peroxides,6 suggesting
radical-mediated activation of NF-B as a possible mediator of
the biological effects of Ox-LDL. Some indirect support for this
hypothesis comes from studies demonstrating that NF-B
activates transcription of VCAM-17 and
E-selectin8 9 genes in endothelial cells
and that an increased endothelial expression of VCAM-1
is the first identifiable response of the vessel wall to
diet-induced hypercholesterolemia in
experimental animals.10
The transcription factor AP-1 consists of homo- or heterodimers of the proteins encoded by the fos and jun gene families and is believed to regulate genes involved in the control of cell growth and differentiation.11 Exposure to H2O2 results in a marked accumulation of c-jun and c-fos mRNA but only a weak stimulation of the DNA binding capacity of AP-1.5
Proliferation of SMCs in the arterial intima is one of the
major factors responsible for the development of atherosclerotic
lesions.12 This proliferation is believed to be initiated
by growth factors secreted by activated SMCs,
macrophages, and endothelial cells within the
developing lesion and may be further enhanced by the presence of
Ox-LDL.13 The observation that SMC DNA synthesis is
stimulated by radical generators such as H2O2
and xanthine/xanthine oxidase14 suggests that lipid
oxidation products may act as mediators of the
mitogenic activity of Ox-LDL. In the present study we
have analyzed the effects of native and Ox-LDL and several
lipid oxidation products on the DNA binding activity of
transcription factors NF-B and AP-1.
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Methods |
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Materials
L-
-LysoPC (egg yolk) was purchased from Sigma.
[
-32P]dATP (185 TBq/mmol) was from Amersham.
Antibodies against NFKB1 (p50), RelA (p65), and the
oligonucleotides for mobility shift assays were
obtained from Santa Cruz Biotechnology, Inc.
25-Hydroxycholesterol,
7-hydroxycholesterol, and
cholestan-5,6-epoxy-3ß-ol purified by
high-performance liquid
chromatography15 were a generous gift of
Dr Ingemar Björkhem, Karolinska Institute, Stockholm, Sweden.
Cell Culture
Human arterial SMCs originally isolated by explant
technique were kindly provided by Dr Ulf Hedin (Karolinska Institute).
The cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% FCS, penicillin (50 U/mL), and streptomycin (50
µg/mL). The cells showed positive -actin immunoreactivity
(HHF35 antibody was kindly provided by Dr Allan Gown, University of
Washington, Seattle). Cells in the 12th through the 18th passages were
used in the experiments. Prior to experiments, confluent cells in
100-mm dishes were serum-starved two times in Ham's F-12 medium
for 24 hours. RPMI-1640 medium was used for treatments with
H2O2 because this medium does not contain
transition metal ions, which catalyze the decomposition of
H2O2 into hydroxyl radicals.
Preparation of Nuclear Extracts
Cells in 100-mm plastic dishes were rinsed with ice-cold PBS
and harvested in 5 mL PBS by scraping. Nuclear extracts were prepared
essentially as described by Alksnis et al.16 Briefly, the
cells were washed with 1 mL PBS and resuspended in 100 µL hypotonic
buffer (in mmol/L: HEPES 10, pH 7.3, KCl 10, MgCl2 1.5, DTT
1, and PMSF 1). After centrifugation, cells were lysed
by resuspension in 300 µL lysis buffer (10 mmol/L HEPES, pH 7.3, 10
mmol/L KCl, 1.5 mmol/L MgCl2, 0.4% Nonidet P-40, 1
mmol/L DTT, 1 mmol/L PMSF, 1 µg/mL leupeptin, and 15 µg/mL
aprotinin). After a 10-minute incubation at 4°C nuclei were collected
by centrifugation for 1 minute at 8000g, and
the pellets were washed once in 1 mL of 20 mmol/L KCl buffer (20 mmol/L
HEPES, pH 7.3, 22% glycerol, 20 mmol/L KCl, 1.5 mmol/L
MgCl2, 0.2 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L PMSF,
1 µg/mL leupeptin, and 15 µg/mL aprotinin). The isolated nuclei
were resuspended in 15 µL of 20 mmol/L KCl buffer, and 60 µL of 0.6
mol/L KCl buffer (20 mmol/L HEPES, pH 7.3, 22% glycerol, 0.6 mol/L
KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 1 mmol/L
DTT, 1 mmol/L PMSF, 1 µg/mL leupeptin, and 15 µg/mL aprotinin) was
added. Nuclear proteins were extracted by incubation on ice for 30
minutes. After centrifugation for 15 minutes at
8000g, the supernatant containing nuclear proteins was
transferred to a precooled microcentrifuge tube, and an
aliquot of the extract was diluted 40 times with 484 mmol/L KCl buffer
(mixture of those above) for protein assay. Protein concentration (in
micrograms per milliliter) was determined
spectrophotometrically17 according to the following
equation: Protein Concentration=184xA(230 nm)-81.7xA(260 nm), where
A is absorbance.
EMSA
Equal amounts of protein from nuclear extracts (1 to 3 µg)
were incubated on ice with 2 µg poly(dI-dC) and 1 µg
acetylated bovine serum albumin in binding buffer
(giving the final concentrations stated below) for 10 minutes. The
oligonucleotide probe (50 000 cpm in 5 µL) was
added, and the reaction mixture (25 µL) was incubated for 30 minutes
at room temperature. Final concentrations in binding reactions were as
follows: 10% glycerol, 10 mmol/L HEPES, pH 7.9, 60 mmol/L KCl, 5
mmol/L MgCl2, 0.5 mmol/L EDTA, 1 mmol/L DTT, and 1
mmol/l PMSF. DNA-protein complexes were separated from unbound DNA
probe on native 7% polyacrylamide gels in
low-ionic-strength buffer (22.3 mmol/L each Tris and borate and
0.5 mmol/L EDTA, pH 8). The sequences of the double-stranded
oligonucleotide probes labeled with T4 kinase and
[-32P]dATP were as follows: B consensus, 5'-AGT TGA
GGG GAC TTT CCC AGG C-3'; B mutant, 5'-AGT TGA GGC GAC
TTT CCC AGG C-3'; and AP-1 consensus, 5'-CGC TTG ATG AGT CAG CCG
GAA-3'.
Preparation of LDL
Blood was drawn from fasting, healthy volunteers, and plasma was
recovered by centrifugation at 1400g for 20
minutes at 1°C. The isolated plasma was adjusted to d=1.10
kg/L by the addition of NaCl. A density gradient consisting of 3 mL of
1.10 kg/L–density plasma and 3 mL of 1.065, 1.020, and 1.006 kg/L NaCl
solutions, respectively, was then formed in cellulose nitrate tubes
(Ultraclear tubes, Beckman). The gradient was centrifuged in a
Beckman L8-55 ultracentrifuge at 40 000 rpm in a Beckman
SW-40 swinging-bucket rotor at 20°C overnight.18 The
VLDL and IDL fractions were aspirated from the top 3 mL, and LDL was
harvested from the next 4 mL of the tube. EDTA and excess salt were
subsequently removed by dialysis against EDTA-free PBS. The protein
content of the LDL preparation was determined as described by Lowry et
al.19 Endotoxin levels in both LDL and Ox-LDL were
below 5 ng/mg LDL protein as determined by Limulus assay
(Pharmacia).
Oxidation of LDL
Copper oxidation was performed by incubating the LDL at 37°C
in 5% CO2 in the presence of 5 µmol/L CuSO4
for varying times, typically 15 hours.20 Total amounts of
aldehydes in Ox-LDL were determined by
colorimetric assay (Bioxytech S. A.). The
aldehyde content of LDL oxidized for 0, 2, 6, or 24 hours was 8.6, 8.6,
13.1, and 15.2 nmol/mg LDL protein, respectively.
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Results |
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Activation of NF-
B and AP-1 in SMCs by
H2O2 and LPSSerum-starved SMCs contained small or undetectable amounts of active NF-
B. Exposure of SMCs to H2O2 in
serum-free RPMI-1640 medium resulted in a dose-dependent
activation of NF-B (Fig 1
). The DNA binding activity
of AP-1 was also increased by H2O2, but
not as strongly as that of NF-B (data not shown). Maximal activation
of both transcription factors was obtained at an
H2O2 concentration of 400 µmol/L. EMSAs
revealed two distinct bands that were specific for NF-B (Fig 2). The addition of excess unlabeled mutant B
oligonucleotide (with one point mutation in the binding
site) to the binding reactions resulted in the disappearance of
unspecific binding activity, whereas specific bands were unaffected.
When unlabeled probe was used as a competitor, the specific complexes
were more susceptible to competition than the unspecific ones (Fig 2).
NF-B–specific complexes with higher mobility (in the lower band)
were retarded by antibodies against NFKB1 (p50). Antibodies directed
against RelA (p65) retarded the slower migrating complex as well,
leaving a minor portion of the complexes with higher mobility
unaffected. Taken together, these data suggest that the major component
of NF-B in SMCs consists of heterodimers between p50 and p65. There
seemed to be small amounts of p50 homodimers as well as smaller
quantities of a complex that did not react with antibodies to p50 or
p65. The supershift assays also suggested the existence of at least one
additional NF-B–related complex in the extracts. Incubation of SMCs
with LPS resulted in an activation of both NF-B and AP-1 (Fig 3). Maximal activation of NF-B was obtained at LPS
concentrations between 500 and 750 ng/mL, whereas AP-1 required higher
concentrations for maximal binding activity.
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Effect of Ox-LDL on NF-B Activity in SMCs
The effect of Ox-LDL on NF-B activity was analyzed by
using either native LDL or LDL oxidized by exposure to copper for 2 to
16 hours. None of the preparations of native or Ox-LDL stimulated the
activity of NF-B in quiescent SMCs (Fig 4). In
contrast, Ox-LDL inhibited LPS-induced activation of NF-B. The
inhibitory effect increased with the extent of oxidative
modification; LDL oxidized by incubation with copper for 16 hours
completely abolished the activation of NF-B caused by 1 µg/mL LPS
(Fig 4) as well as that induced by H2O2 (data
not shown). The inhibitory effect of Ox-LDL was
concentration dependent. A lower concentration of copper-oxidized
LDL, 10 µg/mL, neither inhibited nor activated NF-B as
determined by EMSA (Fig 5 and data not shown).
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Effect of Ox-LDL on AP-1 Activity in SMCs
At a concentration of 50 µg/mL Ox-LDL activated AP-1
(Fig 6). AP-1 activation by Ox-LDL was not significantly
affected by the low LPS concentration (5 ng/mL) that in itself did not
activate AP-1 in these cells. When CuSO4 and PBS
were added to the culture medium at concentrations similar to that
resulting from the addition of copper-oxidized LDL, neither NF-B
nor AP-1 were affected (Figs 5
and 6 and data not shown).
Analysis of the influence of degree of oxidation revealed that
LDL only marginally modified by exposure to copper for 2 hours was as
effective in activating AP-1 as LDL extensively modified by incubation
with copper for 16 hours (Fig 7). The nuclear extracts
used to analyze AP-1 activity were the same as those used for
NF-B analysis (Fig 4), in which the biological activities of
the different preparations of copper-oxidized LDL were clearly
different.
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Effect of LysoPC on NF-B and AP-1
Phosphatidylcholine is the major phospholipid in LDL. During
oxidation of LDL up to approximately 50% of this phospholipid can be
converted into lysoPC.20 Studies in our laboratory have
demonstrated that lysoPC is responsible for most of the
mitogenic activity of Ox-LDL (A. Stiko, et al, unpublished
data, 1994). Incubation of SMCs with lysoPC resulted in a
dose-dependent activation of AP-1 with a maximal effect obtained at
a concentration of 15 µg/mL (Fig 8). Only minor
amounts of active NF-B were detected in cells exposed to lysoPC (Fig 9). At higher concentrations of lysoPC,
serum-induced NF-B binding activity was slightly inhibited (Fig 9).
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Effect of Oxysterols on NF-B
Ox-LDL at a protein concentration of 50 µg/mL can be expected to
contain 1 to 12 µg oxysterols/mL.20 At a concentration
of 5 µg/mL all of the three oxysterols
(cholestan-5,6-epoxy-3ß-ol,
7-hydroxycholesterol, and
25-hydroxycholesterol) inhibited LPS-induced NF-B
activity (Fig 10), but this inhibition was weaker than
that obtained by 50 µg/mL LDL oxidized by exposure to copper for 16
hours (Fig 4). As measured by laser densitometry, treatment of cells
with oxysterols resulted in a 50% inhibition of the LPS-induced
activation of NF-B.
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Discussion |
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The radical-sensitive transcription factor NF-
B may
function as a mediator of the biological effects of oxidized
lipoproteins. Experimental studies provide some support for this
hypothesis. LDL mildly oxidized by exposure to soybean
lipoxygenase activates NF-B in cultured
endothelial cells.21 In rabbits fed a
cholesterol-enriched diet, activation of VCAM-1
expression appears to be the initiating factor responsible for the
recruitment of circulating monocytes and the formation of early
inflammatory lesions.10 Diet-induced
hypercholesterolemia is associated with an
increased endothelial production of superoxide
radicals.22 Taken together with the findings that
activation of endothelial VCAM-1 expression is
effectively blocked by antioxidants7 and that the promoter
of the VCAM-1 gene contains two NF-B binding sites, these
observations suggest that hypercholesterolemia
is associated with a radical-dependent, NF-B–mediated
activation of endothelial VCAM-1 expression. However,
Maier et al23 report that neither NF-B nor any of the
adhesion molecules (VCAM-1, E-selectin, intracellular adhesion
molecule–1, or P-selectin) were activated by Ox-LDL despite
the fact that Ox-LDL caused an increase in monocyte binding to
endothelial cells.
We have shown in this study that LDL oxidized by exposure to copper
sulfate did not activate NF-B in SMCs. Instead, at a
concentration of 50 µg/mL Ox-LDL, a marked inhibition of NF-B
activation could be observed. Simultaneously, Ox-LDL
stimulated the DNA binding activity of AP-1. LPS-mediated activation of
NF-B was inhibited by three different oxysterols, suggesting that
these substances may be partially responsible for the
inhibitory effect of Ox-LDL.
In contrast to its effect on NF-B, Ox-LDL was found to stimulate the
DNA binding activity of AP-1. This effect was dependent on
concentration and to a lesser extent on the degree of oxidation,
suggesting that the activation was due to a substance that was formed
during oxidation of LDL. Several lines of evidence suggest that this
substance may be lysoPC. LysoPC stimulated AP-1 activity in a
dose-dependent manner, and oxidation of LDL results in the
formation of lysoPC at concentrations equivalent to those found to
activate AP-1. For example, if Ox-LDL contains 400 nmol
lysoPC/mg protein,20 the lysoPC concentration in medium
containing Ox-LDL at a concentration of 50 µg protein/mL will be 10
µg/mL. LysoPC also activates protein kinase C, a potent
mediator of AP-1 activation.24 25 26 Preliminary studies in
our laboratory have demonstrated that lysoPC is a potent mitogen for
SMCs (A. Stiko, et al, unpublished data, 1994). Therefore, it is likely
that the mitogenic activity of Ox-LDL is at least partially
explained by a lysoPC-dependent activation of AP-1. Several other
observations also indicate that lysoPC may play a role in
atherogenesis. LysoPC is abundantly present in atherosclerotic
plaques,27 functions as a selective chemoattractant for
mononuclear leukocytes,28 induces expression of VCAM-1 in
endothelial cells,29 and stimulates the
synthesis of heparin-binding epidermal growth factor–like
growth factor in endothelial cells30 and
monocytes.31 Whether these effects are mediated by
activation of AP-1 remains to be determined.
The possibility that the inhibitory effect of Ox-LDL on
NF-B activation is due to general cytotoxicity should be taken into
account. However, the parallel activation of AP-1 and the
identification of single-lipid molecular species (lysoPC and
oxysterols) that are capable of activating AP-1 or inhibiting
activation of NF-B argue against this. Furthermore, no cell
detachment could be observed after treatment with Ox-LDL for up to 6
hours.
In a study on cultured endothelial cells, Parhami et
al21 have shown that LDL minimally oxidized by incubation
with soybean lipoxygenase enhanced NF-B
activity. To analyze how the extent of oxidation influences the
effects of Ox-LDL, we exposed SMCs to LDL incubated with copper for 2,
4, 6, and 16 hours. No stimulatory effect on NF-B activity could be
observed with the partially oxidized LDL preparations obtained by a
shorter exposure to copper, but these preparations were less potent
inhibitors of NF-B than fully oxidized LDL. Other
researchers20 have determined the peroxide content of
Ox-LDL. If these peroxides are not considerably more potent
activators of NF-B than exogenous
H2O2, their concentration is probably
too low to induce NF-B activation. LDL oxidized with copper for 4
hours should contain a maximum amount of lipid
peroxides,20 but we could not detect NF-B activation
with any of the copper-oxidized LDL preparations oxidized for 2, 4,
or 6 hours.
Among the genes that could be affected by altered levels of AP-1
and NF-B are the inflammatory cytokines tumor necrosis
factor– and IL-1ß. Even though NF-B regulates the expression
of tumor necrosis factor– and IL-1ß, a number of
reports32 33 34 35 suggest that AP-1 sites are more important
for the activation of these genes. AP-1 may be involved in the
transcriptional regulation of several other cytokines as
well,32 including IL-2, IL-3, IL-6, colony stimulating
factor–1, and transforming growth factor–ß1. It is also interesting
to note that the mitogenic response of fibroblasts to tumor
necrosis factor– requires active AP-1.36 In
bacteria the NF-B–related, radical-activated
transcription factor oxyR controls the expression of antioxidant
enzymes.37 38 If NF-B plays a similar role in human
SMCs, the inhibitory effect of Ox-LDL could impair the
survival of cells in lesions under oxidative stress. However, further
studies to clarify which SMC genes are controlled by NF-B are
required before the putative role of Ox-LDL–mediated inhibition of
NF-B activation can be evaluated.
In summary, the present study shows that oxidative
modification of LDL is associated with the formation of substances that
inhibit the activation of NF-B but stimulate the activity of AP-1 in
human SMCs. These effects may be of importance in atherogenesis, but
their possible pathophysiological role remains
to be determined.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grants from the Swedish Medical Research Council (8311), the King Gustaf V 80th Birthday Fund, the Nordic Academy for Advanced Study (94.30.024/00), the Nanna Svartz Fund, the Swedish Heart and Lung Foundation, and the Wallenberg Foundation.
Received February 10, 1995; accepted June 22, 1995.
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