Articles |
B by Oxidized Low-Density Lipoprotein
From the Institute of Clinical Chemistry and Pathobiochemistry (K.B., T.E., U.K., M.P., S.P., M.H., D.N.) and the Department of Internal Medicine I (F.J.N.), Klinikum rechts der Isar, Technical University Munich (Germany); the Institute of Clinical Chemistry (A.G., A.K.W.), Klinikum Grosshadern, Ludwig-Maximilians University Munich (Germany); the Institute of Biochemistry (C.K.), Albert-Ludwigs University, Freiburg, Germany; the Departments of Immunology and Vascular Biology (N.M.), the Scripps Research Institute, La Jolla, Calif; and Tularik Inc (P.A.B.), South San Francisco, Calif.
Correspondence to Dr Korbinian Brand, Institute of Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar, Technical University Munich, Ismaninger Str 22, D-81675 München, Germany.
| Abstract |
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B (NF-
B)/Rel transcription
factors may be involved in atherosclerosis, as is
suggested by the presence of activated NF-
B in human
atherosclerotic lesions. The aim of the present study was to
investigate the effects of oxidized LDL (oxLDL) on the NF-
B system
in human THP-1 monocytic cells as well as adherent monocytes. Our
results demonstrate that short-term incubation of these cells with
oxLDL activated p50/p65 containing NF-
B dimers and induced
the expression of the target gene IL-8. This activation of NF-
B was
inhibited by the antioxidant and H2O2 scavenger
pyrrolidine dithiocarbamate and the proteasome inhibitor
PSI. The oxLDL-induced NF-
B activation was accompanied by an initial
depletion of I
B-
followed by a slight transient increase in the
level of this inhibitor protein. In contrast, long-term
treatment with oxLDL prevented the lipopolysaccharide-induced
depletion of I
B-
, accompanied by an inhibition of both NF-
B
activation and the expression of tumor necrosis factor-
and
interleukin-1ß genes. These observations provide additional evidence
that oxLDL is a potent modulator of gene expression and suggest that
(dys)regulation of NF-
B/Rel is likely to play an important role in
atherogenesis.
Key Words: nuclear factor-
B oxidized LDL reactive oxygen intermediates I
B-
monocytes macrophages
| Introduction |
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B (NF-
B)/Rel is
present in the cytosol in an inactive state bound to
inhibitory proteins collectively termed
I
B.1 2 The prototypic NF-
B dimer consists of the
subunits p50 and p65, although other subunits such as c-Rel have been
described.3 4 Several I
B proteins have been identified,
including I
B-
and I
B-ß.5 6 Activation of
NF-
B is induced by a variety of agents including inflammatory or
lymphoproliferative cytokines, reactive oxygen intermediates,
microbial pathogens, and viruses.5 7 This activation of
NF-
B involves phosphorylation of I
B and
proteolysis of this inhibitor by a large protease complex,
the proteasome, followed by nuclear translocation of the
dimer.4 5 In the nucleus, activated NF-
B
interacts with regulatory
B DNA elements in promoters and enhancers,
thereby controlling the inducible gene transcription of a plethora of
genes involved in inflammation and proliferation.5 7
It has been suggested that NF-
B may play an important role in
atherosclerosis by modulating gene expression in cells
participating in lesion formation.8 9 10 We have recently
demonstrated the presence of activated NF-
B in
monocyte/macrophages, smooth muscle cells, and
endothelial cells of human atherosclerotic lesions in
vivo.11 A variety of genes are induced in the
atherosclerotic lesion and/or proposed to be involved in
atherogenesis12 that are known to be regulated by NF-
B
in cultured cells, including the genes encoding
TNF-
5 13 ; IL-1,14 IL-6,5 and
IL-85 15 ; the granulocyte/macrophage-colony
stimulating factors (G-CSF, M-CSF, and GM-CSF)5 16 17 ;
monocyte chemotactic protein-1 (MCP-1)5 18 19 ; tissue
factor20 21 22 23 ; several adhesion
molecules5 24 25 26 27 ; and c-myc.28
oxLDL has been identified as a major component of the early as well as
advanced atherosclerotic lesion, and numerous studies have demonstrated
a variety of effects of this lipoprotein on different cell
functions9 29 30 including gene expression in several cell
types involved in atherogenesis.31 32 33 34 35 36 37 Recent studies
indicate that NF-
B is involved in mediating the effects of oxLDL on
gene expression: However, cultured human endothelial
cells are the only cell type in which an oxLDL-induced activation of
NF-
B has been so far described.38 39 In contrast,
recent reports have found an inhibition of LPS-induced NF-
B
activation by long-term treatment with oxLDL in macrophages and
smooth muscle cells.40 41 42
The aims of the present study were, therefore, to test whether
oxLDL can activate NF-
B in cells of the monocytic lineage
under certain conditions and to investigate the effects of oxLDL on the
NF-
B system more thoroughly in order to understand the underlying
mechanisms that produce the apparently conflicting results in the
literature described above. Using human THP-1 monocytic cells and ex
vivo isolated adherent monocytes, we demonstrated that short term
exposure to oxLDL activated NF-
B and associated target gene
expression, whereas after long-term incubation with these lipoproteins
an inhibition of the NF-
B system was observed. Both opposing effects
were further characterized using specific antibodies and
inhibitors and by investigation of the fate of the NF-
B
inhibitor protein I
B-
. A potential role of aberrant
NF-
B/Rel regulation by oxLDL in the pathology of
atherosclerosis is discussed.
| Methods |
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Lipoprotein Isolation, Modification, and Characterization
LDL (d=1.019 to 1.063 g/mL) was isolated from
human plasma of normolipidemic healthy volunteers by sequential
ultracentrifugation as described33 and
stored in PBS containing 2 mmol/L EDTA. Immediately before
oxidation, the EDTA was removed from LDL by passing the lipoprotein
through a PD 10 column (Pharmacia). LDL was oxidized in Ham's F-10
medium by exposure to 5 µmol/L CuSO4 at
37°C.44 33 Slightly modified LDL was also obtained by
long-term storage at 4°C34 or exposure of the LDL to
monocytic cells.44 Equivalent effects were found with
dialyzed and nondialyzed preparations (data not shown). For the
presented experiments, the preparations were dialyzed before
they were added to the cultured cells. Acetylated LDL was
prepared by treatment of LDL with acetic anhydride as described
earlier.45 The protein concentration was determined by the
Lowry method45 and the cholesterol content by
the cholesterol-assay from Boehringer Mannheim.
Thiobarbituric acid reactive substances (TBARS) were measured as
described33 46 and the initial TBARS value of native LDL
was <0.1 nmol malondialdehyde equivalents/mg of protein (7
preparations). Unless otherwise stated, mildly to moderately oxidized
forms of oxLDL were used for the experiments which were modified by
short time exposure to CuSO4 (TBARS values ranging from
1.94 to 5.37 nmol/mg). Precautions taken to prevent endotoxin
contamination during lipoprotein isolation and oxidation and
experimental procedures included the use of pyrogen-free sterile water,
reagents, and culture dishware. Endotoxin contamination was screened by
the limulus amoebocyte lysate assay (Labortechnik Peter Schultz) and
the Kinetic-QCL-test (BioWhittaker) and only lipoprotein preparations
with an endotoxin content of <10 pg/mL were used for the
experiments. As an additional control, some preparations were passed
through a column containing the Affinity Pak Detoxi-Gel, which removes
endotoxin.
Exclusion of Toxic Effects of Lipoproteins
A potential toxic effect of LDL and oxLDL on the cells was
investigated by trypan blue exclusion, which demonstrated that >98%
of the cells were trypan blue negative in the presence of the
lipoproteins. In addition, toxicity of the reagents on the cells was
monitored by the WST-1 test (Boehringer Mannheim), which
confirmed that the lipoproteins were not toxic under the conditions
used in our experiments (Table
).
Furthermore, the adherence of monocytes in the presence of LDL or oxLDL
was not affected up to 48 hours as determined by counting the cells
that remained attached (data not shown).
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Electrophoretic Mobility Shift Assay
Nuclear extracts from 5x106 cells were prepared and
analyzed as described previously.22 Protein
concentrations were determined by the Bradford method (BioRad). The
prototypic immunoglobulin
-chain oligonucleotide was
used as a probe (5'-CAGAGGGACTTTCCGAGA-3')5 and labeled by
annealing of complementary primers followed by primer extension with
the Klenow fragment of DNA polymerase I (Boehringer Mannheim)
in the presence of [
-32P]dCTP (>3000 Ci/mmol; DuPont)
and deoxynucleoside triphosphates (Boehringer Mannheim).
Nuclear extracts (5 µg protein) were incubated with radiolabeled DNA
probes (
10 ng; 105 cpm) for 30 minutes at room
temperature in 20 µL of binding buffer (20 mmol/L
Tris-HCl, pH 7.9; 50 mmol/L KCl; 1 mmol/L
dithiothreitol; 0.5 mmol/L EDTA; 5% glycerol; 1
mg/mL BSA; 0.2% NP-40; 50 ng of poly(dI-dC)/µL). Samples were
run in 0.25x TBE buffer (10x 890 mmol/L Tris; 890
mmol/L boric acid; 20 mmol/L EDTA, pH 8.0) on
nondenaturing 4% or 6% polyacrylamide gels at 125 V. Nuclear
extracts from LPS-stimulated THP-1 cells and Hela cells were used as
positive controls. As an additional control, samples were incubated
with an excess (10x, 100x) of nonlabeled
B
oligonucleotide, which completely abolished binding of
the radiolabeled oligonucleotide to the nuclear
proteins. To control the nuclear protein content, the nuclear extracts
were incubated with a blunt end double-stranded Sp-1
oligonucleotide that was labeled with
[
-32P]ATP (>5000 Ci/mmol, DuPont) and T4
polynucleotide kinase (Boehringer Mannheim). Gels
were dried and analyzed by autoradiography.
Supershift Analysis
The nuclear extracts were incubated with 2 µL of
appropriate TransCruz gel supershift antibodies (Santa Cruz
Biotechnology) per 20 µL of reaction volume at 4°C or room
temperature for 2 hours. The following rabbit polyclonal supershift
antibodies were used: anti-p50 (recognizing part of the basic nuclear
location sequence and the N-terminal adjacent 11 amino acids of the
p105 precursor), anti-p65 (raised against a peptide corresponding to
amino acids 3-19 within the N-terminal region) and antic-Rel
(recognizing amino acids 152-176 within the N-terminal domain). This
incubation step was followed by EMSA as described above.
Immunocytochemistry
Immunocytochemistry was performed to examine the labeling for
activated NF-
B within cells. The antibody used was
-p65MAb (11 Boehringer-Mannheim), which
recognizes an epitope on the p65 subunit of NF-
B that is masked by
the bound inhibitor I
B. Therefore, this antibody reacts
only with the activated I
B released form of NF-
B. After
incubation with lipoproteins, the cells were harvested by
centrifugation, washed three times with PBS, and bound
to adhesion slides (BioRad). After fixation with reagent A of the Fix
and Perm kit (An der Grub) at a dilution of 1:2 in PBS, nonspecific
antibody binding was blocked with 0.2% bovine serum albumin
(BSA) and the cells incubated for 2 hours at 4°C with the
-p65MAb
antibody at a dilution of 1:500 in PBS with 0.25% reagent B (Fix and
Perm kit) to aid permeabilization. The cells were then incubated with a
biotinylated goat anti-mouse secondary antibody (Dianova) followed by
ABC (avidin biotin horseradish peroxidase complex) (Zymed) both for 30
minutes at room temperature, before visualization with benzidine
dihydrochloride (BDHC) (0.01% wt/vol). The slides were dehydrated and
mounted in DePex (Fluka) before photography.
Transfection of THP-1 Cells
A luciferase reporter plasmid (pGL2 Basic, Promega) containing
420 bp of the 5'-upstream region of the IL-8 gene (pGL2 IL-8) was
transiently cotransfected with a Renilla luciferase control
plasmid (pRLtK, Promega) into THP-1 cells using a DEAE-dextranbased
protocol.22 Cells were plated out after transfection to a
density of 2x106/3 ml in a 6-well plate and kept at
37°C, with 5% CO2. After 2 days the cells were incubated
for 5 hours with LDL, oxLDL, or without lipoproteins. A plasmid lacking
the 5'-upstream region of the IL-8 gene was used as a specificity
control (pGL2 Basic, Promega). Subsequent to stimulation the cells were
harvested by centrifugation and washed once with PBS.
Lysis was performed in 1x Passive Lysis Buffer (Promega) for 15
minutes at room temperature. The luciferase activity in the resulting
protein lysates was measured using the Dual Luciferase Reporter Assay
system (Promega), recording the firefly luciferase activity
produced by the experimental plasmids and the Renilla
luciferase activity produced by the constitutively active cotransfected
control plasmid. The results are expressed as normalized (against the
value for lysis buffer alone) firefly luciferase RLU divided by the
normalized RLU values obtained for the Renilla luciferase,
giving comparative data that account for any differences in
transfection efficiency.
Polyacrylamide Gel Electrophoresis and Western Blot
Analysis
Cytosolic extracts were isolated as described
earlier.47 Electrophoresis was performed with 9%
polyacrylamide gels (0.1% SDS) and carried out at 140 mA for
1.5 hours (separating gel) in a cooled system as previously
described.43 The proteins were transferred to a
nitrocellulose membrane using the wet blotting technique. After
transfer, the nitrocellulose membranes were incubated with a polyclonal
antibody raised against the carboxy terminal domain of the
inhibitor I
B-
(Santa Cruz Biotechnology), followed by
a peroxidase-conjugated polyclonal goat anti-rabbit IgG antibody
(Dianova). Antibody binding to I
B-
was visualized on x-ray film
using the enhanced chemiluminescence technique (Amersham). The protein
size was confirmed by molecular weight standards.
Northern Blot Analysis
Total RNA was extracted from 5x106 cells by means
of the micro RNA isolation kit (Stratagene). Northern blotting was
performed essentially as described.21 Five micrograms
total RNA was electrophoresed through a denaturing 1.2% formaldehyde
gel and capillary blotted overnight onto nylon membranes
(Boehringer Mannheim). Hybridization was carried out overnight
at 42°C with probes that were labeled using the Multiprime DNA
labeling system (Amersham). The blot was washed with increasingly
stringent concentrations of SSC at 52°C and exposed to
autoradiography film (Du Pont). To account for
variability in sample loading, the blot was rehybridized with a GAPDH
cDNA probe.
Determination of IL-8
Concentrations of IL-8 in cell supernatants were measured by
sandwich-type immunoassay (Quantikine, R&D systems).
| Results |
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B
B was
measured by EMSA. In our previous dose-response experiments, it had
been shown that this concentration of lipoprotein was effective for the
modulation of NF-
B target gene expression in THP-1 cells and
adherent monocytes.15 33 Exposure of cells to
copper-modified oxLDL for 1 hour increased NF-
B activation in THP-1
monocytic cells (Fig 1A
B (data not shown). Little or no NF-
B
activation was observed in cells incubated with LDL (Fig 1A
B activity by oxLDL was
measured.
|
The nuclear appearance of oxLDL-activated NF-
B was also
examined by immunocytochemistry. For this purpose, we used
-p65MAb,
a monoclonal antibody that selectively detects activated
NF-
B.11 In unstimulated cells, little staining was
detected, indicating the presence of only low amounts of active
p65-containing NF-
B dimers (Fig 1B
). In the presence of oxLDL
(12-hour incubation), we observed an increased nuclear staining of
cells, suggesting increased activation and nuclear translocation of
NF-
B, which corroborates our results from EMSA analysis. A
staining pattern similar to that of the control was observed in the
presence of LDL (data not shown). No staining was detected in the
absence of the primary antibody or when an isotype control was used
(data not shown).
NF-
B Activation by oxLDL and Associated Target Gene Expression
in Adherent Monocytes
Next, we examined whether oxLDL activates NF-
B in ex
vivo isolated human adherent monocytes. Human peripheral
blood monocytes were isolated, adhered overnight, and subsequently
incubated with the lipoproteins for 4 hours. As can be seen in Fig 2A
, incubation with 80 µg/mL of oxLDL but not LDL
activated NF-
B in these
cells. We have recently shown that
oxLDL induces the expression of IL-8 in adherent
monocytes.15 Therefore, in the same set of experiments,
the production of the NF-
B target gene IL-85
was monitored. In the presence of oxLDL, a significant increase in the
synthesis of IL-8 compared with the control or the LDL-treated sample
was observed under the same conditions in which NF-
B activation was
detected (Fig 2
, A and B). A similar induction of IL-8
production by oxLDL was observed in THP-1 cells (data not
shown).
|
IL-8 PromoterDependent Transcription Is Induced by oxLDL
To investigate whether oxLDL specifically induced IL-8
promoter-dependent transcription in our system, THP-1 cells were
transiently transfected with a luciferase reporter construct containing
420 bp of the 5'-upstream region of the IL-8 gene. Incubation with
oxLDL but not LDL significantly induced the transcription of this
construct (Fig 3
). A control plasmid
lacking the IL-8 5'-region was not induced by oxLDL.
|
OxLDL Induces p50/p65 Containing NF-
B Dimers
To identify the NF-
B subunits in the protein/DNA complexes
induced by oxLDL, polyclonal antibodies against the NF-
B subunits
p50, p65, and c-Rel were used for supershift analysis of
nuclear extracts from THP-1 cells (Fig 4A
) and adherent monocytes (Fig 4B
). Incubation with anti-p50 almost
completely abrogated or supershifted the protein/DNA complexes in EMSA
induced by oxLDL, indicating that the majority of the dimers contained
a p50 subunit. Likewise, a similar effect was observed with anti-p65,
whereas no inhibition was observed with antic-Rel. In conclusion,
oxLDL appears to induce predominantly p50/p65 containing NF-
B dimers
in human monocytic cells.
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Effects of the Antioxidant PDTC and the Proteasome Inhibitor
PSI
Various compounds were tested for their ability to modulate
oxLDL-induced activation of the NF-
B system. To demonstrate an
involvement of ROI in oxLDL-induced signaling and NF-
B activation,
the cells were incubated with the antioxidant and
H2O2 scavenger PDTC.48 49 As seen
in Fig 5A
, PDTC blocked the activation of NF-
B by oxLDL in a
dose-dependent manner. Next, we
tested the proteasome inhibitor PSI47 to
investigate whether I
B-
proteolysis was involved in oxLDL-induced
NF-
B activation. In cells pretreated with PSI for 1 hour, the
oxLDL-induced NF-
B activation was strongly inhibited (Fig 5B
). In
the same experiments, the level of I
B-
was examined in cytosolic
extracts by Western blot analysis. The data show that the
oxLDL-induced activation of NF-
B at 1 hour was accompanied by a
depletion of I
B-
, which was prevented by PSI in a similar dose
range to that in which NF-
B activation was inhibited (Fig 5B
). Under
the experimental conditions used, the toxicity of PDTC and PSI was
excluded by the WST-1 assay (data not shown).
|
In contrast, neither the xanthate D609, an inhibitor of
phosphatidylcholine-specific phospholipase C, which blocks TNF-induced
NF-
B activation,50 nor antibodies against the LPS
receptor CD 14 had an effect on oxLDL-induced NF-
B activation (data
not shown).
Time Course of oxLDL-Induced NF-
B Activation and I
B-
Depletion
In several studies, cells were incubated with oxLDL for a longer
period of time before gene expression was examined.36 37
Therefore, time course experiments were performed in which the cells
were cultivated for up to 24 hours with oxLDL, and the activation of
NF-
B and the level of I
B-
was monitored over time. Incubation
with oxLDL induced the activation of NF-
B, which reached a maximal
level at 1 hour, remained elevated at 6 hours, and returned to baseline
by 24 hours (Fig 6
). Under the same
conditions, oxLDL caused an initial depletion of I
B-
by 1 hour
followed by a transient slight increase in the level of this
inhibitor by 6 hours. In the presence of LDL or in
untreated cells, the I
B-
levels remained constant during the
incubation intervals (data not shown).
|
Loss of Inducible NF-
B Activation and Target Gene Expression
After Long-term Exposure to oxLDL
It has been reported that a longer preincubation with oxLDL
inhibits the inducible expression of the NF-
B target genes TNF-
and IL-1ß.36 37 To test whether long-term exposure to
lipoproteins can alter the induced NF-
B activation in our system,
the cells were cultured in the absence or presence of LDL or oxLDL for
24 hours followed by a 1-hour stimulation with 1 µg/mL LPS, a
very potent stimulus for NF-
B activation in cells of the monocytic
lineage. In cells preincubated with LDL, the LPS-induced NF-
B
activation was not affected (Fig 7A
). Preincubation with oxLDL,
however, significantly reduced the activation of NF-
B by LPS. Little
or no NF-
B activity was observed in cells exposed to LDL or oxLDL
for 24 hours (data not shown). In the same extracts, we also
investigated the binding of nuclear proteins to an Sp-1
oligonucleotide, which was not affected by long-term
treatment with oxLDL (Fig 7A
).
|
Next, we examined whether long-term treatment with oxLDL affects the
expression of the NF-
B target genes TNF-
and IL-1ß. The cells
were preincubated with either LDL or oxLDL for 24 hour followed by
stimulation with LPS for 3 hours, and total RNA was harvested for
Northern blot analysis. As expected, stimulation with LPS
induced mRNA expression for the two NF-
B target genes TNF-
and
IL-1ß (Fig 7B
). Exposure of cells to oxLDL but not LDL significantly
inhibited the LPS-induced expression of both genes. These data
demonstrate an inhibition of NF-
B by long-term exposure to oxLDL
associated with a negative regulatory effect on NF-
B target gene
expression in monocytic cells.
Long-term Exposure to oxLDL Prevents the Efficient
Activation-Induced Depletion of I
B-
The LPS-induced NF-
B activation and the expression of TNF-
and IL-1ß was inhibited in cells exposed to oxLDL for 24 hours (see
Fig 7B
). Therefore, the fate of the inhibitor I
B-
was
examined in cells preincubated with oxLDL for 24 hours and then
stimulated with LPS for 1 hour. As expected, incubation with LPS leads
to a rapid proteolytic removal of I
B-
within 1 hour in the
absence of lipoproteins (Fig 8
).
This effect, however, was not evident in cells pretreated with oxLDL.
In contrast, the LPS-induced removal of I
B-
was only slightly
affected by the presence of LDL (Fig 8B
). Therefore, preincubation with
oxLDL appears to prevent an efficient depletion of I
B-
after
stimulation of cells.
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| Discussion |
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B system.
In our study, a short time exposure to mildly oxLDL activated
transcription factor NF-
B in human THP-1 cells and adherent
monocytes, whereas a longer preincubation (24 hours) with these
lipoproteins inhibited the LPS-induced NF-
B activation. In
endothelial cells it has been shown that short-term
exposure to minimally or mildly oxLDL activates
NF-
B.38 39 In human and mouse macrophages, a
long-term incubation (22 to 24 hours) with mildly and highly oxLDL
inhibits this transcription factor.40 42 In one of these
studies, the appearance of an oxLDL-induced NF-
B band was observed
that was not characterized further.40 In human smooth
muscle cells, mildly and highly oxLDL had no effect on the DNA-binding
activity of NF-
B.41 However, a 2.5-hour preincubation
with extensively oxLDL significantly inhibited the LPS-induced
activation of this transcription factor, whereas mildly oxLDL showed
only a weak effect.41 All these data, taken together with
our results, indicate that various cell types in culture apparently
react differently to oxLDL and/or that the biological effects of mildly
oxLDL and highly oxLDL are different. For instance, the longer
preincubation period required to inhibit the LPS-induced NF-
B
activation in our system compared with the relatively short exposure
time found to be effective in smooth muscle cells41 could
be due to differences between monocytic and smooth muscle cells but
also a result of the fact that we used mildly oxLDL.
The oxLDL-induced activation of NF-
B in our study was accompanied by
a dramatic increase in the production of the NF-
B target
gene, IL-8.5 Using reporter constructs, we demonstrated
that oxLDL significantly induced IL-8 promoter-dependent transcription.
These data suggest that the NF-
B activated by oxLDL induces
transcription of the IL-8 gene, leading to the upregulation of IL-8
expression in monocytic cells. In addition, it has been demonstrated
that oxLDL stimulates or enhances the expression of the
NF-
Bregulated genes IL-1ß31 and tissue
factor33 in cells of monocytic origin.
ROI such as H2O2 have recently been implicated
as one of the intracellular second-messenger molecules that induce
NF-
B activation.5 7 48 51 Cell treatment with
H2O2 has been demonstrated to activate
NF-
B and induce
B-dependent gene expression.49 Cell
lines stably overexpressing the H2O2 degrading
enzyme catalase are deficient in activation of NF-
B in response to
TNF and okadaic acid.52 In contrast, stable overexpression
of cytoplasmic superoxide dismutase, which enhances the
production of H2O2 from superoxide,
potentiated NF-
B activation.52 In our study, the
activation of NF-
B by oxLDL could be inhibited by the antioxidant
and H2O2 scavenger PDTC in a dose-dependent
manner. Therefore, it is interesting to hypothesize that oxLDL-induced
signaling leads to the production of ROI followed by the
activation of NF-
B.
The oxLDL-induced activation of NF-
B was accompanied by a depletion
of I
B-
, both of which were inhibited by the proteasome
inhibitor PSI. This suggests that exposure to oxLDL induces
I
B-
proteolysis leading to NF-
B activation. The initial
depletion of I
B-
was followed by a slight and transient increase
in the level of this inhibitor in our experiments. It
should be mentioned that a number of functional NF-
B regulatory
sequences have been found in the I
B-
promoter and that the gene
encoding this inhibitor is itself regulated by
NF-
B.5 6 Therefore, the increase in the level of
I
B-
in cells exposed to oxLDL is likely to be the result of
NF-
Bmediated expression of the I
B-
gene. Long-term exposure
to oxLDL for 24 hours inhibited the LPS-induced NF-
B activation and
target gene expression in our study. Under these conditions, our data
demonstrated that the LPS-induced depletion of I
B-
was not
evident in cells preexposed to oxLDL for 24 hours, whereas in the
absence of modified lipoproteins, the expected proteolytic degradation
of I
B-
after activation was observed.5 7 This
suggests that long-term incubation with oxLDL inhibits pathways for
NF-
B activation at the level of I
B-
degradation (eg,
inhibition of the proteasome) and/or mechanisms located upstream of
this protease step.53
Recently, we have shown the presence of activated NF-
B in
some but not all macrophages present in the atherosclerotic
lesion.11 How is this possible in view of the presence of
oxLDL in the lesion, since we have shown in the present study that
the long-term exposure of macrophages to oxLDL actually
inhibits the NF-
B system? Several potential explanations arise:
Despite a significant inhibition of LPS-induced NF-
B activity by
long-term exposure to oxLDL in our study, a modest activation of this
transcription factor above the base line level remained under these
conditions (ie, in the presence of oxLDL plus LPS). The inhibition of
NF-
B activation by long-term incubation with oxLDL may also
represent an autoregulatory mechanism53 and allow
restimulation after a certain time span. Furthermore, it is not known
whether oxLDL is able to inhibit the activation of NF-
B by all of
the numerous known stimuli for this transcription factor in the
atherosclerotic lesion including TNF-
, IL-1ß, growth factors,
thrombin, and fibronectin,8 9 10 which may induce different
signaling pathways. Furthermore, macrophages should also be
able to phagocytose the oxLDL12 29 and therefore remove
these lipoproteins from their close environment, thereby preventing a
long exposure to oxLDL that presumably would be highly toxic. There are
also lipid-poor atherosclerotic regions12 in which the
macrophages would be less exposed to oxLDL. Finally, the
various subpopulations of macrophages that exist in sites of
chronic inflammatory processes such as atherosclerosis
may respond differently to oxLDL.9 12 29 In conclusion, as
in any environment of chronic inflammation, there may be both
activating and inhibiting forces in the atherosclerotic lesion which
positively as well as negatively (dys)regulate the NF-
B-system.
Future experiments should address the question to what extent both
activation and inhibition of NF-
B affect the process of
atherogenesis.
| Selected Abbreviations and Acronyms |
|---|
|
| Acknowledgments |
|---|
Received September 30, 1996; accepted May 2, 1997.
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