 |
INTRODUCTION |
The chronic inflammatory-fibroproliferative process of
atherosclerosis is triggered or modulated by various substances
accumulating in the damaged vessel wall, which have been shown to exert
specific effects on transcriptional systems or signal transduction
cascades (1). One of these substances is oxidized low density
lipoprotein (ox-LDL)1 that
has been found to display both positive and negative effects on gene
expression (2-4). Ox-LDL represents a complex mixture of various
components including lipid hydroperoxides, oxysterols, lysophosphatidylcholine, and aldehydes such as 4-hydroxynonenal (HNE)
(5, 6). The specific substances within the ox-LDL molecule that are
responsible for its effects on signaling/transcriptional regulation and
the mechanisms involved are only partially known.
The pleiotropic transcription factor NF-
B has been suggested to play
an important role in gene regulation during inflammatory and immune
reactions in the atherosclerotic lesion (3, 7-10). NF-
B exists as a
dimeric complex that is present in the cytosol in an inactive state
bound to inhibitory proteins, collectively termed I
B (11-13).
Several I
B proteins have been identified, including I
B-
(10,
14) and the more recently cloned I
B-
(15) and -
(16). A
variety of agents including inflammatory or lymphoproliferative
cytokines and microbial pathogens (12, 17) induce the activation of
NF-
B which is presumably facilitated by a network of kinases, some
of which have been recently cloned, including I
B kinase (IKK)-
and -
(18-22), NF-
B-inducing kinase (23), and mitogen-activated
protein kinase/ERK kinase kinase-1 (24). Activation of these kinases
leads to the phosphorylation of I
B which is subsequently degraded in
a ubiquitin-dependent step by the proteasome, a
multicatalytic high molecular weight protease system (14, 25-27). The
removal of the inhibitor I
B allows the translocation of the thus
activated NF-
B dimer into the nucleus. NF-
B regulatory sequences
have been found in promoters or enhancers of a variety of genes,
e.g. coding for the proinflammatory cytokines tumor necrosis
factor (TNF) and interleukin (IL)-1, chemokines such as IL-8 and
monocyte chemotactic protein-1 (MCP-1), and diverse adhesion as well as
immunoregulatory molecules (11-13).
Several laboratories, including ours, have shown that ox-LDL inhibits
the LPS-induced activation of NF-
B (4, 28, 29) and the expression of
the NF-
B target gene products TNF, IL-1
/
, and MCP-1 in
monocyte/macrophages (30-32). One report describes the impairment of
NF-
B activation by ox-LDL in vascular smooth muscle cells (33). The
suppressive activity of ox-LDL on IL-1 and TNF expression was acquired
only after extensive oxidation and was localized in the extractable
lipid component (29-31, 33). HNE is one of the most prominent aldehyde
substances generated during extensive lipid peroxidation of ox-LDL (5,
34, 35). Furthermore, HNE-modified lysine residues have been identified in the atherosclerotic lesion (36), and a variety of HNE effects in
different cell types have been described (37-40). The aim of this
study was to investigate whether HNE is able to modulate the activation
of the NF-
B system by different stimuli or alter the expression of
the NF-
B target gene product TNF. Furthermore, we examined to what
extent this aldehyde affects
B- as well as TNF and IL-8
promoter-dependent transcriptional activity. We also characterized the effects of HNE on the stimulus-induced proteolysis of
I
B-
, -
, and -
, on the three major peptidase activities of
the proteasome, as well as on the phosphorylation status of I
B-
,
in order to obtain more insight into the mechanisms involved.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture Conditions and Reagents--
THP-1 monocytic cells
(DSM, Braunschweig, Germany) were maintained in suspension in RPMI 1640 (Glutamax-1, low endotoxin) containing 7% fetal calf serum (FCS)
(Myoclone super plus, low endotoxin), 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc., Eggenstein, Germany) as
described (41). For the experiments, the cells were plated at a density
of 3 × 106 per well in 6-well culture dishes.
Peripheral blood mononuclear cells were isolated from blood samples of
normal donors by the Ficoll-Paque method as described (42). Monocytes
were isolated from mononuclear cells by adherence to achieve a purity
of approximately 90% as determined by flow cytometry. The adherent
monocytes were cultured overnight in the same medium as THP-1 cells
with 10% FCS before the experiment was started. Mono Mac 6 cells (43) were maintained in 24-well plates at a density of 2 × 105 per ml in RPMI containing 2 mM
L-glutamine, 200 units/ml penicillin, 200 µg/ml
streptomycin, 1× non-essential amino acids (all from Life
Technologies, Inc.), 1% OPI supplement containing oxalacetic acid,
sodium pyruvate, and insulin (Sigma, Deisenhofen, Germany) and 10% FCS
(Myoclone). 4-Hydroxynonenal was synthesized at the Institute of
Biochemistry, University of Graz, Austria (5, 34), and supplied to our
laboratory for use in the present study. LPS (Escherichia
coli 0111:B4), FITC-LPS, IL-1
, TNF, phorbol 12-myristate
13-acetate (PMA), and okadaic acid (OA) were purchased from Sigma. The
proteasome inhibitor PSI was purchased from Calbiochem (Bad Soden,
Germany). Endotoxin contamination was screened by the limulus
amoebocyte lysate assay (BioWhittaker, Walkersville, MD), and only
reagents with an endotoxin content of <10 pg/ml were used in the
experiments. A potential toxicity of the cell culture conditions
applied was monitored by cell morphology and count, trypan blue dye
exclusion, and the WST-1 cell toxicity test (Boehringer Mannheim,
Mannheim, Germany).
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared and analyzed as described previously (4, 44).
The prototypic immunoglobulin
-chain oligonucleotide was used as a
probe 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
(>3,000 Ci/mmol; NEN Life Science Products, Brussels, Belgium) and
deoxynucleoside triphosphates (Boehringer Mannheim). Nuclear extracts
(5 µg of protein) were incubated with radiolabeled DNA probes (~10
ng; 105 cpm) for 30 min at room temperature in 20 µl of
binding buffer (20 mM HEPES, pH 7.9, 50 mM KCl,
1 mM dithiothreitol, 0.5 mM EDTA, 10%
glycerol, 1 mg/ml bovine serum albumin, 0.2% Nonidet P-40, 50 ng of
poly(dI-dC)/µl). Samples were run in 0.25× TBE buffer (10× TBE is
as follows: 890 mM Tris, 890 mM boric acid, 20 mM EDTA, pH 8.0) on nondenaturing 4% polyacrylamide gels.
The binding of Sp-1 and AP-1 was also analyzed by EMSA using specific
consensus oligonucleotides (Promega, Heidelberg, Germany) that were
labeled with [
-32P]ATP (>5,000 Ci/mmol, NEN Life
Science Products) and T4 polynucleotide kinase (Boehringer Mannheim).
Gels were dried and analyzed by autoradiography.
Transfection of THP-1 Cells--
The following reporter plasmids
were utilized in transfection studies: 3×
B. luci, a firefly
luciferase reporter plasmid containing 3 copies of a prototypic
(5'-GGGACTTTCC-3')
B site (45); TNFkop.luci, comprising 1108 base
pairs of the TNF promoter region (45); pGL2-IL-8, containing 420 base
pairs of the IL-8 promoter region (4) or pGL2basic (Promega) lacking
any insert. These individual plasmids were transiently co-transfected
with a constitutively active Renilla luciferase control
plasmid (pRLtk, Promega) into THP-1 cells using a DEAE-dextran-based
protocol (4, 44). After transfection, cells were plated out at a
density of 2 × 106/3 ml RPMI with 7% FCS in a 6-well
plate and incubated for 2 days. After this time, the cells were first
preincubated with HNE for 1 h followed by a 5-h LPS stimulation.
Subsequent to stimulation the cells were lysed and the luciferase
activity was determined using the Dual Luciferase Reporter Assay system
(Promega). The results are expressed as firefly luciferase relative
light units (RLU) divided by the RLU values obtained for the
Renilla luciferase.
Determination of TNF and LDH--
The concentration of TNF
protein in the medium taken from experimental cultures was measured by
sandwich type immunoassay, according to the manufacturer's
instructions (Coulter Immunotech, Hamburg, Germany). The activity
(units/liter) of LDH in cytosolic extracts was measured in an automated
colorimetric analysis system (Boehringer Mannheim/Hitachi 737) at
37 °C.
Polyacrylamide Gel Electrophoresis and Western Blot
Analysis--
Cytosolic extracts were isolated as described earlier
(45). Electrophoresis was performed with 12.5% polyacrylamide gels (0.1% SDS) as described previously (41). The proteins were transferred to a nitrocellulose membrane using the wet blotting technique. After
transfer, the nitrocellulose membranes were incubated with polyclonal
antibodies against the carboxyl-terminal domains of the inhibitors
I
B-
, -
(Santa Cruz Biotechnology, Heidelberg, Germany), and
-
(a kind gift from Prof. N. Rice, (NCI-Frederick Cancer Research
and Development Center, Frederick, MD) or with a monoclonal antibody
against
-actin (Sigma). To detect I
B phosphorylation we used a
polyclonal phospho-specific anti-I
B-
-antibody (Calbiochem) which
detects I
B-
only when activated by phosphorylation at Ser-32.
This incubation was followed by the appropriate horseradish peroxidase-conjugated secondary antibody (Dianova, Hamburg, Germany). Antibody binding to I
B proteins was visualized on x-ray film using
the Western blot Chemiluminescent Reagent Plus (NEN Life Science
Products). The protein size was confirmed by molecular weight standards
(Amersham Pharmacia Biotech, Braunschweig, and Bio-Rad, Munich, Germany).
Proteasome Assay--
Purified yeast 20 S proteasomes (kindly
provided by Prof. R. Huber, Max-Planck-Institute of Biochemistry,
Martinsried, Germany) (46) were incubated with either HNE or
N-Ac-Leu-Leu-norleucinal in 20 mM Tris-HCl, pH
7.4, for 1 h at 37 °C. The assay was started by addition of
fluorogenic substrates (Bachem, Heidelberg, Germany; dissolved in Tris
buffer, 1% Me2SO) for chymotrypsin-like
(Suc-Leu-Leu-Val-Tyr-AMC, 8 µM), trypsin-like
(Bz-Phe-Val-Arg-AMC, 8 µM), or peptidylglutamyl peptide
hydrolyzing (Cbz-Leu-Leu-Glu-
NA, 40 µM) activities,
and conducted at 37 °C (where AMC is 7-amido-4-methylcoumarin;
NA is
-naphthylamide; Suc is succinyl; Bz is benzoyl; Cbz is
benzyloxycarbonyl). The rates of substrate hydrolysis were determined
using a luminescence spectrophotometer (Perkin-Elmer, Weiterstadt, Germany).
LPS Binding and Flow Cytometry--
Mono Mac 6 cells, grown in
LPS-negative medium as described, were incubated with or without HNE at
106 cells per ml for 1 h at 37 °C. After washing,
the cells were incubated with and without LPS at 10 µg/ml and 5 min
later FITC-LPS was added at 1 µg/ml. After overnight incubation at
4 °C, cells were washed, and 104 cells per sample were
analyzed using an EPICS Version 753 flow cytometer (Coulter
Electronics, Krefeld, Germany). The percentage of positive cells was
determined by subtraction of the histogram for the LPS-competed sample
from the histogram of the non-competed sample. Specific mean
fluorescence intensity is the delta mean of the competed and uncompeted
histogram in channels.
Kinase Assays--
Kinase assays were performed essentially as
described (47). Cytosolic extracts were subjected to
immunoprecipitation in TNT buffer (200 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1 mM
dithiothreitol, 0.5 µM phenylmethylsulfonyl fluoride,
leupeptin, antipain, aprotinin, pepstatin A, chymostatin 0.75 µg/ml
each; Sigma). Unspecific binding was blocked by incubation with 1 µg of normal rabbit IgG (Santa Cruz Biotechnology) and 25 µl of 6% protein A-agarose (Boehringer Mannheim) for 30 min at 4 °C followed by immunoprecipitation for 2 h at 4 °C with 1 µg of
anti-IKK-
antibody (Santa Cruz Biotechnology) and 25 µl of 6%
protein A-agarose. After washing three times with TNT buffer and three
times with kinase buffer (20 mM HEPES, pH 8.0, 10 mM MgCl2, 100 µM
Na3VO4, 20 mM
-glycerophosphate,
50 mM NaCl, 2 mM dithiothreitol, 0.5 µM phenylmethylsulfonyl fluoride, leupeptin, antipain,
aprotinin, pepstatin A, chymostatin 0.75 µg/ml each), the kinase
reaction was carried out in kinase buffer for 30 min at 30 °C in the
presence of 5 µCi of [
-32P]ATP (NEN Life Science
Products) and 500 ng of the substrate GST-I
B-
(Santa Cruz
Biotechnology). Proteins were analyzed on 12.5% polyacrylamide gels
(0.1% SDS), dried, and visualized by autoradiography.
In Vitro OA Stimulation--
For OA stimulation experiments
cytosolic extracts of unstimulated cells were incubated for 30 min at
37 °C with OA (50-200 nM) in the presence or absence of
HNE (25 µM). In addition, 2 mM ATP
(Boehringer Mannheim), 1 mg/ml ubiquitin (Sigma), and 10 mM
creatine phosphate (Boehringer Mannheim) were supplemented. The level
of phosphorylated I
B-
was analyzed by Western blot using an
antibody which detects I
B-
when phosphorylated at Ser-32.
 |
RESULTS |
Selective Effects of HNE on NF-
B Activation--
The initial
experiments were performed to examine whether HNE affects the
activation of NF-
B or other transcriptional systems (Sp-1, AP-1).
THP-1 monocytic cells were preincubated with different concentrations
of HNE and then stimulated for 1 h with LPS (1 µg/ml). The
activation of NF-
B was determined by EMSA. In the absence of HNE we
observed the expected dramatic activation of NF-
B by LPS (Fig.
1A). This increase was
slightly affected by pretreatment with HNE at 6.25 µM,
significantly decreased by 12.5 and 25 µM, and completely
abolished by 50 µM. No effect of HNE alone on NF-
B
binding activity was observed (25 or 50 µM HNE, no
induction of NF-
B activity above base line). In the same nuclear extracts we also examined the binding of nuclear proteins to
oligonucleotides comprising the Sp-1 consensus sequence, which was
unchanged by HNE treatment (Fig. 1A). The slight increase of
AP-1 binding in the presence of LPS was also not significantly changed
by HNE (25 µM, no inhibition compared with the LPS
control). Furthermore, we determined the effect of HNE ex
vivo in isolated human adherent monocytes. Similarly as observed
above, in these primary monocytic cells the LPS-induced NF-
B
activation was also inhibited by HNE in a dose-dependent
manner (Fig. 1B). In addition, experiments were performed
with monocytic Mono Mac 6 cells, which were also incubated with or
without HNE followed by a 1-h LPS stimulus (100 ng/ml). EMSA showed an
HNE dose-dependent inhibition of LPS-induced NF-
B
activation (50 µM HNE, inhibition below base line; 25 µM HNE, 62% inhibition).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 1.
HNE selectively inhibits
NF- B activation. A, THP-1
monocytic cells were incubated with HNE at the concentrations indicated
or with PSI (10 µM) for 1 h, followed by a 1-h
stimulation with 1 µg/ml LPS. EMSA was performed, and the
brackets indicate NF- B or Sp-1 binding, as shown.
Com, the probes were incubated with an excess (100×) of the
appropriate unlabeled oligonucleotide to abolish specific binding.
B, human adherent monocytes were treated as in A,
with the exception that 100 ng/ml LPS was used. EMSA was carried out,
and the results were analyzed by densitometry. The diagram shows
percent of NF- B activity normalized to Sp-1 binding relative to the
100% LPS control. C, THP-1 cells were pretreated with 25 µM HNE for 1 h and then activated with various
stimuli as follows: LPS (1 µg/ml) or IL-1 (10 pg/ml) for 1 h,
PMA (50 ng/ml) for 4 and 12 h, and TNF (1.6 ng/ml) for 1 h.
Data from EMSA were analyzed densitometrically as described above. The
dashed line indicates the 100% control value obtained with
the respective stimulus in the absence of HNE.
|
|
We then tested whether HNE (25 µM) modulates the
activation of NF-
B by other stimuli such as IL-1
, PMA, and TNF.
Our experiments demonstrated a marked inhibition of NF-
B activation
induced by a 1-h incubation with 10 pg/ml IL-1
(Fig. 1C).
Furthermore, pretreatment with HNE also significantly affected both
short and long term incubation with PMA (50 ng/ml). In contrast, the
activation of NF-
B by treatment with TNF (0.5 to 4.0 ng/ml) for 0.5 or 1 h was not affected by 25 µM HNE (Fig.
1C, and data not shown), which suggests selective and
differential inhibitory actions of HNE.
A potential toxic effect of HNE on monocytic cells was monitored by
cell morphology and count, trypan blue dye exclusion, and the WST-1
cell toxicity test. HNE was not found to be toxic at the concentrations
and conditions applied in our study (50 and 25 µM HNE, no
decrease in metabolic activity after 24 h incubation compared with
the untreated control using the WST-1 test).
HNE Inhibits Temporarily and Is Active When Added Subsequent to the
Stimulus--
The next experiments tested whether the inhibition of
NF-
B by pretreatment with HNE is reversible. THP-1 cells were
pretreated with HNE (25 µM), washed intensively, and
transferred to a new culture dish. At different time points LPS was
added for a 1-h incubation period. When LPS was added immediately or
2 h after removal of HNE a significant inhibition of NF-
B was
detected (Fig. 2A). However,
when cells were activated with LPS 4 h after HNE removal, a
reduction of the inhibitory effect was already observed. The
LPS-induced stimulation of NF-
B at 24 h after the HNE treatment
was no longer impaired, which indicates that the effect of HNE is
temporary and reversible. It should be mentioned that the observation
that addition of LPS does not activate NF-
B even 2 h after HNE
is removed also demonstrates that the effect of this aldehyde is not
due to an interaction with the stimulatory molecule in the
supernatant.

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 2.
Effects of different HNE treatment on
NF- B activation. A, the
inhibitory effect of HNE on NF- B activation is reversible. THP-1
cells were treated with HNE (25 µM) for 1 h,
extensively washed, and stimulated with LPS (1 µg/ml) at the times
indicated. EMSA was carried out, and the brackets indicate
NF- B or Sp-1, respectively. B, HNE remains effective when
added subsequent to LPS stimulation. THP-1 cells were stimulated with
LPS (1 µg/ml), and HNE (25 µM) was added at the times
indicated relative to LPS stimulation. EMSA revealed the NF- B
binding activity of the nuclear extracts (bracket).
Com, competition experiments were performed as described for
Fig. 1A.
|
|
Furthermore, a different incubation condition was used, i.e.
HNE was added together with or varying times (10 min steps) after the
addition of LPS. These experiments showed that HNE (25 µM) was still effective when given to the cells as late
as approximately 20 min after LPS (Fig. 2B).
B- as Well as TNF and IL-8 Promoter-dependent
Transcription Is Prevented by HNE--
In order to examine whether HNE
selectively prevents
B-dependent transcription in our
system, THP-1 cells were transiently transfected with a luciferase
reporter construct bearing three copies of a prototypic
B site (45).
Incubation with LPS dramatically induced the transcriptional activity
of this construct (Fig. 3A). This effect was strongly inhibited by preincubation with HNE. In
contrast, HNE showed no effect on the expression plasmid pRLtk (25 µM, no inhibition of transcriptional activity). A similar approach was used to test if HNE affects the transcription of both the
TNF and IL-8 gene which contain regulatory
B sites in their
respective promoters (12, 48). Preincubation with HNE inhibited the
transcriptional activity of both the TNF and IL-8 promoter construct in
a dose-dependent manner with a marked inhibition with 25 µM (Fig. 3, B and C).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3.
HNE inhibits B- as
well as TNF and IL-8 promoter-dependent transcription.
THP-1 cells were co-transfected with 4 µg of the appropriate reporter
plasmid and 0.2 µg of the constitutively expressed pRLtk
Renilla plasmid, left for 2 days, and then incubated with
HNE for 1 h followed by LPS stimulation for 5 h. The results
are presented as firefly luciferase relative light units
(RLU) divided by the relative light unit values obtained for
the Renilla luciferase. A, the effect of HNE on
the LPS (1 µg/ml)-induced activation of the prototypic B-driven
reporter plasmid 3× B. luci and the control plasmid pGL2 was
monitored. B, TNF promoter construct
(TNFkop.luci)-transfected cells were incubated with increasing
concentrations of HNE and stimulated with 1 µg/ml LPS. The results
are presented as percent sample values of the LPS control (100%).
C, an IL-8 promoter-driven luciferase reporter plasmid
(pGL2-IL-8) was transfected into the cells, which were subsequently
treated with HNE as above followed by LPS (1 µg/ml or 0.1 µg/ml).
Co, control.
|
|
HNE Suppresses the Production of TNF Protein--
Next we
investigated whether the inhibition of transcriptional activity by HNE
has a corresponding impact on protein production. For this purpose we
monitored the accumulation of TNF in the supernatant. Incubation of
cells with LPS induced a dramatic increase in the levels of TNF protein
which was inhibited in a dose-dependent manner by HNE with
a marked effect at 25 and 50 µM and a partial inhibition
at 12.5 µM (Fig.
4A). This was accompanied by
an inhibition of LPS-induced TNF mRNA expression, as demonstrated
by Northern blotting (data not shown). A similar inhibition of TNF
production by HNE was observed ex vivo in isolated human
adherent monocytes (Fig. 4B). The level of the
constitutively expressed intracellular protein LDH was unaffected by
the HNE treatment used in this study (25 and 50 µM, no
inhibition).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
TNF protein production is suppressed by
HNE. THP-1 cells (A) or human adherent monocytes
(B) were treated for 1 h with the indicated
concentration of HNE and subsequently stimulated for 5 h with LPS
(1 µg/ml, THP-1 cells; 100 ng/ml, adherent monocytes). The culture
supernatants were analyzed for TNF content in an ELISA. The data
(mean ± S.D., n = 3) are shown as percent of TNF
protein production in the samples in comparison to the control (LPS)
value. The actual level of TNF measured in LPS-stimulated control
supernatants varied from 90 to 300 pg/ml for THP-1 cells and 2 to 12.45 ng/ml for adherent monocytes.
|
|
Prevention of I
B-
, -
, and -
Proteolysis by HNE Is
Stimulus-dependent--
In the following stage of the
study we investigated whether HNE could affect the activation-induced
proteolysis of the I
B inhibitor proteins which trap the NF-
B
dimer in the cytosol (10, 14). Incubation of THP-1 cells with LPS over
1 h led to a significant proteolysis of I
B-
(Fig.
5A). HNE (25 µM)
when added
1 h relative to LPS significantly prevented the
LPS-stimulated proteolysis of I
B-
. A similar effect of HNE on
LPS-induced I
B-
proteolysis was observed in Mono Mac 6 cells
(data not shown). Pretreatment with the proteasome inhibitor PSI also
inhibited I
B-
degradation (Fig. 5A) as observed
earlier (45). However, addition of PSI resulted in an accumulation of
the phosphorylated form of I
B-
, an effect not seen with HNE.
Consistent with the data observed with EMSA, the TNF-induced I
B-
proteolysis was not impaired by 25 µM HNE, neither after
a 30-min (data not shown) nor after a 1-h incubation interval with this
cytokine (Fig. 5B). Stimulus-induced proteolysis of
I
B-
(15) was also inhibited by HNE as shown by a dose-response
experiment (Fig. 5C). Furthermore, we investigated the fate
of the recently cloned I
B-
(16) following treatment with LPS in
the absence/presence of HNE. In contrast to I
B-
or -
, the
LPS-induced degradation of I
B-
in THP-1 cells occurred at a later
time point, i.e. 90 and 120 min (Fig. 5D). This
decrease of I
B-
was also impaired by 25 µM HNE.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
Degradation of
I B- ,
- , and - is
selectively prevented by HNE. A, THP-1 cells were
incubated with or without HNE (25 µM) for 1 h and
then stimulated for different times with LPS (1 µg/ml). Cytosolic
extracts were analyzed by Western blot analysis. I B- is indicated
by the arrow. The asterisk depicts a 1-h
incubation with the proteasome inhibitor PSI (10 µM),
followed by LPS (1 h). The accumulation of the phosphorylated form of
I B- in the presence of PSI is shown by the double arrow.
B, cells were incubated as in A but with a 1-h
stimulation with either LPS (1 µg/ml) or TNF (1.6 ng/ml). I B-
is shown by the arrow. C, cells were pretreated with
increasing concentrations of HNE followed by LPS stimulation. The level
of I B- (arrow) was monitored by Western blot analysis.
D, THP-1 cells were incubated in the presence/absence of HNE
(25 µM) and then activated with LPS for different time
intervals. I B- is depicted by the arrow.
|
|
Proteolytic Activities of the Proteasome Are Not Affected by
HNE--
Inhibition of I
B proteolysis could be due to prevention of
proteasome peptidase activities (25). Therefore, we examined the
effects of HNE on the three major catalytic activities of this protease
complex. 20 S proteasome particles were preincubated with 100 µM HNE for 1 h. This was followed by addition of
fluorogenic substrates for chymotrypsin-like, trypsin-like, or
peptidylglutamyl peptide hydrolyzing activity. Table
I demonstrates that none of these
activities were significantly impaired by pretreatment with HNE,
whereas the chymotrypsin-like activity was significantly inhibited by
the proteasome inhibitor N-Ac-Leu-Leu-norleucinal. It should
be mentioned that the chymotrypsin-like activity has been previously
identified as mainly responsible for I
B degradation (25). In a
different approach we pretreated the fluorogenic substrates with HNE
before they were added to the assay, which also did not affect the
proteasomal functions tested (data not shown).
View this table:
[in this window]
[in a new window]
|
Table I
Effect of HNE and ALLN on proteolytic activities of the
proteasome
20 S proteasomal particles were incubated with 100 µM
HNE or 10 µM N-Ac-Leu-Leu-norleucinal
(chymotrypsin-like activity) and 100 µM
N-Ac-Leu-Leu-norleucinal (trypsin-like and PGPH activity)
for 1 h, followed by addition of the fluorogenic substrates. The
rate of substrate hydrolysis in the absence of HNE or ALLN,
respectively, was defined as the 100% control value, and data are
presented as mean ± S.D. (n = 2). The
abbreviations are explained under "Experimental Procedures."
|
|
LPS Binding in the Presence of HNE--
In these experiments we
wanted to determine whether pretreatment with HNE affects the binding
of LPS to monocytic cells. Unfortunately, with the sensitivity of
available methods, non-differentiated THP-1 cells do not show
detectable LPS binding (49), and this was confirmed in our studies
using E. coli LPS-FITC (data not shown). In order to study
the effects of HNE on LPS binding, we therefore turned to the more
mature cell line Mono Mac 6 (43). In a representative series of
experiments, LPS binding was detected on 25.7 ± 3.0% of these
cells with an average intensity of 19.1 ± 3.0 channels
(n = 3). Preincubation with even the highest
concentration of HNE used in this study (50 µM) resulted
only in a moderate reduction of LPS binding to 21.8 ± 5.1%
(n = 3; 15% inhibition) and 15.8 ± 4.4 channels
(17% inhibition).
HNE Specifically Blocks Phosphorylation of I
B--
Activation
of NF-
B occurs via phosphorylation of I
B-
at the serines at
position 32 and 36 (50). This has been shown to enable conjugation with
ubiquitin followed by proteasome-mediated degradation of I
B,
resulting in the release of active NF-
B (10, 25). To determine I
B
phosphorylation, we used a phospho-specific anti-I
B-
-antibody
that detects I
B-
only when activated by phosphorylation at
Ser-32. In the first set of experiments we examined the phosphorylation
status of I
B-
over time following LPS stimulation. In
unstimulated cells no signal was detected (Fig.
6A). Activation of cells with
LPS led to an increase of phosphorylated I
B-
reaching a plateau
between 30 and 40 min. This effect was markedly reduced in HNE (25 µM)-pretreated cells (Fig. 6B). An opposite
result was observed in the presence of the proteasome inhibitor PSI,
where a dramatic accumulation of the phosphorylated form of I
B-
was detected following LPS stimulation. The level of
-actin was not
changed under these experimental conditions (Fig. 6B). In
agreement with the results described in Fig. 5B, the
TNF-induced phosphorylation of I
B-
at Ser-32 was not
affected by HNE (Fig. 6C).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 6.
I B-
phosphorylation is inhibited by HNE. A, the
LPS-induced phosphorylation of I B- was investigated using an
antibody that detects I B- only when activated by phosphorylation
at Ser-32 (arrow). THP-1 cells were stimulated for varying
times with LPS (1 µg/ml), and cytosolic extracts were analyzed by
Western blot. The asterisk marks a 1-h incubation with the
proteasome inhibitor PSI (10 µM), followed by LPS.
B, HNE (25 µM) was added to the cells 1 h
prior to LPS stimulation for 30 or 40 min. The cytosolic extracts were
analyzed as in A. In the same extracts the level of
-actin was investigated (arrow). C, cells were
pretreated with HNE as described in B before they were
stimulated with TNF (1.6 ng/ml) for 5 min. The cytosolic extracts were
examined as described above.
|
|
Effect of HNE on I
B Kinase Activity and OA Stimulation--
We
further investigated whether HNE directly affects the IKK-
activity
in LPS-treated cells using I
B kinase assays. Recent studies using
THP-1 cells have shown that LPS-induced IKK-
activity reached a
maximum at 60 min (51). Therefore, cells were stimulated with LPS for
1 h before cytosolic extracts were prepared. IKK-
was isolated
by immunoprecipitation, and the kinase activity was determined by
measuring the phosphorylation status of the substrate GST-I
B-
.
Incubation with LPS induced a marked phosphorylation of GST-I
B-
(Fig. 7A). Different results
were now obtained when HNE was added prior to LPS stimulation compared
with when it was added directly to the kinase assay. Similar to the
results described in Fig. 6, little or no phosphorylation was detected
when HNE was given to the cells prior to LPS addition (Fig.
7A, CE). An opposite effect was seen when HNE was
added directly to the assay in vitro, simultaneously with
GST-I
B-
. Under these conditions, the level of GST-I
B-
phosphorylation induced by LPS was unaffected by the presence
of HNE (Fig. 7A, KA).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of HNE on IKK-
activity and OA stimulation. A, THP-1 cells were
incubated with or without LPS (1 µg/ml) for 1 h. IKK- was
immunoprecipitated from cytosolic extracts, and kinase activity was
determined using GST-I B- as a substrate. CE indicates
a 1-h preincubation of cells with HNE (25 µM) prior to
LPS stimulation. KA indicates an in vitro HNE
treatment (25 µM), which means that this aldehyde was
added after immunoprecipitation directly to the in vitro
kinase reaction together with the GST-I B- as substrate. The
phosphorylated GST-I B- is indicated by the arrow. B,
cytosolic extracts of THP-1 cells were stimulated in vitro
with different concentrations of OA for 30 min at 37 °C in the
presence or absence of HNE (25 µM). The phosphorylation
of I B- was investigated by Western blot using an antibody that
detects I B- only when phosphorylated at Ser-32
(arrow).
|
|
Okadaic acid, a phosphatase inhibitor, is known to stimulate NF-
B
(52). We investigated whether the in vitro effect of this
substance was affected by HNE. Cytosolic extracts from unstimulated cells were treated with or without 25 µM HNE and then
with different concentrations of OA for 30 min. Neither the OA-induced
phosphorylation of I
B-
at Ser-32 nor I
B-
proteolysis were
inhibited by this treatment (Fig. 7B and data not shown).
 |
DISCUSSION |
The presented report demonstrates that HNE, a prominent aldehyde
component of atherogenic ox-LDL (2, 3, 34, 35), specifically and
reversibly inhibits the activation of NF-
B and
B-dependent transcription in a dose range expected to be
found in extensively oxidized forms of LDL (5). Furthermore, TNF and
IL-8 promoter-dependent transcription as well as the
production of TNF mRNA and protein was dose-dependently
reduced in the presence of HNE which implies that the effect of HNE on
the NF-
B system is indeed associated with functional consequences
(12, 48). The inhibitory actions of HNE parallel those of ox-LDL on
NF-
B and target gene expression, including TNF, reported previously in cells of the monocytic lineage (4, 28-32).
HNE inhibited the activation of NF-
B by a variety of stimuli,
i.e. LPS, IL-1
, and PMA. To further elucidate how HNE
modulates cellular function, we used LPS as a stimulus in most of the
experiments. This mediator is a potent activator of monocytic cells
(48) and has been used earlier as a tool to study the effects of ox-LDL on monocyte/macrophage gene expression (4, 28-32). More importantly, certain forms of LPS may have some pathophysiological relevance in
atherogenesis since LPS from Gram-negative Chlamydia
pneumoniae has recently been shown to be present in the
atherosclerotic lesion (53, 54). Furthermore, it has been suggested
that infection of cells with C. pneumoniae modulates gene
expression in this environment (53, 55). Since to our knowledge it is
not possible to get a large amount of C. pneumoniae LPS, we
used the commercially available E. coli LPS. We are aware
that there are differences between both forms of LPS (56). However, it
should be mentioned that LPS from a different strain of
Chlamydia, i.e. trachomatis, has been
shown to significantly activate NF-
B (56).
If one assumes that some forms of C. pneumoniae LPS or other
substances such as IL-1 (1) activate NF-
B and related target cytokine expression in the atherosclerotic lesion, what is then the
pathophysiological significance of the suppressive effect of HNE on
monocyte/macrophage inflammatory gene expression? The following
speculative scenario could be envisaged. An antigen (e.g.
C. pneumoniae)-induced production of cytokine mediators may
be required to allow the coordinated orchestration of immune cell
behavior between monocyte/macrophages and T cells in the atherosclerotic lesion (57) with the aim of removing C. pneumoniae or any other foreign material and finally resolving
inflammation. This cytokine fine-tuned monocyte/macrophage/T cell
interaction may be impaired by ox-LDL-related compounds such as HNE,
allowing the development of a state of chronic, low level inflammation, which is a characteristic feature of the atherosclerotic lesion (1-3).
Our data show that HNE prevented the degradation of the cytosolic
NF-
B inhibitor proteins I
B-
(10) and -
(15) following 1 h of LPS stimulation. It is of note that ox-LDL is also capable of inhibiting LPS-induced I
B-
degradation, as shown earlier by
our laboratory (4). Surprisingly, a significant degradation of the
recently cloned I
B-
(16) was only observed after a longer LPS
incubation period of 90 and 120 min in THP-1 cells in the present
study. This LPS-induced slow decrease was also impaired by pretreatment
with HNE. It should be mentioned that the regulation of I
B-
is
only partially understood in monocytic cells and requires further
investigation. I
B proteolysis is mediated by the proteasome, and the
chymotrypsin-like proteasome activity has been demonstrated to be most
important for I
B degradation (25). Therefore, we tested the effect
of HNE on the three major peptidase activities of this multicatalytic
enzyme complex (26, 27). Our experiments demonstrated that
preincubation with HNE did not modulate these activities.
Stimulus-induced degradation of I
B by the proteasome requires the
phosphorylation of these inhibitor proteins at specific residues (50).
For example I
B-
is phosphorylated following activation at the
amino-terminal Ser-32 and Ser-36 (50). This was the reasoning behind
our examination of whether HNE interferes with the phosphorylation
of I
B. Indeed, using a phospho-specific antibody we were able to
demonstrate that HNE specifically prevented the LPS-induced
phosphorylation of I
B-
. At this point it should be mentioned that
our binding studies showed that even at the highest concentration of
HNE only a marginal reduction in LPS binding was achieved. This
indicates that the profound effect of HNE on NF-
B is not due to an
interference with the LPS-binding properties of the cells. Hence, HNE
appears to inhibit I
B phosphorylation at a signaling stage located
downstream from the LPS receptor level.
Various stimuli activate the NF-
B-system by engaging different
receptor/signaling pathways (58, 59). This involves the independent/simultaneous activation of a network of kinases
constituting several hierarchical modules that ultimately leads to the
phosphorylation of I
B (10, 50). In our study, HNE was able to
inhibit the activation of NF-
B by LPS, IL-1
, and PMA but not by
TNF, which indicates that the signaling pathways induced by the tested
stimuli exhibit different sensitivity to HNE. Interestingly, TNF, as
well as okadaic acid given in vitro, still activated
phosphorylation/proteolysis of I
B-
in the presence of HNE.
Furthermore, kinase assays showed that HNE was correspondingly unable
to inhibit LPS-induced IKK-
activity when added in vitro
directly to the kinase assay step, but when cells were treated with HNE
prior to the assay, the kinase activity was reduced. This could mean
that the signalsome (IKK-
, IKK-
, NF-
B-inducing kinase,
additional proteins), suggested to represent the bona fide I
B kinase
(18-23), may be not directly impaired by this aldehyde but rather a
more upstream receptor proximal step. Several molecules are involved in
NF-
B activation, such as TNF receptor-associated factor 6 (60) or
mitogen-activated ribosomal S6 protein kinase (pp90rsk) (61),
which presumably are not engaged by TNF but may participate in
signaling by other stimuli. For example, dominant-negative pp90rsk has been shown to interfere with phorbol ester-induced,
but not with TNF-induced, degradation of I
B-
in vivo
(61). In this context, it is also interesting to cite recent work which
suggests that LPS specifically induces an IL-1 receptor-like NF-
B
signaling cascade (62). On the other hand, it should be mentioned that TNF activates several (kinase) pathways such as TNF receptor-associated factor 2-, mitogen-activated protein kinase/ERK kinase kinase-1, or
sphingomyelinase-associated cascades (24, 58, 59, 63) which may not be
activated by the other stimuli and not be affected by HNE. These
various pathways offer potential points of divergence in the signaling
events following LPS/IL-1
/PMA or TNF stimulation and thereby the
possibility of a differential effect of HNE.
In conclusion, our data suggest that the aldehyde HNE is one of the
active components in ox-LDL responsible for the inhibitory capacities
of this lipoprotein on the NF-
B system. Treatment with HNE appears
to block selectively signaling events that are required for I
B
phosphorylation, thereby preventing NF-
B activation. The inhibition
of NF-
B-regulated gene expression may contribute at certain stages
of atherogenesis to the low level of chronic inflammation. Besides
atherosclerosis, HNE is potentially involved in other degenerative
diseases such as liver cirrhosis (64) or neurodegenerative processes
including Alzheimer's (65, 66) and Parkinson's disease (67).
Therefore, the inhibition of NF-
B/Rel and regulated gene expression
may be a process relevant to a broader field of chronic
inflammatory/degenerative disease.