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INTRODUCTION |
Aldehydes are products and propagators of oxidative stress (1).
They are reactive electrophiles that form adducts to protein and DNA
that have been detected in tissues from healthy human beings and
individuals with various diseases (2-6). Consequently, aldehydes
modulate the activities of numerous proteins, induce mutations, and
alter cell cycle progression (7-12). For example, malondialdehyde, a
major carbonyl product of lipid peroxidation, is mutagenic and
carcinogenic and induces cell cycle arrest at the G1/S and
G2/M checkpoints (7). The G1/S arrest in human colon and lung cancer cells (RKO and H1299, respectively) is caused by
induction of the cyclin-dependent kinase inhibitor, p21,
whereas the G2/M arrest appears to be due to a reduction in
the level of the cdc2 kinase. Thus, alteration of gene expression
triggered by protein or DNA damage may contribute to the range of
biological effects exerted by aldehydes.
A panoply of pathophysiological responses is also exerted by
4-hydroxynonenal (HNE),1 the
major toxic product of lipid peroxidation (1). HNE reacts with
sulfhydryl and amino groups and leads to inactivation of DNA
polymerases, dehydrogenases, and various transporters, inter alia (13). It also causes cell cycle arrest and apoptosis (8-10). HNE treatment of cells alters the expression of several transcription factors including c-Myc (12), c-Myb (14), and c-Jun (15), suggesting that it may have more global effects on protein expression and cell function. The induction of c-Jun by HNE is associated with activation of JNK kinase and p38 kinase, perhaps by
H2O2 modulation of upstream signaling pathways
(15, 16).
A major signaling pathway associated with inflammation and oxidative
stress is mediated by the transcription factor NF-
B (17-19).
NF-
B consists of heterodimers of two polypeptides, p50 and p65,
which are members of a family of proteins related to the proto-oncogene
c-rel (20, 21). Inactive NF-
B is located in the
cytosol, bound to its inhibitory protein, I
B. Dissociation of
NF-
B from I
B is a critical step in NF-
B activation that leads
to translocation of NF-
B to the nucleus, enabling DNA binding and
transactivation (22). This process is triggered by sequential phosphorylation and ubiquitination of I
B
, followed by digestion of the ubiquinated protein by the proteasome (23-25). The enzyme that
catalyzes the ubiquitination of phosphorylated I
B is constitutively active. Hence, in most cases, the key event for NF-
B activation is
phosphorylation of two serine residues at the N terminus of I
B by
I
B kinase (IKK) (23, 24).
We report here that treatment of RKO and H1299 cells with HNE leads to
a dramatic loss of DNA binding and transcriptional activation by
NF-
B in cells treated with tetradecanoylphorbol acetate (TPA) and
ionomycin (IM). The loss of NF-
B activity is due to stabilization to
the I
B
-NF-
B complex, which results from a decrease in
the rate of turnover of I
B
. The prevention of I
B
turnover
is attributable to the inhibition of IKK caused by direct reaction with
HNE. These findings indicate that HNE is a potent inhibitor of the
NF-
B-dependent cell signaling.
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EXPERIMENTAL PROCEDURES |
Cell Lines, Culture Conditions, and Chemical
Treatment--
Human colorectal carcinoma cells (RKO) were maintained
in McCoy's 5A medium (Hyclone, Logan, Utah). Human large cell lung carcinoma cells (H1299) were maintained in F-12 medium (Hyclone), and
human lymphoma Jurkat T cells were maintained in RPMI (Hyclone). RKO
and H1299 cells were grown in the presence of 10% bovine serum, and
Jurkat T cells were grown in the presence of 10% heat-inactivated fetal bovine serum. All media were supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin. Cells were maintained in 5%
CO2 at 37 °C. RKO and H1299 cells were plated 18-24 h
prior to chemical exposure and were 50-70% confluent at a density of 7 × 105/ml at the time of treatment. HNE (a generous
gift from V. Amarnath, Vanderbilt University) and TPA (Sigma)
were dissolved in 70% ethanol, and IM (Calbiochem, San Diego, CA) was
dissolved in Me2SO. The final concentration of
ethanol or Me2SO in the medium was
0.1%.
Nuclear Extract, Cytoplasmic Extract, and Total Cell
Extract--
Cells were washed twice with ice-cold phosphate-buffered
saline and lysed in buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.2 mM EDTA, 1 mM
Na3VO4, 50 mM NaF, 1 mM
DTT, and the protease inhibitors antipain (5 µg/ml), leupeptin (5 µg/ml), pepstatin A (5 µg/ml), chymostatin (5 µg/ml),
phenylmethylsulfonyl fluoride (50 µg/ml) (Sigma)) for 30 min at
4 °C. After addition of Nonidet P-40 (Roche Molecular Biochemicals)
(final concentration, 0.4%), cell lysates were incubated on ice for 10 min. Nuclear and cytoplasmic fractions were separated by centrifugation
at 1000 × g. The supernatant (cytoplasmic extract) was
cleared by further centrifugation at 10,000 × g in a
microcentrifuge for 10 min (cytosolic extract). The pellets were washed
once with buffer A and resuspended in buffer B (20 mM HEPES
(pH 7.9), 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 1 mM DTT, 25%
glycerol, and protease inhibitors (as above)). The suspension was
agitated for 30 min at 4 °C and centrifuged at 10,000 × g for 10 min. The supernatant containing nuclear proteins was collected. For isolation of total cell extracts, cells were lysed
in kinase lysis buffer (KLB: 40 mM Tris (pH 8.0), 250 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 5 mM EGTA, 10 mM
-glycerophosphate, 10 mM NaF, 0.3 mM Na3VO4,
1 mM DTT, and protease inhibitor mixture tablets (Roche
Molecular Biochemicals)) for 20 min at 4 °C. The cell lysates were
centrifuged at 10,000 × g for 10 min, and the resulting supernatants (total cell extracts) were collected. Protein concentration was determined by the bicinchoninic acid protein assay (Pierce).
Western Analysis--
Cellular protein (30-50 µg) was
resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and
electrophoretically transferred onto polyvinylidene difluoride
membranes (Millipore, Bedford, MA). The membranes were blocked with 5%
nonfat milk in Tris-buffered saline (50 mM Tris·HCl (pH
7.5), 150 mM NaCl) containing 0.1% Tween 20 and then
incubated with anti-I
B
and anti-IKK polyclonal or
anti-phospho-specific Ser-32 I
B
monoclonal (Santa Cruz
Biotechnology, Santa Cruz, CA) antibodies or anti-HNE-Michael adducts
antiserum (Calbiochem). The primary antibody complex was then stained
with horseradish peroxidase-conjugated anti-rabbit or anti-mouse
secondary antibodies (Amersham Pharmacia Biotech). Protein bands were
visualized by enhanced chemiluminescence (ECL Western blotting
detection system, Amersham Pharmacia Biotech).
Electrophoretic Mobility Shift Assay--
The double-stranded
oligonucleotide probes used to assay NF-
B and AP1/c-Jun
binding were 5'-AGT TGA GGG GAC TTT CCC AGG C-3' and 5'-CGC
TTG ATG AGT CAG CCG GAA-3', respectively (Promega, Madison,
WI; bold letters indicate the binding site). DNA probes (3.5 pmol) were
reacted with [
-32P]ATP (10 µCi) in the presence of
T4 polynucleotide kinase (10 units). Labeled probes were
purified on a Sephadex G50 column (Amersham Pharmacia Biotech).
Electrophoretic mobility shift assays were conducted with 20,000 cpm of
oligonucleotide probe per sample (35 fmol of double-stranded oligonucleotide).
To assess NF-
B binding, nuclear extracts (5-10 µg) were incubated
with the labeled DNA probe in the presence of poly(dI-dC) (2 µg),
bovine serum albumin (2 µg), and Nonidet P-40 (0.5%) in 20 µl of
reaction buffer (10 mM HEPES (pH 7.9), 20 mM
KCl, 0.5 mM EDTA, 2.5 mM DTT, and 4% Ficoll)
at room temperature for 10 min. Similarly, AP-1 binding was evaluated
by incubating nuclear extracts (5-10 µg) in reaction buffer with 1.5 mM MgCl2 and 5 mM DTT in the
presence of labeled DNA probe, poly(dI-dC) (2 µg), and bovine serum
albumin (2 µg) at room temperature for 10 min. The specificity of
binding was examined both by competition with unlabeled oligonucleotide
and by supershift assay with respective antibodies. In supershift
experiments, antibodies (1-2 µg) directed against
NF-
B/p65, NF-
B/p50, or AP1/c-Jun (Santa Cruz
Biotechnology) were incubated with nuclear extracts for 45 min at
4 °C before addition of labeled probe. In competition experiments,
antibodies were replaced with a 50-fold molar excess of unlabeled oligonucleotide.
Binding activity was analyzed by electrophoretic mobility shift assay
using a 4-5% polyacrylamide gel and Tris, glycine, EDTA buffer (5 mM Tris (pH 8.4), 9 mM glycine, and 0.2 mM EDTA). Visualization and quantification of radioactive
bands were performed using a PhosphorImager (Molecular Dynamics).
Luciferase Assay--
Cells were plated in 6-well plates at a
density of 4 × 105 cells per well 18-24 h prior to
transfection. Cells were transfected with 0.5 µg of reporter
construct in a pGL2 luciferase-expressing vector (Promega) using
LipofectAMINE reagent (Life Technologies, Inc.). The plasmid was
constructed with 6× NF-
B binding sites upstream of the SV40
promoter. Thus, the luciferase reporter gene was under NF-
B control.
Cells were exposed 18-24 h after transfection to TPA/IM (0.08 µM/2 µM) or TPA/IM plus HNE (10-80
µM) for 6 h and lysed in luciferase reporter lysis
buffer (Promega). Total cell lysates (5-10 µg of protein) were
determined for luciferase activity by the luciferase assay reagent
(Promega) in a Monolight 2010 luminometer (Analytical Luminescence Laboratory).
Kinase Assay--
To determine IKK activity, total cell extracts
(200-250 µg of protein) were rotated with anti-IKK
antibody (2 µg/ml) in KLB containing 0.5 M NaCl for 1 h at
4 °C and then for an additional 2 h with protein A-Sepharose
beads (Amersham Pharmacia Biotech). The immunoprecipitates were washed
twice with KLB, 0.5 M NaCl and twice with kinase
buffer (20 mM HEPES (pH 7.5), 10 mM
MgCl2, 0.1 mM Na3VO4,
10 mM
-glycerophosphate, 1 mM DTT, 50 mM NaCl, 2 mM phenylmethylsulfonyl fluoride,
and protease inhibitors). The washed beads were incubated with 20 µl
of kinase buffer containing 2 µg of GST-I
B
substrate, 50 µM ATP, and 2 µCi of [
-32P]ATP for 30 min at 30 °C. The reactions were stopped by addition of 5× SDS-PAGE
sample buffer. The proteins were resolved by 10% SDS-PAGE and
transferred onto a polyvinylidene difluoride membrane. IKK activity was
evaluated from the formation of
-32P-I
B
-GST by
autoradiography. The levels of IKK
and GST-I
B
were analyzed by
Western blot and ink staining, respectively.
HNE Inhibition of IKK Activity--
Total cell extracts
(200-250 µg of protein) were immunoprecipitated with anti-IKK
antibody and bound to protein A-Sepharose as described above.
Immunoprecipitates served as the enzyme source for testing the effect
of HNE on IKK activity in vitro. Prior to use,
immunoprecipitates were washed twice with KLB, 0.5 M NaCl and twice with kinase buffer with or without DTT (1 mM).
Cleared and washed beads were treated with HNE in 30 µl of kinase
buffer in the presence or absence of DTT (1 mM) for 10 min
at 30 °C. HNE treatment was terminated by adding 300 µl of
ice-cold kinase buffer with 1 mM DTT. Following treatment,
beads were centrifuged and subjected to kinase assay as described.
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RESULTS |
HNE Blocks DNA Binding Activity of NF-
B but Not
AP-1/c-Jun--
To determine whether HNE initiates a cellular
response that suppresses NF-
B DNA binding, we evaluated the effects
of HNE treatment on NF-
B activity in H1299 cells stimulated with TPA and IM. H1299 cells are derived from a large cell lung carcinoma and
are p53-deficient. Fig. 1 demonstrates
that treatment of H1299 cells with TPA/IM resulted in enhanced NF-
B
DNA binding activity in nuclear extracts. Increased activity was
detectable within 5 min, reached a maximum at 15 min, and declined
after 30 min of TPA/IM stimulation. The specificity of the nuclear
binding activity was confirmed by competition of the labeled probe with a 50-fold excess of unlabeled NF-
B oligonucleotide (Fig.
1A, lane 9). No competition was observed with an
excess of an oligonucleotide containing an SP-1 sequence (data not
shown). Treatment of the extracts with either anti-NF-
B p50 or
anti-NF-
B p65/Rel A antibodies yielded supershifted bands (Fig.
1A, lanes 10 and 11). Hence, the
binding is indeed NF-
B-specific. Pretreatment of H1299 cells with 40 µM HNE for 30 min completely prevented the
TPA/IM-dependent increase in NF-
B DNA binding activity.
In fact, HNE pretreatment lowered the basal levels of NF-
B binding
activity 2-fold (Fig. 1A, compare lanes 1 and
5).

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Fig. 1.
Effect of HNE on the binding of nuclear
proteins to oligonucleotides containing NF- B
and AP-1 recognition sequences. H1299 cells, either untreated or
pretreated with HNE (40 µM) for 30 min, were incubated
with TPA plus ionomycin (TPA/IM, 0.08 µM/2
µM) at 37 °C for the time indicated. The binding
activity of the nuclear extracts prepared from these cells was analyzed
by an electrophoretic mobility shift assay using
-32P-labeled NF- B (A) or AP-1
(B) oligonucleotide probes. Equal amounts of nuclear extract
were added to each lane. The specificity of binding was determined
either by adding a 50-fold molar excess of unlabeled oligonucleotides
as competitor or by supershift assays with respective antibodies. The
results are representative of three independent experiments.
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The inhibitory effect of HNE was not restricted to H1299 cells. A
similar inhibition of TPA/IM-stimulated increase in NF-
B binding was
observed in RKO cells. RKO is a human colon carcinoma that is wild-type
for p53 but mismatch repair-deficient. The effects of HNE in RKO cells
were not as dramatic as in H1299 cells, because of the higher basal
level of activity in the former (data not shown).
To determine whether the inhibitory effect of HNE was a nonspecific
effect, we performed similar experiments probing for an alteration in
TPA/IM-stimulated AP-1/c-Jun DNA binding activity. Using the
same preparations of nuclear protein from H1299 cells that were used
for the experiment summarized in the legend to Fig. 1A, we
found that AP-1 binding activity was neither stimulated by TPA/IM nor
inhibited by HNE (Fig. 1B). Binding specificity was
confirmed, as before, by competition with unlabeled probe and
supershift with anti-AP-1 antibody (Fig. 1B, lanes
9 and 10, respectively). The supershift in this
particular experiment was relatively weak. Clearly, concentrations of
HNE that completely prevent DNA binding by NF-
B have no effect on
DNA binding by AP-1.
HNE Blocks NF-
B Transactivation in H1299 Cells--
To
correlate HNE effects on NF-
B transactivation with DNA binding
inhibition, an NF-
B-dependent, luciferase-expressing
vector was employed. Twenty-two h after transient transfection with the luciferase reporter, H1299 cells were stimulated with TPA/IM or treated
with HNE and TPA/IM for 6 h. Control cells were not treated with
HNE or with TPA/IM. Cell extracts were prepared and analyzed for
luciferase activity. TPA/IM treatment induced a 3-fold increase in
luciferase activity relative to untreated cells (Fig.
2). HNE treatment suppressed the
TPA/IM-induced increase in luciferase activity in a
dose-dependent manner, with 20 µM HNE
providing complete suppression and higher doses decreasing luciferase
activity to below unstimulated levels (Fig. 2). Thus, HNE inhibited
both NF-
B DNA binding and NF-
B transcriptional activation.
Parallel experiments with RKO cells produced similar results, as shown in the lower panel of Fig. 2.

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Fig. 2.
HNE blocks NF- B
transcriptional activation. H1299 or RKO cells were transiently
transfected with a luciferase expression vector containing an SV 40 promoter controlled by six NF- B binding sites. Following a 24-h
incubation, cells were treated with TPA/IM (0.08 µM/2
µM) or HNE plus TPA/IM at the concentration
(µM) indicated for 6 h. Control cells were
untreated. Luciferase activity in total cell lysates (5-10 µg) was
analyzed by a luminometer using luciferin as substrate. The results
represent the mean ± S.D. calculated from three independent
experiments.
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HNE Blocks I
B
Degradation in H1299 Cells and Jurkat T
Cells--
NF-
B activation requires degradation of the inhibitory
protein, I
B
(26, 27). Consequently, HNE inhibition of NF-
B DNA
binding and transactivation activities could result from the inhibition
of I
B
degradation. To test this possibility, the effects of
TPA/IM stimulation and HNE treatment on I
B
degradation were
evaluated. Treatment of H1299 or Jurkat T cells with TPA/IM for 0-30
min at 37 °C resulted in a rapid decrease in cellular I
B
protein (Fig. 3). For H1299 cells, the
reduction in the level of I
B
protein appeared maximal by 5 min,
and some increase was evident by 30 min (Fig. 3A,
lanes 1-4). For Jurkat T cells, the reduction in the level
of I
B
protein was detectable at 5 min, with complete
disappearance evident in 20 min (Fig. 3B, lanes 1-4). In Jurkat cells, the TPA/IM-mediated decrease in cellular I
B
concentrations resulted from an induction of I
B
phosphorylation followed by a degradation of phosphorylated I
B
(p-I
B
, Fig. 3B, lanes 1-4). In contrast,
no detectable p-I
B
was found in TPA/IM-treated H1299 cells (Fig.
3A, lanes 1-4). Pretreatment of cells with HNE
prevented the TPA/IM-mediated reduction of I
B
concentration (Fig.
3, A and B, lanes 5-8). In addition,
HNE pretreatment completely abolished the formation of p-I
B
in
Jurkat T cells. Thus, it appears likely that HNE treatment prevents
I
B
degradation by inhibition of I
B
phosphorylation.

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Fig. 3.
HNE blocks
I B degradation by
inhibiting its phosphorylation. A, H1299 cells either
untreated or pretreated with HNE (40 µM) for 30 min were
incubated with TPA/IM (0.08 µM/2 µM) at
37 °C for 0-30 min. B, Jurkat T cells either untreated
or pretreated with HNE (30 µM) for 30 min were incubated
with TPA/IM (0.04 µM/3 µM) at 37 °C for
0-20 min. The levels of I B protein in cytosolic extracts (40 µg/lane) were analyzed by Western blot. p-I B was detected by a
phospho-specific monoclonal antibody that recognized I B
phosphorylated on Ser-32. The results are representative of three
independent experiments.
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HNE Inhibits IKK Activity in Jurkat T Cells--
IKK activity is
required for I
B
phosphorylation (28-30). Thus, one possible
mechanism to explain the inhibitory effect of HNE on TPA/IM stimulation
of NF-
B activity is that HNE inhibits IKK activity. To test this
possibility, Jurkat T cells, with or without a 30-min pretreatment with
HNE, were stimulated with TPA/IM. Total cell extracts were prepared,
and IKK activity was determined using a fusion protein of I
B
and
glutathione S-transferase (I
B
-GST) as substrate.
Kinase activity was evaluated by incorporation of 32P into
the fusion protein substrate. Incubation of the IKK substrate with
TPA/IM-stimulated cell extracts resulted in a
time-dependent increase in I
B
phosphorylation (Fig.
4, lanes 1-4). Pretreatment of cells with HNE significantly inhibited the formation of
32P-labeled I
B
-GST. This suggests that HNE inhibition
of NF-
B activation is due to inhibition of IKK activity.

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Fig. 4.
HNE inhibits IKK activity. Jurkat T
cells either untreated or pretreated with HNE (30 µM) for
30 min were incubated with TPA/IM (0.04 µM/3
µM) at 37 °C for 0-20 min. Total cell extracts (200 µg/lane) were immunoprecipitated with IKK antibody and analyzed
for IKK activity in vitro. The kinase activity associated
with the immunoprecipitate was determined using I B -GST fusion
protein as a substrate. Equal amounts of the I B -GST substrate and
the immunoprecipitated kinase complex were present in the assay, as
confirmed by ink staining and immunoblotting of the membrane for the
I B -GST and the IKK , respectively. Some random variation in the
levels of IKK was observed in individual experiments (e.g.
lanes 6 and 7). The results are representative of two
independent experiments.
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HNE Blocks IKK Activity in Vitro--
To clarify whether HNE
inhibition of IKK activity occurs by direct interaction with IKK, an
in vitro assay for HNE-mediated inhibition of IKK activity
was developed. Immune complexes of IKK were precipitated, then
incubated with HNE in the presence or absence of DTT for 10 min at
30 °C, and assayed for IKK activity, as shown in Fig.
5. Treatment of immune complexes of IKK
with HNE in the absence of DTT caused dose-dependent
inhibition of IKK activity (Fig. 5A). Addition of 30 µM HNE resulted in clear inhibition of IKK activity, and
60 µM HNE completely inhibited activity. In fact, the
higher dose of HNE lowered IKK activity to below basal levels. When
parallel incubations were conducted in which immune complexes were
treated with HNE in the presence of an excess of the HNE scavenging
agent DTT (1 mM), only a modest decline in IKK activity was
detected at the higher HNE concentration.

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Fig. 5.
HNE inhibits IKK activity by directly
reacting with IKK in vitro. Jurkat T cells either
untreated or pretreated with HNE (H, 30 µM)
for 30 min were incubated with TPA/IM (0.04 µM/3
µM) at 37 °C for 10 min. Total cell extracts (250 µg/lane) were immunoprecipitated with IKK antibody and analyzed
for the effect of HNE on IKK activity in vitro.
A, the IKK immune complex was incubated with 30 or 60 µM HNE or vehicle in the absence of DTT at 30 °C for
10 min. B, the IKK immune complex was incubated with 30 or
60 µM HNE or vehicle in the presence of DTT (1 mM) at 30 °C for 10 min. The kinase activities
associated with the immunoprecipitated IKK complex were determined
using I B -GST fusion protein as substrate and are displayed in the
top panels of A and B. Immune
complexes of IKK corresponding to equal volumes of cell extracts were
loaded in each lane. Equal amounts of the substrate (I B -GST) were
present in each assay, as confirmed by ink staining and immunoblotting
of the membranes (second panel of A and
B). Individual samples were divided in two, and separate
PAGE gels were run for Western blotting. After Western transfer, the
blots were visualized with antiserum to IKK or to HNE-modified
protein. The third panel of A and B
represents the detection of IKK molecules with an antiserum against
IKK . The lower panel of A and B
represents the detection of HNE-modified IKK molecules with an
antiserum that recognizes HNE-modified protein conjugates. The amounts
of IKK immune complexes added to each reaction corresponded to equal
amounts of cell lysate. These complexes contained comparable amounts of
IKK protein, as judged by the Western blots in A and
B, lanes 1-3. Incubation with HNE may alter
immune reactivity; so the amounts of IKK detected in A and
B, lanes 4-5, may not accurately reflect IKK
content. The results are representative of three independent
experiments.
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Western blots were performed to probe for the modification of IKK
protein by HNE. Incubations of immune complexes of IKK with HNE
produced bands that migrated more slowly than IKK as well as bands that
migrated at the anticipated size for a dimer of IKK subunits (~220
kDa) (Fig. 5A, lower two panels, lanes
4-5). Thus, incubation mixtures of IKK with HNE contained
HNE-modified IKK molecules, some of which migrated as cross-linked
protein dimers. Comparison of the kinase assay bands in Fig.
5A with the Western blots in the lower panels indicates that
the formation of the HNE·IKK conjugates correlated with the
loss of IKK activity. When parallel incubations of immune complexes of
IKK and HNE were conducted in the presence of an excess of DTT, only
trace amounts of slower migrating forms of IKK were detected; no higher
molecular size HNE·IKK complexes were evident on gel electrophoresis
(Fig. 5B, lower two panels, lanes
4-5). These results demonstrate that HNE reacts covalently with
IKK, which prevents I
B
degradation and NF
B activation.
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DISCUSSION |
In the present studies, we show that TPA/IM stimulates I
B
phosphorylation and subsequent degradation, resulting in NF-
B activation. This finding is consistent with previous observations that
NF-
B activation is responsive to a wide range of activators that
lead to phosphorylation and degradation of I
B
(19, 26, 27, 31,
32). Our experiments demonstrate that pretreatment of human cancer
cells or Jurkat T cells with HNE leads to the inhibition of the NF-
B
signaling pathway. HNE prevents I
B
phosphorylation and subsequent
degradation, reducing NF-
B DNA binding activity and NF-
B
transactivation. These results are in good agreement with the findings
that HNE modulates NF-
B activation by inhibiting I
B
phosphorylation and subsequent proteolysis in human monocytic cells
(33).
Interestingly, the complete process of I
B
phosphorylation and
subsequent degradation following treatment of cells with TPA/IM was
only observed in Jurkat T cells. Phosphorylation of I
B
was not
observed in H1299 cells even though its TPA/IM-stimulated degradation
was obvious (Fig. 3). Three possibilities may explain the inability to
detect p-I
B
in H1299 cells. TPA/IM-induced phosphorylation of
I
B
may occur at a residue other than Ser-32 or Ser-36, so that
the phosphorylated protein may not be recognized by the antibody
employed in these studies. This possibility has been documented with
anoxia, which stimulates phosphorylation of I
B
at Tyr-42 and
NF-
B activation without proteasome-mediated degradation of I
B
(34). A second possibility is that activation of NF-
B in H1299 cells
results from phosphorylation-independent I
B
degradation. For
example, UV irradiation leads to I
B
degradation without
phosphorylation in HeLa cells, 293 cells, and human fibroblasts (35,
36). Finally, the kinetics of I
B
phosphorylation and p-I
B
degradation in H1299 cells may prevent a detectable steady-state concentration of p-I
B
from accumulating.
Phosphorylation of I
B requires IKK activity (22). IKK is a complex,
which contains two catalytic subunits, IKK
(IKK1) and IKK
(IKK2),
along with a regulatory protein, IKK
(37-40). In our experimental
conditions, both IKK
and IKK
were immunoprecipitated by
anti-IKK
antibody (data not shown). Thus, the IKK activity represented the combination of IKK
and IKK
. A variety of stimuli modulate the signal transduction pathways that lead to activation of
upstream kinases including NF-
B-inducing kinase and
mitogen-activated protein kinase kinase kinase 1. These kinases are
responsible for phosphorylation and activation of IKK (29, 30, 41). HNE
did not inhibit any of these upstream kinases, and in fact, a brief
survey indicated that it stimulated the activity of ERK1, ERK2,
JNK1, and JNK2 (data not shown). This is consistent with a
previous finding of stimulation of p38 kinase activity by HNE (42).
The effects of HNE are directly on IKK activity and appear to result
from covalent modification of IKK protein. Aspirin, salicylate, and
sulindac inhibit IKK
activity by competing for binding to ATP (43,
44), whereas anti-inflammatory cyclopentenone prostaglandins inhibit
NF-
B activation by covalently modifying Cys-179 on the activation
loop of IKK
, leading to substantially reduced IKK
activity (45).
It is well known that HNE can rapidly react with proteins containing
sulfhydryl groups by Michael addition; so it is possible that HNE
inhibits IKK activity by direct reaction with a cysteine residue (16,
46). To test this possibility, we conducted an in vitro
assay to assess the effect of HNE on IKK activity and protein
modification. Our results demonstrated that HNE induced the loss of IKK
activity concomitant with the formation of higher molecular size forms
of IKK (Fig. 5A). A prominent band was detected at a
molecular size corresponding to cross-linked homodimers or heterodimers
of IKK subunits. The higher molecular size band on SDS-PAGE gels
reacted with antibodies specific for IKK
and with antibodies
specific for a Michael addition product of HNE with protein residues.
This is consistent with the formation of an HNE-mediated cross-link of
IKK protein subunits. The activation domains of IKK
or IKK
are
believed to be located in close proximity to each other in the IKK
complex, which might place the cysteine residues of the two activation
domains close enough to enable cross-link formation (22, 45).
The importance of the reaction of HNE with cysteine residues is
suggested by the observation that treatment with DTT inhibited HNE-induced cross-link formation and loss of enzyme activity. DTT is a
dithiol that is used as a reducing agent to protect free protein thiols
from oxidation; it is commonly added to enzyme assays or purification
buffers for this purpose. DTT also reacts with
,
-unsaturated
carbonyl compounds such as HNE. Its inclusion in the present
experiments abolished modification and inhibition of IKK in cell-free
extracts. We believe this accounts for previous reports that HNE does
not inhibit IKK activity (33).
We demonstrate here that the key target in HNE modification of NF-
B
activity is IKK. Inhibition of IKK activity by this major product of
lipid peroxidation occurs through covalent modification of the
constituent proteins. Because NF-
B stimulates transcription in
response to oxidative stress, HNE modification may limit the magnitude
of this transcriptional response. A similar role was recently
proposed for 15-deoxyprostaglandin J2, which is a
decomposition product of prostaglandin D2, a product of
arachidonic acid metabolism in inflammatory cells (45, 47).
Furthermore, a related reaction with IKK may account for the previously
noted inhibition of NF-
B by acrolein (48). HNE is structurally
related to 15-deoxyprostaglandin J2 and acrolein, because
it contains an
,
-unsaturated carbonyl compound capable of
reacting as a bifunctional electrophile. In this way, it may serve as
an endogenous factor that regulates the inflammatory response
associated with oxidative stress.