Toxicology Program, Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Texas Houston Health Science Center, Houston, Texas 77030
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ABSTRACT |
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Acrolein is an environmental pollutant that is
known to suppress respiratory host defense against infections; however,
the mechanism of the decrease in host defense is not yet clear. We have
previously reported that acrolein inhibited endotoxin-induced cytokine
release and induced apoptosis in human alveolar macrophages, suggesting
that the inhibition of cytokine release and/or cytotoxicity to alveolar
macrophages may, in part, be responsible for acrolein-induced immunosuppression in the lung. Because nuclear factor-B (NF-
B) is
an important transcription factor for a number of cytokine genes and is
also an important regulator of apoptosis, the effect of acrolein on
NF-
B activity was examined by electrophoresis mobility shift assay.
Acrolein caused a dose-dependent inhibition of endotoxin-induced
NF-
B activation as well as an inhibition of basal level NF-
B
activity. Because I
B is a principal regulator of NF-
B activity in
the nucleus, changes in I
B were determined by Western blotting.
Acrolein-inhibited I
B phosphorylation leads to an increase in
cellular I
B levels preventing NF-
B nuclear translocation and is
likely the mechanism of acrolein-induced inhibition of NF-
B
activity. The role of basal level NF-
B in acrolein-induced apoptosis
was also examined. An NF-
B inhibitor (MG-132) also induced apoptosis
in human alveolar macrophages, suggesting that a certain basal level
NF-
B activity may be required for macrophage cell survival. Taken
together, our results suggest that the acrolein-inhibited
endotoxin-induced NF-
B activation decreased the basal level NF-
B
activity, which may be responsible for the inhibition of cytokine
release and the induction of apoptosis in human alveolar macrophages.
nuclear factor-B; I
B; apoptosis; cytokine; MG-132
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INTRODUCTION |
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ACROLEIN (CH2CHCHO) is a volatile, highly inflammable liquid with a pungent, lacrimatory, and irritating odor. It is an air pollutant generated by incomplete combustion or pyrolysis of organic materials such as fuels, wood, synthetic polymers, food, and tobacco (3, 39). Because of the source distribution, the general population is exposed to acrolein mainly in indoor and, to a lesser extent, outdoor air. However, acrolein has been identified as the noncancer hazardous air pollutant of greatest health concern (11).
Acrolein has been reported to be a highly selective toxin of the respiratory tract for humans and experimental animals (1, 39). Acute inhalation of acrolein vapor has been reported to cause degeneration of the respiratory epithelium in rats, hamsters, and rabbits (39). In addition, short-term acrolein inhalation exposure decreased bactericidal activity within the respiratory tract in experimental animals (2, 3). Acrolein has also been proposed to contribute to deficiencies in lung host defense against nonspecific respiratory infections in cigarette smokers (15, 16, 21). However, only limited information is available on the mechanism of acrolein-induced injury, especially on decreased microbiocidal activity and immunosuppression in the lung.
Alveolar macrophages (AMs) are the key lung cells involved in
nonspecific host defense and are considered to play a central role in
the regulation of the immune response to inhaled pathogens and
development of inflammation (14, 20). The adverse effects of acrolein
on AMs have been studied in a number of animal models and include
inhibition of macromolecule synthesis (23), ATPase activity, and
phagocytosis (27). In addition, acrolein has been reported to modulate
arachidonic acid metabolism (18) and change phagocytic and enzymatic
patterns (33) in macrophages. Previously, Li et al. (25)
reported that acrolein caused a dose-dependent inhibition in the
release of interleukin (IL)-1, IL-12, and tumor necrosis factor (TNF)-
in vitro from endotoxin-stimulated human AMs.
Acrolein also caused a dose-dependent induction of a stress response,
apoptosis, and necrosis in human AMs (25). It is possible that the
inhibition of cytokine release and cytotoxicity to AMs by acrolein may
contribute to the immunosuppression in the lung by acrolein. However,
the molecular mechanism of acrolein-induced inhibition in cytokine
release and induction of apoptosis is yet not clear.
In general, macrophage cytokine release is predominantly regulated by
the transcription rates of cytokine genes. Nuclear factor-B (NF-
B) is a primary transcription factor for inflammatory cytokines such as IL-1
, IL-12, and TNF-
(9). NF-
B is known to be
activated by a wide range of stimuli, including bacterial endotoxin,
mitogens, viral proteins, ionizing radiation, and ultraviolet radiation (5, 34). NF-
B activity is negatively regulated by a family of
inhibitor proteins known as I
B. Activation of NF-
B requires that
I
B be phosphorylated, allowing the NF-
B dimer to dissociate from
I
B. Dissociated NF-
B can translocate into the cell nucleus, bind
to its target DNA sequence, and activate the transcription of its
target genes (9, 36). After phosphorylation of I
B, it is
ubiquitinated and degraded (9, 36).
In addition to being an important transcription factor for a
number of cytokine genes, NF-B is also important for cell
survival. Mice lacking Rel A [Rel A(
/
);
p65 subunit of NF-
B] died at the embryonic stage due to
extensive apoptosis in the liver (8). Fibroblasts and macrophages from
Rel A(
/
) mice are sensitive to TNF-
-induced cell
death, and reintroduction of Rel A to Rel A(
/
)
fibroblasts enhanced cell survival (7). Furthermore, a study (38) has
shown that the activation of NF-
B protected cells from apoptosis
induced by TNF-
and ionizing radiation, whereas the inhibition of
NF-
B activity increased sensitivity to TNF-
-induced apoptosis
(37, 38).
The important roles of NF-B in regulating inflammatory cytokine gene
expression and in protecting cells from apoptosis makes it a logical
target to examine acrolein-induced inhibition of cytokine release and
induction of apoptosis. Therefore, in this study, the effects of
acrolein on the regulation of NF-
B activity and the role of NF-
B
on cell survival were examined in human AMs in vitro.
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METHODS |
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Materials. Acrolein and
fetal bovine serum (FBS) were obtained from Sigma (St. Louis, MO).
Medium 199 was obtained from GIBCO BRL (Life Technologies,
Gaithersburg, MD). DNA oligonucleotides for the NF-B consensus
sequence 5'-AGT TGA GGG GAC TTT CCC AGG C-3' and antibodies
against I
B-
and phosphorylated I
B-
were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). Antibodies against actin,
horseradish peroxidase-linked anti-rabbit Ig whole antibody (from
donkey), and horseradish peroxidase-linked anti-mouse Ig whole antibody
(from sheep) were obtained from Amersham (Arlington Heights, IL).
Isolation and culture of human AMs. Cells were obtained by bronchoalveolar lavage of healthy volunteers by methods described elsewhere (13, 19, 31). In brief, instillations of 140 ml of sterile saline resulted in recoveries of 80-100 ml of lavage fluid that was kept at 4°C until the cells were isolated from the lavage fluid by a 4°C centrifugation at 500 g (IEC centrifuge). The cell pellet was resuspended in a small volume (1-5 ml) of HEPES-buffered medium 199 (GIBCO BRL) with 10% heat-inactivated FBS and added antibiotics (50 U/ml of penicillin, 50 µg/ml of gentamicin, and 50 µg/ml of streptomycin). All culture media were pretreated with polymyxin B beads, which were then removed before use, ensuring an endotoxin-free medium. Cell counts were then determined with a ZBI Coulter Counter (Hialeah, FL) and yielded an average of ~20 × 106 cells that were ~90% macrophages, 7% lymphocytes, 2% neutrophils, and 1% eosinophils. The viability was >90% as measured by trypan blue exclusion. The cells were cultured in medium 199 with 10% FBS in suspension by slow end-to-end tumbling in sterile polypropylene tubes at 37°C before being harvested.
SDS-PAGE and Western blotting. The
cells were washed once with phosphate-buffered saline (PBS; pH 7.2) and
centrifuged at 500 g for 7 min at
4°C. The cells were then solubilized in Laemmli sample buffer (2%
SDS, 10% glycerol, 60 mM Tris · HCl pH 6.8, 0.1 M
dithiothreitol, and 0.01% bromphenol blue), sonicated for 30 s with an
ultrasonifier cell disrupter (Heat System-Ultrasonics, Farmingdale,
NY), and heated in boiling water for 5 min before storage at
20°C.
Protein samples were analyzed with SDS-gel electrophoresis (12% Ready
Gels, Bio-Rad, Hercules, CA). Total proteins from an equal number of
cells (2 × 105 cells/sample)
were loaded onto each lane. Resolved proteins were transferred to
nitrocellulose membranes (Amersham) with a Trans-Blot Electrophoretic
Transfer Cell (Bio-Rad). The membranes were incubated for 16 h at
4°C in blocking buffer (5% Blotto in 10 mM
Tris · HCl, pH 7.2, and 150 mM NaCl) and were then
incubated with primary antibody (1:200 for IB-
, 1:1,000 for
phosphorylated I
B-
, and 1:2,000 for actin) for 16 h at 4°C.
The membranes were washed extensively with 10 mM
Tris · HCl, 150 mM NaCl, and 0.05% Tween, pH 8.0, incubated with the secondary antibody conjugated to horseradish peroxidase at a 1:10,000 dilution for 1 h at room temperature, and
washed extensively with 10 mM Tris · HCl, 150 mM
NaCl, and 0.05% Tween. Enhanced chemiluminescence detection (Amersham)
followed by autoradiography (Hyperfilm-ECL, Amersham) was used to
visualize the proteins.
Electrophoretic mobility shift assay. Nuclear extracts from AMs were prepared by a modification of the procedure of Misra et al. (28a). In brief, 2.5 × 106 cells were washed twice with 3 ml of PBS, washed once with 3 ml of modified Dignam et al. (13a) solution A (2.5 mM MgCl2 and 10 mM HEPES, pH 8.0), and suspended in 1 ml of solution A. The cell suspensions were then lysed in a glass Dounce homogenizer (Wheaton, Millville, NJ) with 20 strokes of an A-type pestle. The nuclei were placed into 1.5-ml Eppendorf tubes, pelleted at 12,000 g for 10 min, and extracted with 50 µl of modified Dignam et al. solution C (100 mM HEPES, 25% glycerol, 1 mM leupeptin, and 400 mM NaCl, pH 8.0). The nuclear extracts were obtained after centrifugation at 25,000 g for 10 min. Protein concentrations were determined with Bio-Rad protein assay dye reagent, with bovine serum albumin as the standard.
NF-B activity in the nuclear proteins were analyzed by
electrophoretic mobility assay as described by Misra et al.
(28a). Oligonucleotides for NF-
B binding sequence were
5'-end labeled with
[
-32P]ATP by T4
polynucleotide kinase (Promega, Madison, WI). Each assay (20 µl) had
5 µg of nuclear extract, 0.1 ng of
32P-labeled DNA, 1 µg of
poly(dI-dC), 100 mM NaCl, 25 mM HEPES, 6.25% glycerol, and 0.25 mM
leupeptin, pH 8.0. The reaction mixtures were loaded onto 6%
polyacrylamide gels in 0.25× buffer consisting of 22 mM Tris, 22 mM sodium borate, and 0.5 mM EDTA, pH 8.0. After electrophoresis at 10 V/cm, the gels were dried at 70°C under vacuum in a gel dryer and
then exposed to X-ray film.
Apoptosis assays. Macrophage apoptosis induced by acrolein was examined by a combination of morphological differential staining and detection of DNA fragmentation in the cells. For morphological differential staining, 30 × 103 cells were suspended in PBS (pH 7.2) at room temperature for 5 min and cytocentrifuged onto charged microscope slides (Fisher Scientific, Pittsburgh, PA) at 1,500 rpm for 5 min with a Shandon Cytospin 2 centrifuge. The slides were treated with the Leukostat fixation and staining protocol (Fisher Scientific), dried in air, and examined by light microscopy.
To detect nucleosomes in the cytoplasmic fraction, the cells were processed and analyzed with the Cell Death Detection ELISA kit (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's protocol. The assay is based on the quantitative sandwich-enzyme-immunoassay principle with monoclonal antibodies directed against DNA and histone. Cells at a concentration of 1 × 105 from each sample were processed, 5 × 103 cells were used for each reaction, and triplicate reactions were performed for each sample.
To visualize DNA fragmentation, genomic DNA was isolated from treated
cells with the DNA ISOLATOR (Genosys, The Woodlands, TX) and
3'-end labeled with
[-32P]dCTP (ICN) by
incubation of 1 µg of DNA in 50 µl of reaction buffer (50 mM
Tris · HCl, pH 7.6, 10 mM
MgCl2, 200 µM dATP, 200 µM
dGTP, 200 µM dTTP, 2 µl of
[
-32P]dCTP, and 2 U
of Klenow) at 37°C for 30 min. The same amount of
[
-32P]dCTP-labeled
DNA (50 ng) for each sample was loaded onto a 2% agarose gel and run
at 5 V/cm for 5 h in 40 mM Tris-acetate buffer, pH 8.0, with 1 mM EDTA.
The gel was dried at 80°C under vacuum in a gel dryer and then
exposed to X-ray film.
Statistical analysis. Values are presented as means ± SE. Statistical differences between control and treated groups were determined by a one-way analysis of variance followed by Student-Newman-Keuls test. Differences were considered significant at P < 0.05.
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RESULTS |
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Effects of acrolein on lipopolysaccharide-induced
NF-B activity. Li et al. (25) recently
reported that acrolein inhibited endotoxin-induced cytokine release in
human AMs. Because NF-
B is the critical transcription factor for
many inflammatory cytokines, the effects of acrolein on
endotoxin-induced NF-
B activity was examined by electrophoretic
mobility shift assay. As shown in Fig.
1, endotoxin treatment caused
an activation of NF-
B activity and acrolein caused a dose-dependent
inhibition of endotoxin-induced NF-
B activation in human AMs. A 25 µM dose of acrolein completely blocked endotoxin induction of NF-
B
activity. Acrolein treatment (50 µM for 4 h) also caused a decrease
in the basal level NF-
B activity in unstimulated macrophages (Fig.
1). The specificity of NF-
B binding was confirmed by competition and
supershift assay (data not shown). Because NF-
B is the critical
transcription factor that regulates the expression of many inflammatory
cytokine genes, it is likely that the inhibition of endotoxin-induced
NF-
B activation by acrolein may contribute to acrolein-induced
inhibition of cytokine release.
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Effects of acrolein on basal level
NF-B activity. The dose response and
time course for the effect of acrolein on basal (unstimulated) level
NF-
B activity in human AMs were also examined by electrophoretic mobility shift assay. As shown in Fig. 2,
at both 6 and 24 h, 25 and 50 µM acrolein caused almost complete
inhibition of basal level NF-
B activity, whereas 10 µM acrolein
had very little or no inhibition of basal level NF-
B activity (Fig.
2). These results are consistent with those in Fig. 1 and indicate that
acrolein not only blocks endotoxin-stimulated NF-
B activity but can
also decrease basal levels of NF-
B.
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Effect of acrolein on cellular
IB-
level. Considering
that NF-
B activity is regulated by I
Bs and the best-characterized I
B is I
B-
(9, 36), the effect of acrolein on the basal levels
of I
B-
in human AMs was examined by Western blotting. As shown in
Fig. 3, acrolein caused a dose-dependent
increase in I
B-
levels within 1 h. Although no apparent change in
the level of I
B-
was evident at 10 µM, large increases in
I
B-
were evident at 25 µM and higher doses of acrolein. Based
on these findings, it is likely that the increase in cellular I
B-
levels may be responsible for the inhibition of NF-
B activity by
acrolein.
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Effect of acrolein on
IB-
phosphorylation.
The increase in cellular I
B-
levels could be caused by an
increase in I
B-
synthesis and/or a decrease in I
B-
degradation. Experiments with an inhibitor of protein synthesis
indicated that an acrolein-induced increase in I
B-
is independent
of protein synthesis (data not shown), suggesting that the decrease in
I
B-
degradation is responsible for the increase in I
B-
.
Degradation in I
B is regulated by phosphorylation: I
Bs are first
phosphorylated, then ubiquitinated and degraded during NF-
B
activation (4, 6, 36). Therefore, the effect of acrolein on I
B-
phosphorylation was examined. As shown in Fig.
4, acrolein caused a dose-dependent
decrease in phosphorylated I
B-
in human AMs within 1 h. Decreased
phosphorylation was evident with even the lowest dose of acrolein
examined (10 µM). This result suggests that the decrease in
phosphorylated I
B-
levels in the cells is likely responsible for
acrolein-induced decreases in I
B-
degradation and increases in
cellular I
B-
levels in human AMs.
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Effects of NF-B inhibition on AM cell
survival. Inhibition of NF-
B activity has been
demonstrated to cause increased sensitivity to TNF-
-induced
apoptosis (37, 38), suggesting that the level of NF-
B is important
for cell survival. Our results demonstrate that acrolein decreased
basal level NF-
B activity in human AMs (Fig. 2) and, at similar
concentrations, caused apoptosis in human AMs (25). However, it is not
known whether the inhibition of basal level NF-
B activity is
responsible for acrolein-induced apoptosis. Therefore, the effects of
NF-
B inhibition on human AM cell survival were examined with NF-
B
inhibitors. SN-50 has been reported to inhibit NF-
B nuclear
translocation in endothelial cells (26). MG-132
(Z-Leu-Leu-Leu-H) is a proteasome
inhibitor reported to inhibit NF-
B activity in a number of cell
types (12, 30). The efficacy of these inhibitors on human AM NF-
B
activity was tested by electrophoretic mobility shift assay (Fig.
5). Twenty-four hours after treatment,
MG-132 (5 µM) completely blocked NF-
B activity, whereas SN-50 (20 µM) had no effect on NF-
B activity in human AMs.
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The possibility of induction of apoptosis by NF-B inhibition
was examined by Cell Death ELISA, a procedure that measures cytosolic DNA fragmentation during apoptosis (24). MG-132 caused a
significant increase in cytosolic DNA fragments (Fig.
6), suggesting apoptosis in human AMs. In
contrast, SN-50 had no effect on cytosolic DNA fragmentation, i.e., no
apoptosis in treated cells. The induction of apoptosis by MG-132 was
confirmed by morphological examination (Fig.
7). MG-132, as well as acrolein, caused
morphological changes in human AMs, such as shrinkage of the cytoplasm,
nuclear condensation, and fragmentation, that were characteristic of
apoptosis. The apoptotic DNA fragmentation induced by MG-132 and
acrolein was confirmed by DNA gel electrophoresis. Both MG-132 and
acrolein caused internucleosomal DNA fragmentation, resulting in a DNA ladder of multiples of 180-200 bp (Fig.
8). The formation of internucleosomal fragments in DNA from MG-132- and acrolein-treated cells confirmed that
cells were dying by an apoptotic mechanism. The induction of apoptosis
by the NF-
B inhibitor MG-132 suggests that basal level NF-
B
activity may be required for human AM cell survival.
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DISCUSSION |
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In the present study, we provide new information on the molecular
mechanism by which acrolein may be causing alterations in host defense
and apoptosis. Central to both effects is the ability of acrolein to
block the nuclear activity of NF-B. This study, therefore, not only
provides new information on the molecular targets of acrolein in human
cells, which would be exposed to acrolein in the ambient environment or
through cigarette smoke, but also confirms the importance of NF-
B in
macrophage cytokine release. In addition, the work provides evidence
for the important role of NF-
B in regulating apoptosis of human AMs.
We demonstrated that acrolein inhibits endotoxin-induced NF-B
activation in human AMs. NF-
B is a primary transcription factor critical for regulating the immune response to many pathogenic signals,
and activation of NF-
B is required for inflammatory cytokine release
by AMs during infection (5, 29). Consequently, inhibition of
endotoxin-induced activation of NF-
B may be responsible for
acrolein-induced inhibition of macrophage cytokine release (25). Also,
it is well known that NF-
B plays a central role in the regulation of
gene expression in T and B cells (10, 17). Therefore, acrolein
inhibition of NF-
B activity in B and T cells present in the lung may
also contribute to acrolein-induced immunosuppression in the lung. It
is most likely that it is the combined effects of acrolein on
suppression of cytokine release from a number of immune regulatory
cells with the effects of acrolein on specific cell functions that
contributes to the overall immunosuppressive effects of acrolein
exposure. However, it is important to point out that our experiments
were performed under in vitro settings. It is possible that the
mechanism(s) of acrolein-induced immunosuppression in the lung in vivo
may differ from the mechanism(s) characterized under the in vitro conditions.
It has been reported that endotoxin-stimulated release of TNF-,
IL-1
, and IL-12 from human AMs had different sensitivities to
inhibition by acrolein, with TNF-
release being the least-sensitive response (25). Endotoxin-stimulated TNF-
release has been reported to involve both transcriptional and translational activation (22). On
endotoxin stimulation, TNF-
transcription is increased 5- to
50-fold, whereas its translation was increased >100-fold. This could
account for the decreased sensitivity of endotoxin-induced TNF-
release to acrolein treatment compared with that of IL-1
and IL-12.
Also, the regulation of different cytokine gene transcription involves
NF-
B as well as other transcription factors such as Sp1, Ets,
ATF-2/c-Jun, and NF-IL-6 (36). Consequently, the interaction of NF-
B
and other transcription factors will determine the final rates of
transcription of specific cytokines. Therefore, the difference in
acrolein sensitivity of endotoxin-inducted cytokine release could be
accounted for by other transcription factors working with NF-
B that
may or may not be affected by acrolein treatment.
NF-B has been reported to protect cells against apoptosis induced by
TNF-
, ionizing radiation, and the chemotherapeutic compound
daunorubicin (38). Correspondingly, inhibition of NF-
B activity
increased the sensitivity to TNF-
-induced apoptosis in a number of
cell types (7, 37, 38). In our study, we demonstrated that acrolein
decreased the basal level NF-
B activity in human AMs. The
relationship between a decrease in the basal level NF-
B activity to
acrolein-induced apoptosis cannot be established equivocally from these
results. However, it suggests that the inhibition of basal level
NF-
B activity may be responsible for acrolein-induced apoptosis
because the NF-
B inhibitor MG-132 also induced apoptosis in human AMs.
It is likely that acrolein and MG-132 inhibit NF-B activity by
different mechanisms. We propose, as described below, that an
acrolein-induced decrease in NF-
B activity is through the blocking
of I
B-
phosphorylation. In contrast, it has been reported that
MG-132 decreases NF-
B by blocking proteasome degradation of I
B
and increasing levels of phosphorylated I
B-
(12, 30). Nevertheless, the fact that both acrolein and MG-132 caused apoptosis suggests that basal level NF-
B activity may be important (or required) for AM cell survival.
The possibility that basal level NF-B activity is required for cell
survival is intriguing, even though previous studies (8,
32) on the role of basal level NF-
B activity on cell survival have
not provided compelling evidence. It has been reported that Rel
A(
/
) cells or cells lacking p50 are viable in culture (8,
32). However, because NF-
B is a family of dimeric transcription factors (4, 36), the full impact of deficiencies of one Rel protein on
cell survival may be masked by the redundancy of the NF-
B/Rel
protein family. It is worth noting that mice lacking Rel A have
excessive apoptosis in the liver (8). Cell lines transfected with dominant negative I
B-
(I
B-
M) or treated
with MG-132 were also viable in culture (37, 38). It is possible that
permanently differentiated cells such as AMs are more sensitive to the
effects of NF-
B inhibition. Further studies will be needed to
elucidate the role of basal level NF-
B activity on AM cell survival.
The present study provides a mechanism to explain decreased NF-B
activity by acrolein. The regulation of NF-
B activity is centered on
phosphorylation, ubiquitination, and/or degradation of I
B (4, 6, 28,
35, 36). The phosphorylation of I
B is catalyzed by the I
B kinase
(IKK) complex. For example, the signal for receptor-mediated NF-
B
activation is transduced by the TNF receptor-associated factor (TRAF)
family of adapter proteins such as TRAF2 and TRAF6. The resulting
activation of mitogen-activated protein kinase kinase kinases, such as
NF-
B-inducing kinase and mitogen-activated protein/extracellular
signal-regulated kinase kinase-1, by TRAF leads to the
phosphorylation of IKK
and IKK
and the activation of IKK. The
activation of IKK leads to the phosphorylation of I
B, which signals
the ubiquitination and degradation of I
B. The I
B-free NF-
B is
translocated into the nucleus where it activates the transcription of
its target genes (28, 35). Our study suggests that the inhibition of NF-
B activity by acrolein is associated with a decrease in I
B-
phosphorylation, resulting in an increase in I
B-
levels. It is
possible (yet to be confirmed) that acrolein has similar effects on
other members of the I
B family. We propose that the decrease in
I
B phosphorylation and increase in I
B levels are likely the cause
of acrolein-induced inhibition of NF-
B activity. However, it is not
clear if the inhibition of I
B-a phosphorylation by acrolein is
through direct inhibition of IKK or through inhibition of upstream
regulators of the NF-
B activation pathway.
In summary, our results demonstrate that acrolein caused a
dose-dependent inhibition of endotoxin-induced NF-B activation. The
inhibition of endotoxin-induced NF-
B activation is likely responsible for acrolein-induced inhibition of endotoxin-induced cytokine release. Also, our results demonstrate that acrolein caused an
inhibition of basal level NF-
B activity, and the inhibition of I
B
phosphorylation is likely the mechanism of acrolein-induced decrease in
NF-
B activity. Furthermore, the inhibition of basal level NF-
B
activity may be responsible for acrolein-induced apoptosis. The
requirement of basal level NF-
B activity for human AMs may have
implications on other cell types.
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ACKNOWLEDGEMENTS |
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This work was supported by the National Center for Research Resources Grant M01-RR-02558.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Holian, Dept. of Internal Medicine, Univ. of Texas Medical School, 6431 Fannin, MSB 1.276, Houston, TX 77030 (E-mail: aholian{at}heart.med.uth.tmc.edu).
Received 9 June 1998; accepted in final form 11 May 1999.
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