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Division of Clinical Immunology and Allergy, Department of Medicine, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Macrophages are a PAH target in the respiratory tract for inhaled PAH. It has been demonstrated that a variety of xenobiotics, including PAH, polychlorinated biphenyls, and halogenated aromatic hydrocarbons, exerts effects on macrophages, including pulmonary alveolar macrophages (10, 12, 13, 14, 15, 16, 17, 18, 19). These effects include induction of oxidative burst activity (10, 12), increased expression of MHC-II gene products (13), induction of cytochrome P4501A1 (CYP1A1) activity (12, 16, 18), and conversion of benzo(a)pyrene to redox-active quinones and other DNA adducts (16, 17, 19). Macrophages, including pulmonary alveolar macrophages, play an important role in allergic inflammation through their effects on Ag presentation, expression of costimulatory molecules that activate T cells, and production of cytokines and chemokines that enhance IgE production (20, 21, 22, 23, 24, 25, 26, 27). Recently, Prell and Kerkvliet have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) inhibits CD86 expression on Mac1+ cells, and suggested that a major target for dioxin in the immune system is APCs, including macrophages (28).
While metabolic pathways for PAH are well described in hepatocytes, not
much is known about PAH pathways for cellular activation in immune
cells, including macrophages. The best-characterized PAH metabolic
pathway involves PAH interaction with the aromatic hydrocarbon receptor
(AhR), which translocates to the nucleus and transcriptionally
activates genes that express the xenobiotic response element (XRE)
(29, 30, 31, 32). Genes that utilize an XRE include CYP1A1, which, in turn, is
responsible for the conversion of PAH to oxidatively labile metabolites
that damage cellular DNA, proteins, and lipids (29, 30, 31, 32, 33, 34). Some PAH
metabolites, e.g., quinone derivatives, participate in additional
pathways such as 1-electron reductions that yield reactive oxygen
species (ROS) (19, 35, 36, 37). In addition to the role of the XRE, more
recent studies in hepatocytes have focused on the role of PAH and their
quinone derivatives on cellular activation via the generation of
oxidative stress (35, 36, 37, 38). An important genetic response element (RE)
that is affected by oxidative stress is the antioxidant or electrophile
response element (ARE or EpRE) (35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45). The ARE, with the core
sequence GTGACNNNCA (39, 40, 41), has been linked
to the expression of genes that encode phase II drug metabolizing
enzymes, e.g., glutathione S-transferase (GST) and
nicotinamide-adenine dinucleotide phosphate (NADPH):quinone
oxidoreductase (NQO1) (Fig. 1
). A major effect of phase II enzymes is
to protect cells against toxic effects of xenobiotics and their
oxidatively labile products (45, 46, 47, 48, 49). Recently, it has been shown that
ARE elements are found in the promoters of other critical cellular
genes, including the IL-6 gene (50).
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) (36, 40, 43). AP-1-like sequences also appear in the
ARE of rat and murine phase II enzyme promoters (Fig. 1) (36, 53, 54).
One hypothesis is that AP-1 proteins play a role in the activation of
the ARE (36, 42, 43, 53, 54). This notion is contentious, however, as
some studies have shown that the ARE can be activated independent of
AP-1 protein(s) (39, 40, 41, 45). Whatever the relationship between ARE and
AP-1 proteins, the molecular events leading to the activation of these
RE are sensitive biochemical tools to study immune cellular activation
by PAH. One of the regulatory pathways for AP-1 proteins are the MAPK
cascades that affect transcriptional activation as well as expression
of AP-1 proteins (55). We are particularly interested in the c-Jun
N-terminal kinases (JNK), also known as stress-activated protein
kinases, and the p38 MAPK cascade because both pathways play a role in
cellular responses to environmental stress, including exposure to toxic
drugs and chemicals, LPS, inflammatory cytokines, oxidative stress, or
hyperosmolarity (55, 56, 57, 58). There have been no systematic studies in macrophages of activation of the ARE or the AP-1 pathway by PAH. We sought to determine whether a representative PAH, ß-naphtoflavone (BNF), and a representative quinone derivative, tert-butylhydroxyquinone (tBHQ), can induce MAPK activation in the macrophage cell lines, THP-1 and RAW264.7 (29, 30, 31, 43). In addition, we investigated the effects of these chemicals on ARE activation. Our data show that BNF and tBHQ induce JNK and p38 MAPK activation in parallel with the generation of AP-1 electrophoretic mobility shift complexes. While these chemicals induced AP-1 interactions with the hARE, hARE reporter gene activity could be activated independent of AP-1 protein binding. These data indicate that ARE and AP-1 response elements may play important roles in macrophage activation by PAH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 was purchased from Irvine Scientific (Santa Ana, CA).
DMEM, penicillin-streptomycin, and L-glutamine were
purchased from Life Technologies (Baltimore, MD). The polyclonal rabbit
IgG anti-JNK2 Ab and polyclonal rabbit IgG anti-phospho-p38 Ab
was purchased from New England Biolabs (Beverly, MA). A p38 MAPK Ab
that recognizes nonphosphorylated epitopes and that can be used for
immunoblotting and immunoprecipitation was bought from Santa Cruz
Biotechnology (Santa Cruz, CA). The anti-phosphotyrosine Ab, 4G10,
was purchased from Update Biotechnology (Lake Placid, NY), while
anti-pan-Fos and anti-pan-Jun antisera were from Santa Cruz
Biotechnology. tBHQ was purchased from Aldrich (Milwaukee, WA). BNF,
LPS (O55:B5 serotype), phenanthrene, and N-acetylcysteine
(NAC) were purchased from Sigma (St. Louis, MO). Silica gel TLC plates
were purchased from VWR Scientific (San Francisco, CA). Wild-type and
hARE core mutant reporter plasmids (hARE-tk-CAT and hAREcm-CAT,
respectively) were obtained from Dr. Anil Jaiswal (Fox Chase Cancer
Center, Philadelphia, PA) (Fig. 1) (38, 42). The cDNAs for DA-MEKK1
(MEKK
) and DN-MEKK1 (MEKK K432 M) were a gift from Dr. G. Johnson
(National Jewish Center for Immunology and Research, Denver, CO) (59).
[14C]Chloramphenicol was from Amersham (Arlington
Heights, IL), while [
-32P]ATP and
[
-32P]dCTP were from NEN (Boston, MA).
Cell culture and stimulation
THP-1 cells were obtained from American Type Culture Collection (Manassas, VA). The cells were cultured at 37°C in a 5% CO2 atmosphere in RPMI 1640 supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% glutamine. RAW264.7 cells were generously provided by Dr. Steven Smale (University of California, Los Angeles). These cells were cultured in DMEM containing 10% FBS, 1% penicillin-streptomycin, and 1% glutamine.
BNF, tBHQ, and phenanthrene were dissolved in DMSO, while TCDD was dissolved in ethanol. Before use, the chemicals were made up in culture media at the indicated concentrations, keeping the final carrier concentration at 0.1%. LPS was dissolved in PBS before adding it to the culture medium.
Transfections
RAW264.7 cells were washed once in PBS and resuspended in DMEM with 20% FCS at a final concentration of 7 x 106 cells/200 µl. These cells were incubated in a 0.4-cm cuvette together with 20 µg plasmid and 30 µl PBS at room temperature for 10 min. Cells were transfected at 260 V and 975 µF in a Bio-Rad (Richmond, CA) Gene Pulser. The transfected cells were rested at room temperature for 10 min, washed once in PBS, and placed in six-well plates in complete medium (see above).
Construction of a hARE-CAT construct with mutation of the AP-1 site
The hARE promoter was excised from the hARE-tk-CAT vector
(pBLCAT backbone) with XbaI, followed by BamHI
digestion to provide an overhanging site on the 3' end. A hARE promoter
with a mutagenized AP-1 site (Fig. 1) was ligated into the double
digested plasmid. The correct orientation and presence of the
mutagenized sequence were confirmed by DNA sequence analysis. The
resulting plasmid was designated hARE(mAP1).
CAT assay
RAW264.7 cells transfected with hARE-tk-CAT or mutant versions
thereof (Fig. 1) were allowed to rest for 24 h before stimulation
with 50 µM BNF, 50 µM tBHQ, and 10 µg/ml LPS for an additional
24 h. The cells were harvested and washed once in PBS. Cell
pellets were resuspended in 100 µl 1x reporter lysis buffer
(Promega, Madison, WI) and incubated at 4°C for 30 min while shaking.
The lysates were freeze thawed once on dry ice and spun at 14,000
x g for 10 min. A total of 50 µg of protein per
stimulation was heat inactivated at 65°C for 4 min. Each sample was
then coincubated with 25 µg N-butyryl-CoA (Promega) and
0.25 µCi [14C]chloramphenicol in a 250 mM Tris/HCl
buffer for 2 h at 37°C. The reaction was stopped with 500 µl
ethyl acetate and centrifuged at 14,000 x g for
30 s. The top organic layer was removed, dried, and resuspended in
15 µl ethyl acetate and spotted on silica TLC plates. The TLC plates
were placed in a TLC tank that was equilibrated previously with 145 ml
chloroform and 5 ml methanol. The TLC plates were dried and exposed to
radiographic film or a PhosphorImager screen (Molecular Dynamics,
Sunnyvale, CA).
MAPK assays
Following stimulation of 3 x 106 cells
for the indicated time period, JNK kinase activity was assessed as
previously described (60). Briefly, 100 µg cellular protein was
coincubated with GST-c-Jun(1–79), immobilized on glutathione beads.
After washing of the beads, the phosphorylation reaction was initiated
by the addition of [-32P]ATP at 20°C for 15 min
(60). The p38 MAPK assay followed the same procedure, except that the
kinase was purified by an anti-p38 MAPK Ab, with GST-ATF2 acting as
substrate (61). Phosphorylated substrates were resolved on 10% SDS
polyacrylamide gels that were dried and autoradiographed.
JNK2 immunoprecipitation and Western blotting
Aliquots of 7 x 106 THP-1 cells were stimulated with 50 µM BNF and 50 µM tBHQ for the indicated time periods. Cells were lysed in 200 µl lysis buffer containing 50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 2 mM PMSF, 10 µg/ml leupeptin, 2 U/ml aprotinin, and 1 mM Na3VO4. A total of 200 µg of lysate protein was incubated with 0.5 µg anti-JNK2 Ab for 1 h at 4°C. After absorption to protein A-Sepharose beads for 1 h, the beads were washed and boiled in SDS sample buffer. Proteins were resolved on a 10% polyacrylamide gel, transferred to an Immobilon-P membrane, and overlaid with anti-phosphotyrosine (4G10) Ab, as previously described (62).
Electrophoretic mobility shift assays (EMSA)
A total of 1 x 107 RAW264.7 cells was
stimulated with 50 µM BNF or 50 µM tBHQ for the indicated time
period. Nuclear extraction was performed, as previously described, with
a few modifications (40). Briefly, cells were pelleted and resuspended
in lysis buffer containing 0.1% Nonidet P-40, 50 mM Tris-HCl, pH 8, 10
mM NaCl, and 5 mM MgCl2 for 1 min. The process was repeated
with the same lysis buffer containing 0.5% Nonidet P-40 for 1 min.
Nuclear proteins were eluted in an extraction buffer containing 500 mM
NaCl, 20 mM HEPES, pH 7.9, 1 mM EDTA, and 20% glycerol. For
DNA-protein binding, 10 µg of nuclear protein was incubated together
with 105 cpm 32P-labeled probe in the presence
of a binding buffer and 3 µg poly(dI/dC) for 20 min. Probes used in
this study include a: 1) AP-1 consensus oligonucleotide,
5'-GATCCGTGACTCAGCGCG-3'; 2) human ARE oligonucleotide (Fig. 1); and 3)
a mutant hARE oligonucleotide in which 3 bp in the AP-1 consensus site
have been changed (see Fig. 1). For cold competition, 100-fold excess
ununlabeled probe was incubated for 15 min at room temperature with the
above mixture before addition of the labeled probe. For Ab supershift,
0.5 µg anti-pan-Fos or anti-pan-Jun antisera were added
to the binding reaction for 15 min before addition of the labeled
probe. Shift complexes were electrophoresed on a 6%
polyacrylamide-glycerol gel, and the dried gel was exposed to
autoradiographic film.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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tBHQ is mechanistically representative of the quinone products
that form when PAH are metabolized by cytochrome P4501A1 (CYP1A1) (46).
Although tBHQ is classified as a phenolic antioxidant, its breakdown
actually generates ROS, which leads to cellular activation and
generation of nuclear responses in hepatocytes (35, 51). One type of
nuclear response is AP-1 protein expression, which affects AP-1
response elements (36, 43, 44, 46, 51, 52, 53, 54). We were interested to
determine whether tBHQ could activate JNK, since this cascade regulates
the expression and transcriptional activation of AP-1 proteins (55).
Treatment of the human macrophage cell line, THP-1, and the murine
macrophage cell line, RAW264.7, with tBHQ induced JNK activation, as
determined by in vitro kinase assay (Fig. 2, A and B).
JNK activation commenced within 30 min of adding the chemical, peaked
within 60 min, and returned to near baseline in about 4 h (Fig. 2, A and B). The magnitude of the response
was more robust in THP-1 than in RAW264.7 cells (Fig. 2, A
and B). Compared with a potent JNK stimulus, e.g.,
LPS (63), tBHQ induced a response of almost similar magnitude in THP-1
cells, but elicited only 50% of the LPS response in RAW264.7 cells
(Fig. 2, A and B).
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C). The response
to BNF in RAW264.7 cells was much stronger and achieved 31% of the
LPS-induced response (see left-hand panel, Fig. 3A). Compared with the
brief activation of JNK by tBHQ (Fig. 2, A and
B), BNF-induced JNK activation was discernible for up
to 22 h after stimulation (Fig. 2C). Taken
together, these data indicate that tBHQ and BNF can be added to
the growing list of compounds and environmental stress stimuli that
activate the JNK cascade (56, 57, 58).
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, A and
B). Exposure to 20 mM NAC abrogated subsequent BNF-
and tBHQ-induced JNK activation (Fig. 3, A and
B), while decreasing the LPS response by 50% (Fig. 3A). During the performance of these experiments, we
noted that NAC lowered resting JNK activity, suggesting that continuous
production of ROS may drive basal JNK activity in macrophages. We also
determined the effects of tBHQ and BNF stimulation upon tyrosine
phosphorylation of JNK (Fig. 3C). JNK activity is
induced by phosphorylation of threonine and tyrosine residues in the
TPY allosteric effector site of that kinase (55, 56, 57). While resting
cells lacked tyrosine phosphorylation of the 55-kDa JNK isoform (Fig. 3C, lane 2), BNF and tBHQ induced
phosphorylation of p55 JNK (lanes 3 and
4). Equal amounts of the 55-kDa JNK isoform were
being immunoprecipitated, as confirmed by anti-JNK immunoblotting
(not shown). tBHQ and BNF induce p38 MAPK activity in an antioxidant-sensitive fashion in THP-1 and RAW264.7 cells
In addition to JNK, a second MAPK, p38 MAPK, plays a role in
macrophage activation by LPS and other forms of acute cellular stress
(58). To determine whether tBHQ and BNF induce p38 MAPK activation in
THP-1 and RAW264.7 cells, we used an in vitro immune complex kinase
assay to measure the activity of p38 MAPK (Fig. 4, A and B).
The magnitude of the response to tBHQ and BNF amounted to 9 and 20%,
respectively, in THP-1, and 45 and 31%, respectively, in RAW264.7
cells (Fig. 4, A and B).
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A). We examined the
role of oxidative stress in p38 MAPK activation, and found that prior
exposure of RAW264.7 cells to 20 mM NAC abrogated BNF- and tBHQ-induced
p38 MAPK activity, while decreasing the LPS response by 43% (Fig. 5B). Similar to what was seen for JNK, NAC lowered
basal p38 MAPK activity, suggesting that ROS may sustain basal p38 MAPK
activity in macrophages (Fig. 5B).
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Based on the effects of MAPK cascades on the expression and
transcriptional activation of AP-1 proteins, we sought to determine
whether tBHQ and BNF could induce AP-1 mobility shift complexes in
macrophages. RAW264.7 cells were treated with 50 µM tBHQ or BNF, as
indicated in Figure 6, and nuclear
extracts were incubated together with a labeled AP-1 consensus
oligonucleotide. BNF induced a discernible increase in AP-1 shift
complexes within 2 h; this effect was sustained during the entire
observation period of 14 h (Fig. 6, lanes 2–5).
The addition of a 100-fold excess unlabeled AP-1 probe abrogated
protein binding to the labeled probe (Fig. 6, lane
6). Prior treatment of nuclear extracts with
anti-pan-Fos or anti-pan-Jun antisera decreased the abundance
of the shift complexes, demonstrating that these complexes contain
proteins from both major AP-1 protein families (Fig. 6, lanes
7 and 8). Similar results were obtained with
nuclear extracts from tBHQ-treated cells, except that the relative
abundance of the shift complexes declined within 6 h (Fig. 6, lanes 9–17). These results are in agreement with the
differences observed in the kinetics of JNK activation (Fig. 2). Taken
together, our data indicate that BNF and tBHQ induce heterodimeric
Fos/Jun complexes in macrophage cell lines. This agrees with the
ability of these chemicals to induce AP-1 shift complexes in
hepatocytes (36, 44, 52, 53, 54).
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The hARE sequence in the NQO1 promoter
(GTGACTCAGC) contains a consensus AP-1 sequence
(underlined) (38, 40, 43). Utilizing the labeled hARE oligonucleotide
shown in Figure 1, together with nuclear extracts from tBHQ- or
BNF-treated hepatocytes, results in at least two shift complexes
(39, 40, 41). To determine whether RAW264.7 cells contain hARE-binding
factors, we used the same labeled hARE probe together with nuclear
extracts from BNF- or tBHQ-treated RAW264.7 cells. BNF treatment
resulted in the occurrence of a new shift complex, designated comp 1,
in addition to two shift complexes (comp 2 and 3) seen in unstimulated
cells (Fig. 7, lanes 1 and
2). BNF also induced an increase in the abundance of
comp 2 (Fig. 7, lanes 1 and 2). While all
three complexes were effectively inhibited by a molar excess of
unlabeled hARE (lane 4), an unlabeled AP-1
consensus oligonucleotide competed for comp 2 only, suggesting that
this complex contains AP-1 proteins (lane 5).
Supershift analysis showed that comp 2 could be inhibited by an
anti-pan-Jun Ab (lane 6). tBHQ-treated
cells contained two complexes (comp 2 and 3), one of which (comp 2) was
induced by tBHQ (lane 3).
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), with nuclear extracts from RAW264.7 cells,
resulted in a single shift complex that comigrated with comp 3 of the
consensus hARE probe (lanes 7–11). While cold
competition with hARE(mAP1) blocked comp 3 formation, a molar excess of
a consensus AP-1 probe had no effect (lanes 10 and
11). Taken together, these data indicate that tBHQ
and BNF induce AP-1 binding to the hARE. These results are in agreement
with data in hepatocytes, which indicate that while the slower
migrating complex contains PAH- and tBHQ-inducible AP-1 proteins, the
faster migrating complex represents a noninducible, AP-1-independent
complex (39, 40, 41). Nguyen and Pickett suggested that the latter complex
represents a hARE-specific transcription factor (39, 40, 41). BNF, tBHQ, and LPS induce hARE reporter gene activity
ARE activation in hepatocytes is dependent on the generation of
ROS (35, 36). Since macrophages generate ROS in response to xenobiotics
(10, 12) as well as ligation of various membrane receptors, we sought
to determine whether these stimuli induce hARE activation in RAW264.7
cells. RAW264.7 cells were chosen for their ease of gene transfection
compared with THP-1 cells. We used a copy of the hARE linked to a CAT
reporter (hARE-tk-CAT; Fig. 1) to study ARE activation during treatment
with BNF, tBHQ, and LPS (38). Constitutively active CMV-CAT
served as a positive control, while a hARE mutant, hAREcm-CAT, which
contains an altered ARE core sequence, served as negative control (Fig. 1). Treatment with BNF and tBHQ induced a 3- and 6.9-fold increase,
respectively, in hARE-CAT activity in the intact reporter gene (Fig. 8A, lanes
2–4). The magnitude of these responses is comparable with
hARE-CAT responses in hepatocytes (38). Interestingly, LPS induced a
6.7-fold increase in CAT activity, implying that an LPS-binding
receptor such as the CD14 receptor (66) may activate a genetic response
element that has classically been linked to chemical effects. No CAT
activity was expressed in hAREcm-CAT-transfected cells (Fig. 8A, lanes 6–9).
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is whether the
AP-1 sequence in the unmodified hARE affects the transcriptional
activity of this reporter. For that reason, we subcloned the hARE(mAP1)
sequence, which disrupts the consensus AP-1, but not the hARE, into the
tk-CAT reporter (Fig. 1). This mutant reporter was transfected into
RAW264.7 cells in parallel with the unmodified hARE-CAT construct, and
cells were stimulated with the same stimuli depicted in Figure 8A (Fig. 8B). Compared with the hARE core
mutant, the hARE(mAP1) mutant showed definitive expression of CAT
activity (Fig. 8B, lanes 6–9).
These data show that although the hARE sequence overlaps with the AP-1
site, an intact AP-1 site is not necessary for generation of hARE-CAT
activity. These data support the existence of AP-1-independent,
ARE-specific transcription factors in macrophages (39, 40, 41, 45). Constitutive activation of the JNK cascade by DA-MEKK1 interferes in expression of hARE-CAT activity
The data in Figures 6 through 8 suggest that BNF and tBHQ
may regulate both hARE and AP-1 response elements. While hARE-specific
transcription factors remain to be identified, previous studies have
shown that the hARE response pathway is regulated by ROS (35, 36).
Since ROS play a role in PAH-induced JNK activation in macrophages
(Figs. 2 and 3), and the hARE contains an overlapping AP-1 sequence
(Fig. 1), we sought to determine whether the JNK cascade regulates
hARE-CAT activity. We used a dominant active (DA) Jun kinase kinase
kinase, MEKK, to activate the JNK cascade RAW264.7 cells (59).
First, we determined whether DA-MEKK1 was expressed in transfected
cells by using anti-MEKK1 immunoblotting (Fig. 9A). Compared with
mock-transfected cells or cells transfected with an empty vector,
MEKK-transfected cells expressed the dominant active MAPKKK (Fig. 9A). Moreover, we confirmed that JNK activity was
increased in DA-MEKK1 compared with untreated or empty
plasmid-transfected cells (Fig. 9B). Subsequently, we
cotransfected hARE-CAT with 1) an empty vector, 2) DA-MEKK1, or 3)
dominant negative or kinase inactive (KI) MEKK1 [pSR-MEKK(K432
M)] (59). Compared with BNF-, tBHQ-, or LPS-inducible reporter gene
activity in empty vector (Fig. 9C, lanes
1–4) or KI-MEKK1-expressing cells (lanes
9–12), the dominant active MEKK1-transfected cells showed
neither basal nor inducible CAT activity (lanes
5–8). These data indicate that although the hARE interacts
with AP-1 proteins, the JNK pathway that regulates AP-1 proteins exerts
an inhibitory effect on hARE transcriptional activity. To show that the
inhibitory effect of DA-MEKK1 on the hARE is mediated via the internal
AP-1 sequence, we cotransfected MEKK1 with the hARE(mAP1) reporter and
did not find interference in the tBHQ- or BNF-inducible hARE responses
(not shown). Taken together, this suggests that while BNF and tBHQ
induce two distinct biochemical events in macrophages, i.e., JNK and
hARE activation, the former exerts a negative regulatory effect on
the hARE.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The induction of
these responses in human and murine macrophage cell lines was dependent
on the generation of oxidative stress (Figs. 3 and 5). An additional
response pathway in macrophages is activation of the antioxidant
response element independent of the effect of chemicals on AP-1
proteins (
Figs. 7–9). At the level of the human ARE, JNK activation
suppressed hARE reporter gene activity (Fig. 9). These data establish
two important and novel biochemical activation pathways for PAH and
their derivatives in the immune system, and provide us with potential
targets by which to interfere in the effects of environmental
pollutants on allergic inflammation. Our understanding of the molecular mechanisms by which PAH and related xenobiotics impact the immune system is rudimentary compared with the understanding of the metabolic pathways that mediate the effects of such chemicals in hepatocytes. Best characterized is the pathway that is mediated by the AhR (29, 30, 31, 32). After entry into the cytosol, polycyclic aromatic and halogenated hydrocarbons are bound by the 95-kDa ligand-binding subunit of the AhR, which translocates to the nucleus, where it associates with the aryl hydrocarbon receptor nuclear translocator protein (29, 30, 31, 32). The AhR/aryl hydrocarbon receptor nuclear translocator complex acts as a transcriptional activator of genes that express the XRE, leading to the expression of CYP1A1 and other phase I drug-metabolizing enzymes. The XRE pathway is functional in macrophages and other immune cells, and has been best studied in the context of immunosuppression by polycyclic aromatic hydrocarbons and dioxin (28, 67, 68, 69). Recent findings of impaired lymphocyte development in AhR knockout mice also suggest that physiologic AhR ligands exist that play a role in the function of the immune system (70).
Our studies focused on the activation of two additional pathways that
have been linked to PAH stimulation, namely the AP-1 (Fig. 6) and the
ARE response pathways (
Figs. 7–9). PAH-induced ARE activation is
dependent on prior XRE activation and expression of CYP1A1 activity.
CYP1A1 is able to convert PAH to redox active compounds such as
quinones (46, 53). One hypothesis is that ARE activation depends on the
generation of oxidative stress by quinone derivatives (35, 36). PAH
that activate XRE and ARE response elements induce both phase I
(cytochromes P450) and phase II (detoxifying) enzymes, and are known as
bifunctional inducers (46, 47, 48). In contrast, compounds such as quinones
and other phenolic antioxidants that induce phase II enzyme expression
without affecting the activity of phase I enzymes are known as
monofunctional agents (46, 47, 48). Our data show that BNF and tBHQ induce
ARE reporter gene activity (Figs. 8 and 9), suggesting that the phase
II enzyme detoxification pathway is important in macrophages. In
addition, ARE consensus sequences have been found in the promoters of
the IL-6, P450 aromatase, ferritin-L, collagenase, and tyrosinase
genes, and the ARE pathway may therefore also be involved in these
cellular responses (50). Future studies will address which macrophage
genes are activated via this route by PAH. It is interesting that
bacterial LPS induced ARE-CAT activity (Fig. 8A), as
this suggests that a LPS-binding receptor may act as an ARE inducer.
Although there are no reports linking membrane receptors to the ARE
pathway, it is possible that the CD14 and other macrophage receptors
that induce ROS may induce cellular activation via an ARE (66). We are
in the process of exploring the effects of those receptors on the ARE
response pathway.
The activation of AP-1 response elements can contribute to a wide range
of biologic responses. PAH-induced NQO1 or glutathione
S-transferase (GST-Ya) gene expression in hepatoma cells is associated
with an increase in AP-1 activity (36, 44, 52, 53, 54). tBHQ and BNF induce
the expression of c-Jun, Jun B, Jun D, Fra1, and Fra2 (36, 43, 44, 52, 53, 54). In addition, Ainbinder et al. (54) showed that BNF and tBHQ
induce GST-Ya gene expression (Fig. 1) by activating a signaling
pathway that involves AP-1 proteins, Ras, and protein tyrosine kinases.
Other groups have reported that PAH can induce Ha-Ras,
c-myc, and protein tyrosine kinase activities (71, 72, 73). We
focused on MAPK activation, since these cascades are functionally
related to Ras and AP-1 proteins. While we failed to obtain
extracellular signal-regulated kinase (ERK) activation (not shown),
both JNK and p38 MAPK activities were increased in human and murine
macrophage cell lines upon exposure to BNF and tBHQ (
Figs. 2–4). The
kinetics of activation was faster and the intensity of the kinase
responses more robust with tBHQ compared with BNF treatment (Figs. 2 and 4). This may be due to direct engagement of the stress kinase
pathway by tBHQ, while BNF may require conversion to quinone products
before acting as a stress stimulus. Our results are in agreement with
the ability of butylated hydroxyanisole, a tBHQ homologue, to activate
the JNK cascade in Jurkat T cells (74). MAPK promote AP-1 activation by
two independent but complementary actions. The first is transcriptional
activation of AP-1 proteins, e.g., phosphorylation of the N-terminus of
c-Jun or ATF2 by JNK (56, 57). The second is increased expression of
AP-1 proteins by transcriptional activation of the promoters of AP-1
proteins, e.g., activation of the modified AP-1 RE in the c-Jun
promoter by phosphorylated c-Jun and ATF2 (56, 57). Moreover, p38 MAPK,
which also targets ATF2, acts synergistically with JNK to activate the
modified AP-1 response element in the c-jun promoter (75).
Taken together, our data provide an explanation for the reported
increase in AP-1 activity during BNF or tBHQ exposure (36, 43, 44, 52, 53, 54). Not all tBHQ-induced effects on the AP-1 RE are stimulatory
because Yoshioka et al. have shown that while tBHQ induces
fra1 and fra2 expression, it was a poor inducer
of c-fos (44). Because Fra1 and Fra2 tend to form
transcriptionally neutral AP-1 complexes, these may compete with
transcriptionally active c-Fos complexes, and thereby interfere in AP-1
activation (44). This notion is in keeping with our data that show that
AP-1 activation suppresses hARE-CAT activity (Fig. 9). The biologic
significance of AP-1 interference in hARE activation remains to be
determined.
The issue as to whether AP-1 proteins are involved in ARE activity is
controversial. One hypothesis is that the ARE is composed of two
adjacent AP-1 sites (Fig. 1) that are activated by chemically induced
AP-1 proteins (42, 53, 54). An opposing view holds that the ARE is
activated by a unique set of transcription factors, independent of the
effect of AP-1 proteins (38, 39, 40, 41, 45). While Pickett et al. (40) have
defined the ARE core as GTGACNNNGC,
Wasserman and Fahl defined the core sequence (underlined) as
TMANNRTGAYNNNGCRWWW (M = A
or C; R = A or G; Y = C or T; W = R or T; S = G or
C; 50). Our studies on hARE activation in macrophages showed that
mutation of the AP-1 consensus sequence at a site that does not overlap
with the hARE core does not affect ARE activation (Fig. 8B). However, mutation of bases in the hARE core
interfered in basal and inducible ARE activity (Fig. 8B). Gel-shift assays also showed that, while the
wild-type hARE yields at least two shift complexes, a constitutive
shift complex remained when the AP-1 sequence was disrupted (Fig. 7).
This complex may represent the ARE-specific transcription factor
postulated by Pickett et al. (41). Our study does not rule out a role
for AP-1 proteins in the extended ARE region, because our
oligonucleotide probes and reporter gene constructs did not include a
complete copy of the upstream AP-1-like element (Fig. 1) (36).
Taken together, our data show that a PAH and a representative quinone product induce oxidative stress in macrophages. Oxidative stress leads to the activation of stress-activated protein kinases that are involved in AP-1-mediated gene responses. Another response pathway linked to PAH in macrophages is activation of the antioxidant RE, which also contributes to gene expression. These activation responses by PAH in inhaled particulates are most likely an important contributor to airway inflammation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andre Nel, University of California, Los Angeles, School of Medicine, Department of Medicine, CIA, 52-175 CHS, 10833 Le Conte Ave., Los Angeles, CA 90095. E-mail address:
3 Abbreviations used in this paper: DEP, diesel exhaust particles; Ahr, aromatic hydrocarbon receptor; AP-1, activator protein-1; ARE, antioxidant response element; ATF2, activating transcription factor 2; BNF, ß-napthoflavone; CAT, chloramphenicol acetyltransferase; comp, complex; CYP1A1, cytochrome P4501A1; GST, glutathione S-transferase; hARE, human antioxidant response element; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NAC, N-acetylcysteine; NQO1, nicotinamide-adenine dinucleotide phosphate (NADPH):quinone oxidoreductase; PAH, polycyclic aromatic hydrocarbon; RE, response element; ROS, reactive oxygen species; tBHQ, tert-butylhydroxyquinone; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; XRE, xenobiotic response element.
Received for publication December 1, 1997. Accepted for publication March 16, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mediates recruitment of neutrophils and esosinophils during airway inflammation. J. Immunol. 54:5411.
in bronchoalveolar lavage fluid of allergic asthmatic patients. Am. J. Respir. Crit. Care Med. 153:1398.[Abstract]
B, and glutathione S-transferase gene expression. J. Biol. Chem. 271:13422. 1, formation of inositol 1,4,5-trisphosphate, and mobilization of intracellular calcium. Proc. Natl. Acad. Sci. USA 90:6105.This article has been cited by other articles:
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 |
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  A. Natsch and R. Emter Skin Sensitizers Induce Antioxidant Response Element Dependent Genes: Application to the In Vitro Testing of the Sensitization Potential of Chemicals Toxicol. Sci., March 1, 2008; 102(1): 110 - 119. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Huc, X. Tekpli, J. A. Holme, M. Rissel, A. Solhaug, C. Gardyn, G. Le Moigne, M. Gorria, M.-T. Dimanche-Boitrel, and D. Lagadic-Gossmann c-Jun NH2-Terminal Kinase-Related Na+/H+ Exchanger Isoform 1 Activation Controls Hexokinase II Expression in Benzo(a)Pyrene-Induced Apoptosis Cancer Res., February 15, 2007; 67(4): 1696 - 1705. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Wan and D. Diaz-Sanchez Phase II Enzymes Induction Blocks the Enhanced IgE Production in B Cells by Diesel Exhaust Particles. J. Immunol., September 1, 2006; 177(5): 3477 - 3483. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Ogborne, S. A. Rushworth, and M. A. O'Connell {alpha}-Lipoic Acid-Induced Heme Oxygenase-1 Expression Is Mediated by Nuclear Factor Erythroid 2-Related Factor 2 and p38 Mitogen-Activated Protein Kinase in Human Monocytic Cells Arterioscler Thromb Vasc Biol, October 1, 2005; 25(10): 2100 - 2105. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Teske, A. A. Bohn, J. F. Regal, J. J. Neumiller, and B. P. Lawrence Activation of the aryl hydrocarbon receptor increases pulmonary neutrophilia and diminishes host resistance to influenza A virus Am J Physiol Lung Cell Mol Physiol, July 1, 2005; 289(1): L111 - L124. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. J. Yin, J. Y. C. Ma, J. M. Antonini, V. Castranova, and J. K. H. Ma Roles of Reactive Oxygen Species and Heme Oxygenase-1 in Modulation of Alveolar Macrophage-Mediated Pulmonary Immune Responses to Listeria monocytogenes by Diesel Exhaust Particles Toxicol. Sci., November 1, 2004; 82(1): 143 - 153. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Ryan, M. F. Smith Jr., M. K. Sanders, and P. B. Ernst Reactive Oxygen and Nitrogen Species Differentially Regulate Toll-Like Receptor 4-Mediated Activation of NF-{kappa}B and Interleukin-8 Expression Infect. Immun., April 1, 2004; 72(4): 2123 - 2130. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Bea, F. N. Hudson, A. Chait, T. J. Kavanagh, and M. E. Rosenfeld Induction of Glutathione Synthesis in Macrophages by Oxidized Low-Density Lipoproteins Is Mediated by Consensus Antioxidant Response Elements Circ. Res., March 7, 2003; 92(4): 386 - 393. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. S. Gilmour, I. Rahman, K. Donaldson, and W. MacNee Histone acetylation regulates epithelial IL-8 release mediated by oxidative stress from environmental particles Am J Physiol Lung Cell Mol Physiol, March 1, 2003; 284(3): L533 - L540. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Rudra-Ganguly, S. T. Reddy, P. Korge, and H. R. Herschman Diesel Exhaust Particle Extracts and Associated Polycyclic Aromatic Hydrocarbons Inhibit Cox-2-dependent Prostaglandin Synthesis in Murine Macrophages and Fibroblasts J. Biol. Chem., October 11, 2002; 277(42): 39259 - 39265. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kawasaki, H. Takizawa, K. Takami, M. Desaki, H. Okazaki, T. Kasama, K. Kobayashi, K. Yamamoto, K. Nakahara, M. Tanaka, et al. Benzene-Extracted Components Are Important for the Major Activity of Diesel Exhaust Particles . Effect on Interleukin-8 Gene Expression in Human Bronchial Epithelial Cells Am. J. Respir. Cell Mol. Biol., April 1, 2001; 24(4): 419 - 426. [Abstract] [Full Text]
|
|
|
|
|
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N. Li, M. I. Venkatesan, A. Miguel, R. Kaplan, C. Gujuluva, J. Alam, and A. Nel Induction of Heme Oxygenase-1 Expression in Macrophages by Diesel Exhaust Particle Chemicals and Quinones via the Antioxidant-Responsive Element J. Immunol., September 15, 2000; 165(6): 3393 - 3401. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Fahy, H. Hammad, S. Sénéchal, J. Pestel, A.-B. Tonnel, B. Wallaert, and A. Tsicopoulos Synergistic Effect of Diesel Organic Extracts and Allergen Der p 1 on the Release of Chemokines by Peripheral Blood Mononuclear Cells from Allergic Subjects . Involvement of the MAP Kinase Pathway Am. J. Respir. Cell Mol. Biol., August 1, 2000; 23(2): 247 - 254. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. K. Kerzee and K. S. Ramos Activation of c-Ha-ras by Benzo(a)pyrene in Vascular Smooth Muscle Cells Involves Redox Stress and Aryl Hydrocarbon Receptor Mol. Pharmacol., July 1, 2000; 58(1): 152 - 158. [Abstract] [Full Text]
|
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|
|
|
|
T. S. Hiura, M. P. Kaszubowski, N. Li, and A. E. Nel Chemicals in Diesel Exhaust Particles Generate Reactive Oxygen Radicals and Induce Apoptosis in Macrophages J. Immunol., November 15, 1999; 163(10): 5582 - 5591. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-W. Kwon, S. Ueda, M. Ueno, J. Yodoi, and H. Masutani Mechanism of p53-dependent Apoptosis Induced by 3-Methylcholanthrene. INVOLVEMENT OF p53 PHOSPHORYLATION AND p38 MAPK J. Biol. Chem., January 11, 2002; 277(3): 1837 - 1844. [Abstract] [Full Text] [PDF]
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