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Department of Internal Medicine, University of Iowa College of Medicine and Veterans Affairs Medical Center, Iowa City, IA 52243
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Abstract |
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B and AP-1. In this study we evaluated the role of
asbestos, a prototypic model of chronic fibrotic lung disease, in Ref-1
expression and activity. We found that incubation with low
concentrations of crocidolite asbestos (0.5–1.25 µg/cm2)
resulted in an increase in nuclear Ref-1 protein after 5 min, with a
persistent elevation in protein up to 24 h. Additionally, an
increase in nuclear Ref-1 could be induced by treating the cells with
an oxidant-generating stimulus (iron loading plus PMA) and inhibited by
diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. The
asbestos-induced accumulation of nuclear Ref-1 was associated with an
increase in AP-1 DNA binding activity. These findings suggest that an
exposure associated with fibrotic lung disease, i.e., asbestos,
modulates accumulation of nuclear Ref-1 in macrophages, and that this
effect is mediated by an oxidant stimulus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A characteristic feature of many of the chronic lung disorders, including pulmonary fibrosis, is that the types of macrophages in the lung change and become more monocyte-like, both phenotypically and functionally (7, 8). We have shown that normal alveolar macrophages express very little protein kinase C-induced DNA binding activity of the transcription factor, AP-1, as compared with monocytes. This decrease in AP-1 binding activity is due to a defect in redox regulation of AP-1 proteins that occurs secondary to a reduction in the amount of the redox active protein, redox factor (Ref)3-1 (9). We also showed that GM-CSF, a mediator known to regulate alveolar macrophage function, also regulates amounts of Ref-1 in these cells. As a composite, these studies suggest that Ref-1 may play an important role in alveolar macrophage phenotype and function (10).
AP-1 is a transcription factor comprised of homo- or heterodimers of Fos and Jun family members. These complexes bind to specific DNA sequences in the promoter-regulatory region of various genes (11, 12). AP-1 activity is regulated at many levels, including transcription of genes that code for the AP-1 proteins, message stability, and translation of the mRNAs. AP-1 activity is also regulated by the composition of the AP-1 complexes, phosphorylation of the proteins, and redox regulation of specific cysteine residues in the AP-1 proteins (13, 14, 15). The redox status of the AP-1 protein complex is an important determinant of the binding of AP-1 to DNA and is regulated by the nuclear protein, Ref-1 (14). This dual-function protein acts as both a base excision repair protein and a redox regulatory protein. The redox function is further controlled by thioredoxin, which activates Ref-1 to reduce conserved cysteine residues on Fos and Jun, allowing AP-1 DNA binding to occur (13).
Asbestos-related pulmonary fibrosis continues to be an important and significant cause of interstitial lung disease, despite the recognition of the health hazards of asbestos inhalation and efforts to reduce exposure. New cases continue to be identified, in part because of the latency period between exposure and development of asbestos-related lung disease (16). Asbestos is a collective term for naturally occurring mineral silicates of which there are two types: serpentine and amphibole. Serpentine asbestos fibers are curly-stranded structures of which chrysotile is an example, while amphibole asbestos (crocidolite, amosite) are rod-shaped, straight fibers (17). The different structural properties of these fibers are relevant to their pathogenicity in asbestos-related lung disease. Amphibole fibers are often regarded as more toxic, as they tend to accumulate in the lung parenchyma and are less effectively cleared as compared with chrysotile asbestos (17, 18, 19, 20). Crocidolite is an iron-rich fiber (Na2[Fe3+]2[Fe2+]3[OH]2[Si8O22]) which can catalyze redox reactions and generate reactive oxygen species (ROS) via the Fenton reaction. This property is thought to be important as a causative factor in the pathogenesis of asbestosis (21).
There are many studies supporting the hypothesis that ROS are involved in the development of asbestos-mediated cellular injury. Goodglick and Kane (22) showed that murine macrophages release hydrogen peroxide in response to crocidolite asbestos, resulting in cell death. In an in vivo model, Schapira et al. (23) demonstrated that intratracheal asbestos instillation resulted in the generation of HO· in rat lungs. Further support for the role of ROS in asbestos cytotoxicity includes the observation that antioxidants such as catalase and superoxide dismutase attenuate asbestos-induced cytotoxicity in a number of pulmonary target cells (22, 23, 24, 25, 26, 27), and antioxidant enzyme expression is up-regulated by asbestos exposure in vitro (26) and in vivo (28).
Asbestos has been shown to increase a number of inflammatory mediators
and transcription factors (29, 30, 31, 32, 33, 34). Asbestos treatment
up-regulates the early response protooncogenes, c-fos and
c-jun, which comprise the AP-1 transcription factor complex
(35), and increases AP-1 and NF-B DNA binding and
transcriptional activation (31, 32, 33, 34). It is postulated that
asbestos, through the modulation of a variety of cell signaling
pathways, may promote expression of genes important in inflammation and
cell proliferation, thus contributing to the pathogenesis of
asbestos-induced lung disease.
Other investigators have also examined the effect of asbestos on Ref-1 amounts and AP-1 activity. Fung et al. (36) showed that mesothelial cells treated with crocidolite asbestos exhibit marked increases in Ref-1 mRNA and protein. These investigators were specifically interested in mechanisms of asbestos-induced DNA repair, hypothesizing that Ref-1, as an important component of the base excision repair process, might be induced by asbestos. Based on previous studies linking Ref-1 and AP-1 modulation by asbestos and the role of reactive oxygen intermediates in asbestos-induced cytotoxicity, we hypothesize that crocidolite asbestos increases nuclear Ref-1 in alveolar macrophages, resulting in increased AP-1 DNA binding, and that this occurs via an oxidant-mediated process.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alveolar macrophages were obtained from bronchoalveolar lavage as previously described (9). Briefly, normal volunteers with a lifetime nonsmoking history, no acute or chronic illness, and no current medications underwent bronchoalveolar lavage. The lavage procedure used five 20-ml aliquots of sterile, warmed saline in each of three segments of the lung. The lavage fluid was filtered through two layers of gauze and centrifuged at 1500 x g for 5 min. The cell pellet was washed twice in HBSS without Ca2+ and Mg2+ and suspended in complete medium (RPMI tissue culture medium (Life Technologies, Gaithersburg, MD) with 5% FCS (HyClone Laboratories, Logan, UT) and 80 µg/ml gentamicin). Differential cell counts were determined using a Wright-Giemsa-stained cytocentrifuge preparation. All cell preparations had between 90 and 100% alveolar macrophages. This study was approved by the Committee for Investigations Involving Human Subjects at the University of Iowa (Iowa City, IA).
Selection of asbestos fibers
Previously well-characterized National Institute of Environmental Health Sciences long crocidolite (37, 38) was kindly provided by Dr. T. Hesterberg (Dupont Haskell Laboratory, Wilmington, DE). Size characterization data are as follows: diameter range, 0.08–1.30 µm; geometric mean diameter, 0.19 µm; length range, 1.3–30.7 µm; geometric mean length, 7.1 µm. The asbestos solutions were prepared in sterile PBS and added to the culture medium before use. The solutions used for these experiments were tested for endotoxin contamination and found to contain <0.08 ng/ml endotoxin. As used in our experiments, final endotoxin concentrations were <0.0005 ng/ml.
Culture of RAW 264.7 cells
RAW 264.7 cells were purchased from American Type Culture Collection (Manassas, VA) and grown in complete DMEM supplemented with 10% FCS, pyruvate, glutamine, and gentamicin. The cells were used at passages 4–11 and were grown to 80% confluence, scraped, and plated 18–24 h before an experiment.
Isolation of nuclear extracts and EMSAs
Alveolar macrophages or RAW cells were treated with crocidolite asbestos (suspended in PBS) at varying concentrations and washed with cold PBS immediately before isolation of protein. The nuclear pellets were prepared by resuspending cells in 0.4 ml of lysis buffer (10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA), placing them on ice for 15 min, and then vigorously mixing after the addition of 25 µl of 10% Nonidet P-40 (Sigma-Aldrich, St. Louis, MO). After a 30-s centrifugation (16,000 x g, 4°C), the pelleted nuclei were resuspended in 50 µl of extraction buffer (50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10% glycerol) and incubated on ice for 20 min. Nuclear extracts were stored at -70°C. The DNA binding reaction (EMSA) was done at room temperature in a mixture containing 5 µg of nuclear proteins, 1 µg poly(d(I-C)), and 20,000 cpm of 32P-labeled double-stranded oligonucleotide probe for 30 min. The samples were fractionated through a 5% polyacrylamide gel in 1x TBE (6.05 g/L Tris base, 3.06 g/L boric acid, 0.37 g/L EDTA-Na2·H2O). The sequence of the nucleotide was 5'-CGCTTGATGAGTCAGCCGGAA-3' (AP-1). Experiments were repeated three times. For the inhibitor studies, cells were pretreated with 50 µM diphenyleneiodonium chloride (DPI; Calbiochem, San Diego, CA) for 1 h, then with asbestos for 1 h.
Western analysis
For these studies, alveolar macrophages or RAW cells were cultured for varying times with different concentrations of crocidolite asbestos. For the in vitro oxidant studies, RAW cells were plated and cultured as above then preloaded with 0.5 mM FeCl3 (Sigma-Aldrich, St. Louis, MO) for 30 min, rinsed with medium, and treated with PMA (100 ng/ml) for 1 h. At the end of the treatment period nuclear protein (see Isolation of nuclear extracts and EMSAs) or total cellular protein extracts were obtained as previously described (39). An aliquot of the supernatant was used to determine protein concentration by the Coomassie blue method. Equal amounts of protein (10 µg for nuclear extracts and 50 µg for total cellular protein) were mixed 1:1 with 2x sample buffer, loaded onto a 10% SDS-PAGE gel, and run at 80 V for 2 h. Cell proteins were transferred to nitrocellulose (ECL; Amersham, Arlington Heights, IL) for 1 h at 4°C at 100 V and visualized using a Ref-1-specific Ab (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive bands were developed using a chemiluminescent substrate (ECL; Amersham).
Quantitation of ROS using fluorescence spectrometry
Passage 4 RAW 264.7 cells were plated on 48-well plates at 1 x 105 cells/well and cultured overnight at 37°C in a CO2 incubator. The cells were treated with crocidolite asbestos at 0.5 and 5 µg/cm2 for 15, 30, 60, 180, and 360 min. After asbestos treatment, cells were washed with PBS then loaded with the fluorescent probe 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (Molecular Probes, Eugene, OR) at a 10 µM final concentration for 30 min in the dark at room temperature. The cells were washed once with PBS and fluorescence readings were performed using the Wallac Victor2 plate reader (540 nm excitation and 620 nm emission; Wallac, Gaithersburg, MD).
Preparation of adenoviral Ref-1 constructs
Three recombinant adenoviral vectors expressing enhanced green
fluorescent protein (AdEGFP; purchased from the University of Iowa Gene
Transfer Vector Core), human Ref-1 wild-type, or a human
Ref-1 gene mutated at amino acid 65 (Cys to Ala) to
eliminate the redox function of the protein were used for the
adenoviral studies. Hemagglutinin (HA)-tagged Ref-1
wild-type and redox-negative mutants in pcDNA3 vectors were kindly
provided by Dr. M. Kelley (Indiana University School of Medicine,
Indianapolis, IN). The Ref-1 genes were subcloned
into a shuttle vector containing the CMV promoter and first-generation
recombinant adenoviral stocks were generated by the University of Iowa
Gene Transfer Vector Core (40). The particle titers of the
adenoviral stocks were typically 1012 DNA
particles/ml; functional titers were 
4 x
1010 PFU/ml. For the adenoviral infection
experiments, RAW 264.7 cells were mixed with virus at 4000 PFU/cell
and plated on six-well plates in serum-free medium. FBS was added back
to the cultures after 5 h to a final concentration of 10%. The
cells were incubated at 37°C for 40–46 h, preloaded with
Fe2+ for 30 min, and then treated with PMA (100
ng/ml) for 1 h. Cells were rinsed with cold PBS and then harvested
for either total cellular or nuclear protein. Expression of the
HA-tagged protein was confirmed in each experiment using Western
analysis and a high-affinity anti-HA mAb (Sigma-Aldrich).
Statistical analysis
Statistical analysis of the densitometric data was performed by determining the fold increase of all the samples as they relate to the control. Statistical comparisons were performed using a paired t test with a p value of < 0.05 considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In order for Ref-1 to modulate AP-1 DNA binding, it must
translocate to the nucleus. To test the hypothesis that crocidolite
asbestos increases Ref-1 nuclear protein in macrophages, we treated
human alveolar macrophages and RAW 264.7 cells with varying
concentrations of asbestos. Fig. 1
shows
that crocidolite increases nuclear Ref-1 amounts relative to controls
in RAW cells (Fig. 1A) and in human alveolar macrophages
(Fig. 1B). These data demonstrate that crocidolite asbestos
up-regulates the amounts of nuclear Ref-1.
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To examine the time course of increases in nuclear Ref-1 in
response to asbestos treatment, RAW cells were treated for varying
times ranging from 5 to 360 min. Nuclear extracts and total cellular
proteins were isolated in parallel experiments and Western analysis was
performed. Fig. 2 shows that nuclear
Ref-1 amounts are increased above control levels in as little as 5 min
after treatment with asbestos, while total cellular Ref-1 amounts
remain constant. These data suggest that the primary result of asbestos
exposure is to induce Ref-1 translocation from the cytosol to the
nucleus, rather than increasing total Ref-1.
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Previous investigators have shown that cytoplasmic Ref-1 is
translocated to the nucleus in response to oxidant stimuli in Raji
cells (41). The observation that there is rapid nuclear
accumulation of Ref-1 in RAW cells led us to hypothesize that a similar
mechanism may account for our findings. To test this hypothesis we used
a system of iron loading and PMA stimulation as a means to promote
HO· production by the macrophages (42). Fig. 3 shows that the production of oxidants
that occurs in response to PMA stimulation in iron-loaded macrophages
increases nuclear Ref-1 amounts as compared with controls and cells
treated with PMA alone (p < 0.05). Iron
loading alone also results in a significant increase in Ref-1 protein
amounts. Having shown that oxidant production results in the
accumulation of nuclear Ref-1, we next asked whether crocidolite
treatment increased ROS production by macrophages.
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To address the question of whether or not macrophages exposed to
asbestos produced increased amounts of ROS, we used the fluorescent
probe, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, to
indirectly quantitate ROS using a fluorescent spectrometer
(43, 44, 45, 46). Fig. 4 shows
relative fluorescence intensity in live RAW cells treated with asbestos
for 15, 30, and 60 min and shows a significant increase in ROS
production in the cells at 30 and 60 min (p <
0.05). These data demonstrate that crocidolite increases intracellular
ROS in macrophages.
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To establish a link between crocidolite-induced ROS and Ref-1
nuclear localization, we inhibited intracellular oxidant production by
inhibiting NADPH oxidase. We hypothesized that asbestos acts on or
through NADPH oxidase to increase ROS production, which in turn
increases nuclear Ref-1 amounts. To test this we treated RAW cells with
DPI, a potent inhibitor of NADPH oxidase, before asbestos exposure.
Fig. 5 compares control cells pretreated
with and without DPI to those treated with asbestos and shows that the
asbestos-induced increase in nuclear Ref-1 is inhibited by DPI. These
data show that crocidolite-induced Ref-1 nuclear localization depends
on NADPH-linked oxidant production.
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Because nuclear Ref-1 amounts are increased in macrophages treated
with asbestos, we next wanted to determine whether there was a
functional consequence to the increased amounts of Ref-1. To do this,
we evaluated the effect of crocidolite on AP-1 DNA binding, because
Ref-1 is directly responsible for activation of the AP-1 complex. Human
alveolar macrophages were treated with asbestos for 24 h, nuclear
protein was isolated, and gel shift assay was performed using an AP-1
consensus oligonucleotide. Fig. 6A shows that AP-1 DNA binding
is increased in cells treated with asbestos as compared with controls.
PMA, a known activator of AP-1 DNA binding, is used as a positive
control. These data demonstrate that, consistent with its effect on
Ref-1, crocidolite increases AP-1 DNA binding. As a corollary to the
experiment shown in Fig. 5
, we wanted to evaluate the effect of NADPH
oxidase inhibition on AP-1 DNA binding in macrophages. Fig. 6B shows that AP-1 DNA binding is increased in
response to treatment with asbestos and that this increased binding is
diminished in the presence of the NADPH oxidase inhibitor, DPI
(p < 0.01).
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To test the hypothesis that Fe2+ and ROS are
involved in the observed up-regulation of Ref-1 and AP-1 DNA
binding, we used adenoviral vector constructs expressing
Ref-1 wild-type (AdRef-1wt) or Ref-1
mutated in the redox domain to eliminate the redox function of the
protein (AdRef-1redox-). RAW 264.7 cells
were infected with AdEGFP (the control vector), AdRef-1wt,
or AdRef-1redox- and then loaded with
Fe2+ and treated with PMA to generate production
of ROS. Fig. 7 shows that
Fe2+/PMA stimulation results in a marked increase
in AP-1 DNA binding in the AdRef-1wt-infected cells and that
this response is abolished in those cells transduced with the
redox-defective Ref-1 mutant. This experiment demonstrates
clearly that AP-1 DNA binding in macrophages not only requires Ref-1
but also requires, more specifically, the redox function of
Ref-1.
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In Fig. 8, we hypothesize that one
possible mechanism for increased nuclear Ref-1 and AP-1 DNA binding in
asbestos-treated macrophages involves oxidant generation mediated by
Fe2+ containing asbestos fibers and HO·
generation via NADPH oxidase.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Asbestos exposure remains an important cause of interstitial pulmonary
fibrosis. Though the in vivo and in vitro toxicity of asbestos has been
long been recognized, the exact molecular mechanisms by which cellular
injury and subsequent fibrosis occur are not well defined. Asbestos has
been shown to induce cellular injury through genotoxicity, modulation
of cell proliferation, apoptosis, and inflammation (19, 50, 51). One of the more interesting hypotheses emerging from these
studies is the idea that ROS such as hydrogen peroxide, superoxide, and
hydroxyl radicals play an important role in the development of
pulmonary toxicity by asbestos. It is well established that asbestos
fibers can augment the production of ROS in cell-free systems
(21, 50, 51). Free radicals damage cellular proteins,
lipids, and DNA and contribute to cell death and malignant
transformation (17, 19, 51). An important property of
asbestos that contributes to its ability to generate ROS is the
presence of iron, which associates with the fibers. Via the Fenton
reaction, the asbestos-associated ferrous iron oxidizes hydrogen
peroxide to ferric iron and highly reactive hydroxyl radicals
[Fe2+ +
H2O2
Fe3+
HO- + HO·]. Superoxide radicals can reduce ferric iron
back to ferrous iron and continue the chain reaction of ROS production.
The mechanism whereby ROS are generated in response to asbestos
exposure is thought to involve both generation of free radicals via the
Fenton reaction from asbestos-associated iron and generation of
oxidants by macrophages as a result of "frustrated" or incomplete
phagocytosis of the asbestos fibers (22). We hypothesized
that crocidolite asbestos, via a ROS-mediated mechanism, increases
Ref-1 nuclear protein and AP-1 DNA binding in macrophages.
Ref-1 is a ubiquitous bifunctional protein that possesses both redox
regulatory activity and DNA repair activity. In signal transduction,
Ref-1 is important in mediating DNA binding of the AP-1 protein complex
(52). This occurs via a posttranslational mechanism in
which conserved cysteine residues in the DNA binding domains of Fos and
Jun proteins are reduced, allowing DNA binding to occur. We have
previously shown that normal alveolar macrophages express decreased
AP-1 DNA binding compared with blood monocytes. This appears to be due
to a decrease in the amount of Ref-1 (9). Although the
functional consequences of this are unknown, we postulate that this may
decrease the expression of profibrotic genes that are known to be
driven by AP-1. AP-1 DNA binding is important for the expression of a
number of genes whose proteins have been implicated in the development
of fibrosis, including GM-CSF, alveolar macrophage-derived collagenase,
and TGF-
(11, 12, 53). We postulate that certain
stimuli, such as exposure to asbestos fibers, may trigger AP-1 DNA
binding in normal alveolar macrophages, and this may play a role in the
initiation/development of fibrosis in the lung.
A number of prior studies have demonstrated the modulation of Ref-1 expression. Both in vivo and in vitro studies have demonstrated the role of oxidant stress in the up-regulation of Ref-1 mRNA and protein levels (41, 47, 48, 49, 54, 55, 56). Diamond et al. (57) have shown that oxidant stress in the form of heat shock in HeLa and NIH 3T3 cells activates the early response genes, c-Fos and c-Jun, and that this response is dependent upon Ref-1. Other investigators have shown that hormones can up-regulate Ref-1 in specific cell types (58, 59). Finally, Fung et al. (36) examined Ref-1 mRNA and protein amounts in rat pleural mesothelial cells treated with crocidolite asbestos. They found that asbestos exposure increased Ref-1 mRNA, protein, and DNA incision activity and postulated that this may be an important adaptive response to oxidative injury in terms of both DNA repair and signal transduction.
Our studies demonstrate that crocidolite asbestos can increase nuclear Ref-1 protein in human alveolar and murine peritoneal macrophages by a mechanism that involves the production of ROS and is inhibited by blocking the function of NADPH oxidase.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dawn M. Flaherty, Division of Pulmonary, Critical Care, and Occupational Medicine, University of Iowa Hospitals and Clinics, C-33 GH, Iowa City, IA 52242. E-mail address: flahertydm{at}mail.medicine.uiowa.edu
3 Abbreviations used in this paper: Ref, redox factor; ROS, reactive oxygen species; DPI, diphenyleneiodonium chloride; HA, hemagglutinin.
Received for publication July 20, 2001. Accepted for publication April 2, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 gene by transforming growth factor-1 and phorbol ester occurs through the same target sequences. J. Biol. Chem. 264:19373. site. J. Biol. Chem. 272:11840.B (NF-B) DNA-binding activity and NF-B-dependent gene expression in tracheal epithelial cells. Proc. Natl. Acad. Sci. USA 92:8458.1 induces prolonged severe fibrosis in rat lung. J. Clin. Invest. 100:768.[Medline]
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