1 Department of Pediatrics, Duke University Medical Center; 4 Department of Internal Medicine, Duke University Medical Center, Durham 27710; 2 Center for Environmental Medicine and Lung Biology, University of North Carolina, Chapel Hill 27599; and 3 National Health and Environmental Effects Research Laboratory, Office of Research and Development, Environmental Protection Agency, Research Triangle Park, North Carolina 27711
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ABSTRACT |
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Anion exchange protein 2 (AE2) is a
membrane-bound protein that mediates chloride-bicarbonate exchange. In
addition to regulating intracellular pH and cell volume, AE2
exports superoxide (O
superoxide; ion transport; gene regulation; activator protein-1
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INTRODUCTION |
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OXIDATIVE TISSUE INJURY by increased levels of reactive oxygen species (ROS) can be produced during inflammation, ischemia/reperfusion (13, 27), hyperoxia, and hypoxia (5). Normally, ROS generated as by-products of aerobic metabolism are contained by an elaborate system of antioxidant defenses. When the level of ROS generated is greatly increased, these defenses can be overwhelmed, causing a state of oxidative stress.
The most readily produced ROS is superoxide anion
(O
AE2 is a membrane-bound electroneutral chloride-bicarbonate
(Cl/HCO
AE2 mRNA and protein expression and localization have been demonstrated in a number of tissues (15), including the lung (7, 17, 18, 23). The relatively ubiquitous distribution of AE2 among epithelial cells demonstrates some tissue specificity through the expression of at least three isoforms (21). The DNA sequence of AE2 has been identified in the human (20) and the mouse (16) and has been shown to contain several response elements in its promoter region (20). Although the regulation of AE2 has been investigated in the kidney (8) and intestine (6), little is known about the regulation of AE2 expression in the lung.
Given the involvement of AE2 in superoxide exchange, we hypothesized that the expression of AE2 in the lung is regulated by oxidative stress. We tested this hypothesis in vitro by evaluating AE2 mRNA and protein expression in differentiated human bronchial epithelial (HBE) cells after exposure to hydrogen peroxide (H2O2). The regulation of AE2 by oxidative stress was further tested in vivo by measuring AE2 protein expression in the lungs of rats exposed to hyperoxia. Finally, we evaluated the role of activator protein-1 (AP-1), a redox-sensitive transcription factor, in regulating the expression of this protein.
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METHODS |
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Cell culture and in vitro exposure. Primary HBE cells were obtained from healthy, nonsmoking adult volunteers after consent was obtained. The protocol and consent form were approved by the University of North Carolina School of Medicine Committee on the Protection of the Rights of Human Subjects. Cells were obtained by cytologic brushing at bronchoscopy and expanded to passage 3 in bronchial epithelial growth medium. They were plated on collagen-coated filter supports with a 0.4-µM pore size (Trans-CLR; Costar, Cambridge, MA) at a density of 1 × 105 and inserted into 12-well culture plates. Cells were maintained in a 1:1 mixture of bronchial epithelial cell basic medium (BEBM) and DMEM-H with SingleQuot supplements, bovine pituitary extracts (13 mg/ml), bovine serum albumin (BSA, 1.5 µg/ml), and nystatin (20 units) with 0.5 ml in the apical chamber and 1.5 ml in the basolateral chamber. Medium was replaced every 48 h. Retinoic acid was added on day 2 to promote differentiation. Air-liquid interface (ALI) was created on day 6 by removing the apical medium. The cells were maintained in the above media until they had achieved uniform differentiation into ciliated, mucus-producing cells, ~9-10 days after creation of ALI.
Differentiated cells were exposed at the apical surface to a single application of H2O2 (100 µM) or to media consisting of a 1:1 mixture of BEBM and DMEM-H without supplements. Placing the oxidant in the apical chamber more closely mimics the in vivo situation and minimizes direct exposure of the transporter to the oxidant, which is located primarily at the basolateral plasma membrane (17, 18). This concentration of H2O2 was chosen because it is typically not associated with alterations in cell viability and replication competence (28). Cells were harvested for measurement of RNA expression after exposure for 0, 4, 12, and 24 h; protein expression after 0, 4, 12, 24, and 36 h; and AP-1 activation after 0, 30, 60, and 120 min.Assessment of cellular injury by carbonyl analysis and lactate
dehydrogenase release.
HBE cells grown at ALI were exposed to a single application of
H2O2 at varying concentrations (100 µM, 1 mM,
5 mM) for 4 h. After treatment with H2O2,
medium was removed, and cells were scraped off the filters into 500 µl of methanol and kept on ice. The cells were incubated briefly with
2,4-dinitrophenylhydrazine (DNPH in acetonitrile), which reacts
selectively with carbonyls. Samples were analyzed for carbonyl content
as an end product of lipid peroxidation after separation from protein
and nucleic acids using a 2690 Separation Module HPLC. Separations were
done on a Waters' Xterra C18 column (2.1 × 15 mm; 3.5 µm).
Carbonyl analysis was performed using a 2487 dual wavelength absorbance
detector (at 365 nm) and a ZMD mass spectrometer (Waters' Associates,
Milford, MA). The solvent system consisted of water-methanol
(9:1, solvent A) and acetonitrile-methanol (9:1,
solvent B) both containing 0.01% formic acid. The
solvent gradient used was 0-10 min at 44.4% solvent
A-55.6% solvent B and then a linear gradient to
11% solvent A-89% solvent B over 30 min at 0.25 ml/min. The mass spectrometry was run in electrospray
ionization negative mode with a capillary voltage of 2 kV, cone voltage
of 18V, extractor voltage of
3V, source block temperature of
150°C, desolvation temperature of 350°C, and radio frequency lens
of 0.3. Selected ion monitoring was done at m/z 223, 237, 251, 265, 279, 293, 307, 321, 335, 349, 363, and 417 ± 2; these values
correspond to the derivatized parent ions for C2-C12, respectively. The
DNPH derivative of cis-11-hexadecenal (m/z 417; Aldrich
Chemical, Milwaukee, WI) was added as an internal standard in some
runs. Data (e.g., peak area) were analyzed using Waters' MassLynx
software (version 3.2).
Evaluation of AE2 localization by immunofluorescence. HBE cells were grown and differentiated at ALI. The filter supports for the cells were excised and embedded in paraffin. Sections of 8 µm were deparaffinized in xylene and hydrated to 70% alcohol. Nonspecific binding sites were blocked with 20% normal goat serum in Tris-buffered saline (TBS)-Triton X-100 (0.25%) for 30 min. The slides were incubated overnight at 4°C with a mouse polyclonal antibody against the COOH-terminal amino acids of AE2 [1:10 dilution with Tris-Triton X-100 (0.25%)]. The primary AE2 antibody was developed in mouse ascites fluid against the rat AE2 COOH-terminal amino acids 1,224-1,237 (CEGVDEYNEMPMPV-COOH) (30) by established methods (22). Antigen-antibody complexes were stained with a FITC-conjugated goat anti-mouse antibody (Molecular Probes, Eugene, OR; 1:200 dilution in Tris-Triton X-100) at pH 7.4 for 1 h at room temperature. Control sections were incubated with only the FITC-conjugated antibody. Nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). The sections were coverslipped using a 1:1 mixture of glycerol in phosphate-buffered saline (PBS).
Western blot analysis of AE2 protein expression.
Cells were washed with ice-cold PBS and lysed with buffer
containing 1% Nonidet P (NP)-40, 0.5% deoxycholate, and 0.1% SDS, and protease inhibitors [Cocktail Set III: 100 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, 80 µM aprotinin, 5 mM
bestatin, 1.5 mM E-64, 2 mM leupeptin, and 1 mM pepstatin A;
Calbiochem, La Jolla, CA]. Cells were harvested and sheared
through a 22-gauge needle, and cellular debris was pelleted by
centrifugation at 500 g for 5 min. The supernatant was
removed. Protein content of the lysate was determined using the
Bradford assay (Bio-Rad, Hercules, CA), and the sample was mixed with
4× sample loading buffer (0.5 M Tris · HCl, pH 6.8, 10%
glycerol, 2% SDS, 0.7 M -mercaptoethanol, and 0.05% bromphenol blue) at a 3:1 ratio.
Analysis of AE2 m RNA expression by RT-PCR. RNA was prepared by lysing cells in buffer containing 4 M guanidine isothiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarkosyl, and 10 mM DTT. After shearing through a 22-gauge needle, the lysate was layered over an equal volume of 5.7 M CsCl, and the total RNA was pelleted by centrifugation for 2 h at 80,000 rpm. Reverse transcription and DNA amplification were performed as previously described (4).
Oligonucleotide sequences used were synthesized using an Applied Biosystems 391 DNA synthesizer (Perkin-Elmer, Foster City, CA) based on the sequences published in GenBank. The following sequences were used: GAPDH sense, 5'-CCATGGAGAAGGCTGGGG-3'; GAPDH antisense, 5'-CAAAGTTGTCATGGATGACC-3'; AE2 sense, 5'-TGTAGCAGCAACCACCTGGAGT-3'; AE2 antisense, 5'-GCAGGAAGAAGGCGATGAAGAA-3'. cDNA was amplified for 26 and 36 cycles for GAPDH and AE2, respectively. PCR products were analyzed by gel electrophoresis through a 2% agarose gel in 0.5× Tris-borate-EDTA buffer. The gels were stained with 1 µg/ml ethidium bromide and photographed under ultraviolet illumination with Polaroid type % P/N film (Polaroid, Cambridge, MA). Bands were quantified using the Kodak 1D Image Analysis software (Eastman Kodak, Rochester, NY). The optical densities for AE2 mRNA bands were normalized against those of the GAPDH bands.Evaluation of AE2 protein induction in vivo after O2 exposure. Animal studies were approved by the Duke University Institutional Animal Care and Use Committee. Sprague-Dawley rats were exposed to 70% O2 for up to 7 days. After euthanasia, the trachea was cannulated and the lungs inflation fixed at 20 cmH20 pressure in 4% paraformaldehyde for immunohistochemistry or flash-frozen for Western blot analysis. AE2 protein expression was analyzed by immunohistochemistry using an established protocol (23). Briefly, paraffin-embedded tissue sections were incubated with the primary AE2 antibody (1:100) followed by a biotinylated goat anti-mouse IgG antibody and labeled with horseradish peroxidase-conjugated streptavidin (Innogenex). Slides were developed with 3,3'-diaminobenzidine and counterstained with hematoxylin. Negative controls were performed with normal mouse serum and antibody co-incubated with AE2 peptide. Tissue sections were examined by light microscopy and photographed at ×132. Western blot analysis was performed as described in Western blot analysis of AE2 protein expression using membrane fractions of the lung protein, isolated by ultracentrifugation at 100,000 g for 1 h at 4°C to enhance detection of AE2 (23).
Isolation of nuclear protein.
After cells were washed with ice-cold PBS, cold cytoplasmic extraction
buffer (CEB; 10 mM Tris · HCl, pH 7.9; 60 mM, 1 mM EDTA, and 1 mM DTT) with protease inhibitors (Cocktail Set III; Calbiochem) was
added to apical chamber. Cells were harvested and transferred to
microcentrifuge tube. They were then allowed to swell on ice for 15 min. NP-40 (Sigma) was added to a final concentration of 0.1%
and the tube vortexed for 10 s. The nuclei were pelleted by
centrifugation at 14,000 g for 40 s. The nuclei were
washed with CEB and centrifuged again at 14,000 g for
30 s. The supernatant was discarded, and the nuclei were incubated for 10 min on ice in nuclear extraction buffer (20 mM
Tris · HCl, pH 8.0, 400 mM NaCl, 1.5 mM MgCl, 1.5 mM EDTA, 1 mM
DTT, and 25% glycerol) with protease inhibitors. The sample was
briefly centrifuged and the supernatant removed. An aliquot was stored
at 80°C for analysis by electrophoretic mobility shift assay
(EMSA). The remainder was processed as described above for Western blot analysis.
Evaluation of DNA binding by EMSA assay.
AE2-specific AP-1 oligonucleotide sequences were synthesized using an
Applied Biosystems 391 DNA synthesizer (Perkin-Elmer). The sequences
were chosen to contain the core AP-1 binding site, GAGTCA, as well as a
number of consecutive nucleotides flanking the potential binding site
within the AE2 promoter (5'-ACTGGAGTCAGCCAGGGTGT-3'). The
probes were labeled by incubating 15 units of T4 polynucleotide kinase
(New England Biolabs, Beverly, MA) and 100 µCi of adenosine 5'-[-32P]triphosphate (ICN, Irvine, CA) with 100 ng of
double-stranded probe at 37°C for 30 min. The mixture was passed
through a desalting column (Nuc Trap; Stratagene, San Diego, CA) to
remove unincorporated 32P. The DNA binding reaction,
consisting of 4 µg nuclear extract, 1.5 µl labeled probe, and 10 µl running buffer (10 mM Tris · HCl, pH 7.5, 50 mM NaCl, 2 mM
EDTA, 1 mM DTT, and 5% glycerol), and 2 µg poly(dI-dC) (Roche
Molecular Biochemical), was performed at room temperature for 25 min.
The samples were separated by electrophoresis on a 4.5% nondenaturing
polyacrylamide gel containing 0.5× Tris-borate-EDTA. The gels were
dried and autoradiographed using a PhosphorImager (Molecular Dynamics,
Sunnyvale, CA). Separate experiments were performed to further
characterize the AP-1 DNA-binding complex. A commercially available
c-Jun gel shift kit was used (c-Jun Nushift kit; Geneka Biotechnology)
according to the supplier's instructions. Briefly, an AP-1 consensus
sequence was labeled as above
(5'-CGCTTGATGAGTCAGCCGGAA-3'). The DNA binding reaction was
performed using 6 µg of nuclear protein in the presence of 2 µg of
phospho-c-Jun (p-c-Jun [Ser63], 200 µg/0.1 ml; Santa Cruz Biotechnology) or c-Jun antibody (c-Jun/AP-1, 200 µg/0.1
ml; Santa Cruz Biotechnology). In addition, the specificity of the DNA
binding was determined using competition with cold, wild-type or
mutated AP-1 oligonucleotides (5'-CGCTTGATGAccCAGCCGGAA-3')
added to the reaction mixture at 100-fold excess. The samples were
separated by electrophoresis on a 4.5% nondenaturing polyacrylamide
gel containing 0.5× Tris-glycine-EDTA and processed as described above.
Evaluation of nuclear levels of phosphorylated c-Jun by Western blot analysis. Equal quantities of nuclear protein were loaded into each lane and separated by SDS-polyacrylamide gel electrophoresis (12%) and electroblotted onto a nitrocellulose membrane. The membrane was blocked with 1% BSA and 1% casein in Tris-buffered saline-Tween 20 (TBS-T) for 1 h at room temperature. This was followed by immunoblotting using a mouse monoclonal antibody against phospho-c-Jun (Ser63) antibody (diluted 1:10,000 in 1% BSA plus 1% casein-TBS-T; p-c-Jun, 200 µg/0.1 ml; Santa Cruz Biotechnology). Antigen-antibody complexes were stained with a horseradish peroxidase-conjugated goat anti-mouse antibody (1:1,000, Santa Cruz Biotechnology) for 1 h at room temperature and developed using enhanced chemiluminescence. Bands were quantified using GeneTools Image Analysis software (Syngene). Protein levels were expressed as percent induction over control.
Statistical analysis. Data are expressed as means ± SE. A minimum of at least three separate experiments was performed for each measurement. Data were compared using one-way analysis of variance followed by the Fisher's protected least square difference test. Significance was assumed at P < 0.05.
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RESULTS |
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AE2 is expressed in differentiated HBE cells.
The constitutive expression of AE2 was confirmed in our differentiated
cell culture model. HBE cells grown at ALI until differentiated into a
mucociliary epithelium expressed AE2 by immunofluorescence. Significant
labeling of the plasma membrane with the AE2 antibody was noted with a
tendency for protein localization at the basolateral plasma membrane in
most of the cells (Fig. 1).
Counterstaining of the nuclei with DAPI suggested significant
perinuclear localization of the AE2 protein as well. Negative controls
produced without the addition of the primary antibody, showed no
fluorescence staining (data not shown).
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Oxidative stress regulates expression of AE2 in airway epithelial
cells.
H2O2, although a relatively weak oxidant, can
rapidly cross cell membranes to react with other ROS and with metal
ions to cause significant cellular injury (11). To
determine whether H2O2-mediated oxidative
stress regulates the expression of AE2, we exposed the apical surface
of differentiated HBE grown at ALI to H2O2 (100 µM). Western blot analysis revealed a single band at 95 kDa (Fig.
2), consistent with the size of the AE2
isoform reported in lung tissue of other species (23).
Constitutive AE2 protein expression was noted before exposure to
H2O2. An increase in protein expression was
seen within 12 h of exposure, with peak expression at 24 h
and return to baseline by 36 h.
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H2O2-induced expression of AE2 is mediated
by transcription factor AP-1.
Oxidative stress can regulate gene expression via the activation of
several transcription factors (9). The promoter region of
the AE2 gene contains multiple potential binding sites for transcription factors (20). We identified a potential DNA
binding sequence for the transcription factor AP-1 at position 1,136 within the 5'-untranslated region of AE2. To determine whether the
activation of transcription factor AP-1 was involved in the regulation
of AE2 by oxidative stress, we evaluated the binding of the
transcription factor to its DNA consensus sequence. Furthermore, we
measured the nuclear translocation of the phosphorylated AP-1 subunit,
c-jun, after exposure to H2O2.
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DISCUSSION |
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This study demonstrates that the expression of AE2 in airway epithelial cells is regulated by oxidative stress and that this regulation appears to be mediated in part by transcription factor AP-1. Increased protein expression is seen both in HBE cells exposed to H2O2 and in airway epithelium in rats exposed to hyperoxia. In the HBE cells this is preceded by a dramatic increase in the transcription of mRNA as demonstrated by RT-PCR. Evidence that AP-1 activity correlates with this regulation is demonstrated by increased binding of the AE2-specific AP-1 sequence after exposure to H2O2, as well as by the increased nuclear translocation of activated c-jun.
AE2 protein expression has been demonstrated previously in lung tissue (7, 17, 23) and in immortalized cell lines (17), although not in primary bronchial epithelial cells until this report. In this study, we show constitutive expression of AE2 in the plasma membrane as well as the perinuclear region of unstimulated HBE cells. AE2 localization (2, 17) and activity (18) have been predominantly described as basolateral in orientation. Recently, AE2 was also found to be associated with the Golgi (12) of a number of cell types. This could explain the presence of AE2 staining in the vicinity of the perinuclear region, although more detailed cellular localization studies will be needed to determine its true location.
The mechanisms regulating AE2 expression in the lung are not well
understood. In other cell models, regulation of AE2 by thyroxine has
been demonstrated in developing enterocytes (5) and by acid/base balance in cortical cells of the kidney (8). It
is unclear whether this represents regulation of cellular homeostasis or a more specific organ function. The transport of
O
The transcription factor AP-1 is known to respond to intracellular concentrations of ROS, including H2O2, to coordinate the induction of a number of antioxidant defenses (3). DNA binding of AP-1 requires the dimerization of c-jun with itself or with a member of the Fos family of binding proteins. Activation of AP-1 then requires the phosphorylation of serine residues within the transactivation domain of c-jun. Given the increased expression of AE2 after stimulation with H2O2 and the presence of an AP-1 consensus binding sequences within the promoter region of AE2, we speculated that AP-1 may be one mechanism by which AE2 is regulated after exposure to oxidative stress. Our investigation revealed that the upregulation of AE2 is preceded by an increase in AP-1 DNA binding and phosphorylated c-jun levels. This result implicates AP-1 in the regulation of AE2, although the substantially more gradual increase in AE2 expression suggests the possibility that posttranscriptional or posttranslational modification may also be involved.
We used primary human airway epithelial cells grown at ALI and allowed to differentiate into mucociliary epithelium as our in vitro model, because these cells display cytological and electrophysiological features that mimic native epithelium (29). This provides a more physiological rationale for studies of ion transport and regulation. Cells grown submerged on plastic lose polarity and varying degrees of their normal differentiated mucociliary features (10) and, therefore, may not respond to oxidant stress in a physiological manner. The use of differentiated cells in culture, however, may explain some of the observed variability in the duration of activation of AP-1. As these cells are obtained from healthy volunteers, it is difficult to know whether there was some degree of activation in the days preceding their collection or if there are differences in individual susceptibility. Despite these inherent problems associated with the use of primary cell cultures, the overall response of AE2 to H2O2 was found to be consistent among individuals.
In conclusion, we have shown that oxidative stress regulates the
expression of AE2 protein in airway epithelial cells of the human and
the rat. In HBE cells, this increase in AE2 expression is preceded by
increases in mRNA, which occurs concurrently with an increase in
activation of AP-1. This may well explain, in part, the mechanism of
regulation by oxidative stress. Increased expression of the AE2 protein
potentially provides the cell greater ability to transport
O
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. L. Turi, Pediatric Critical Care, Duke Univ. Medical Center, Box 3046, Durham, NC 27710 (E-mail: turi0002{at}mc.duke.edu).
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. Section 1734 solely to indicate this fact.
June 5, 2002;10.1152/ajplung.00398.2001
Received 11 October 2001; accepted in final form 23 May 2002.
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