Epithelial mucous membranes are repeatedly exposed to oxidants and xenobiotics. CFTR plays a role in glutathione transepithelial flux and in defining the hydration and viscoelasticity of protective mucus. We therefore hypothesized that CFTR expression and function may be modulated by oxidant stress. A sublethal oxidant stress (tert-butylhydroquinone, BHQ) in CFTR-expressing epithelial cells (T84) induced a significant increase in cellular glutathione that was associated with an increase in expression of the gene encoding the heavy subunit of the rate-limiting enzyme for glutathione synthesis, γ-glutamylcysteine synthetase (γ-GCShs). CFTR gene expression was markedly decreased according to a time course that mirrored the changes in γ-GCShs. Western blot analysis confirmed that the decrease in CFTR gene expression was associated with a decrease in CFTR protein. cAMP-dependent iodide efflux was also decreased by the oxidant stress. Nuclear run-on assays indicated that the oxidant stress had no effect on CFTR gene transcription, but the mRNA stability in the oxidant-stressed cells was markedly reduced. Furthermore, BHQ increased γ-GCShs mRNA while decreasing CFTR mRNA in Calu-3 cells, and taurine chloramine induced similar effects in T84 cells. We conclude that suppression of CFTR expression may represent an adaptive response of mucosal epithelium to an exogenous oxidant stress.
- chloride channels
- epithelial cells
- cystic fibrosis
- reactive oxygen species
the loss of functional CFTR leads to cystic fibrosis, a disease characterized by the obstruction of epithelial tissues that produce mucin-rich secretions (14), probably due to volume depletion of mucus and epithelial surface fluid layers (25, 33, 42). However, the links between CFTR function and normal epithelial physiology are not fully understood (57). CFTR is an anion channel located at the apical surface of epithelial cells, and it regulates the permeability of chloride and bicarbonate through a cAMP-dependent process (2, 22, 43). CFTR also has finite permeability to large organic anions and can mediate membrane permeability of the antioxidant glutathione (26, 29).
Glutathione is an antioxidant tripeptide that plays an essential role in protecting epithelial cells against oxidant stress and xenobiotics (35). The cytoplasmic levels of glutathione rapidly increase in epithelial cells exposed to oxidative stress (46, 47). Glutathione synthesis results from reactions catalyzed by two enzymes, γ-glutamylcysteine synthase (γ-GCS) and glutathione synthetase (7). γ-GCS represents the rate-limiting enzyme in glutathione synthesis (21). This enzyme consists of two subunits, a light and a heavy chain, each encoded by separate genes. The gene encoding the heavy subunit (γ-GCShs) contains regulatory elements in the 5′-flanking region that allow a marked increase in transcription on exposure of epithelial cells to an oxidative stress (36, 51). An example of such an oxidative stress is cigarette smoke, which increases the transcription of γ-GCShs in lung epithelial cells (46, 48, 49). Recently, cigarette smoke was also shown to inhibit chloride secretion in human bronchial epithelium, suggesting that oxidants may modulate CFTR function (27).
In addition to regulating glutathione synthesis, epithelial cells can modulate transmembrane glutathione transport (32). Membrane transport systems for glutathione have been identified in liver and heart cells and are likely to occur in many other tissues such as those affected by cystic fibrosis. Linsdell and Hanrahan (29) provided electrophysiological evidence that CFTR is permeable to glutathione, implicating it in glutathione transport across epithelial cell membranes. This concept is consistent with previous in vivo observations by Roum et al. (52) of decreased glutathione content in the lower respiratory tract extracellular space of patients with cystic fibrosis and subsequent in vitro studies of cell monolayers expressing either wild-type or mutant nonfunctional CFTR, which also demonstrated that CFTR plays an essential role in maintaining high levels of extracellular glutathione (15). Glutathione permeability has been observed in patch-clamp experiments, and glutathione flux through CFTR channels has been demonstrated biochemically (26). In addition, cells expressing wild-type functional CFTR have significantly less glutathione and are more susceptible to oxidant-dependent apoptosis than cells expressing no functional CFTR (24). A large number of studies have clearly indicated that high intracellular glutathione concentrations are essential to protect cells against oxidant injury. One would therefore predict that during an oxidative stress in mucosal tissues, epithelial cells might respond by using mechanisms to increase cellular glutathione such as enhanced glutathione synthesis and decreased glutathione efflux.
In addition to its role in glutathione transport, CFTR is a cAMP-dependent anion channel permeable to chloride and bicarbonate. CFTR also regulates amiloride-sensitive sodium channels and the chloride/bicarbonate exchanger. A decrease in CFTR function increases mucus concentration and decreases mucus pH, the latter of which favors interchain cross-linking of mucins and increased mucus viscosity (42). Because mucins have been shown to scavenge free radicals (12, 17), a decrease in CFTR would likely increase the antioxidant protection provided by mucus.
In the context of the above, we hypothesized that CFTR is a physiologically important regulator of the response to oxidative stress and that its expression in epithelial cells may decrease in the presence of an oxidant burden. To verify this hypothesis, cellular glutathione, expression of the γ-GCShs gene, and expression of CFTR at the gene, protein, and functional levels were characterized in cultured epithelial cell lines exposed to the oxidants tert-butylhydroquinone (BHQ) and taurine chloramine (TauNHCl).
MATERIALS AND METHODS
Cell lines and reagents.
Human colon adenocarcinoma T84 cells [American Type Culture Collection (ATCC) cell line CCL248; Rockville, MD] were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (GIBCO, Invitrogen Life Technologies, Burlington, ON, Canada) supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2.5 μg/ml Fungizone, and 5% fetal bovine serum at 37°C in 5% CO2. The cells were seeded at 6 × 106 cells/well in six-well tissue culture plates (Linbro Chemical, New Haven, CT) and grown to confluence. The cells were treated with 0–1,600 μM BHQ (Sigma-Aldrich, Oakville, ON, Canada) for 0–24 h, according to the protocols described below. TauNHCl was generated by adding taurine (Sigma-Aldrich) and NaOCl at a 2-to-1 ratio to the culture medium as previously reported (6). The human airway epithelial cell line Calu-3 (ATCC HTB 55) was cultured in MEM supplemented with MEM nonessential amino acids (0.1 mM) and sodium pyruvate (1 mM), 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2.5 μg/ml Fungizone, and 15% fetal bovine serum at 37°C in 5% CO2. Calu-3 cells were seeded at 2 × 106 cells/well in six-well tissue culture plates and cultured until confluent. Forskolin, IBMX, dibutyryl cAMP, and mucin from porcine stomach were from Sigma-Aldrich. 51Cr and 125I were from PerkinElmer Life Sciences (Boston, MA). The antibody used for Western blot analysis of CFTR, MAb-450, was a generous gift from T. J. Jensen and J. R. Riordan (S. C. Johnson Medical Research Center, Mayo Clinic, Scottsdale, AZ). This antibody recognizes the R domain of CFTR.
Cytotoxicity and glutathione assays.
To determine the effect of BHQ on cell viability, T84 cells were grown to confluence in 24-well plates and labeled with 5 μCi/well 51Cr for 18 h at 37°C. The cells were then washed three times with Earle’s balanced salt solution (EBSS; GIBCO) and incubated for 7 h at 37°C in 5% CO2 in the presence of 0–1,600 μM BHQ in EBSS. At the end of the incubation period, 100 μl of supernatant was collected from each well to determine radioactivity in a gamma counter (model Wizard 1470; PerkinElmer Life Sciences). An index of cytotoxicity was calculated for each sample by subtracting the radioactivity of untreated cells (background) from test supernatants and dividing this value by the difference in radioactivity between supernatants from cells incubated with 1% Triton X-100 and background. Cellular glutathione was determined with a model DU7 spectrophotometer (Beckman Instruments Canada, Mississauga, ON, Canada) to determine the reduction of DTNB in the presence of glutathione reductase and NADPH as previously described (8). This assay measures both reduced glutathione and glutathione disulfide.
RNA extraction and Northern blot analysis.
Cells were incubated for 0, 2, 4, 6, 18, and 24 h in the presence of 100 (T84) or 300 (Calu-3) μM BHQ before being harvested for Northern blot analysis as previously described (1). Total cell RNA was isolated with a one-step guanidinium-phenol-chloroform extraction procedure (9). RNA was separated by electrophoresis on 1% agarose and transferred onto a hybond-N+ membrane (Amersham, Oakville, ON, Canada) for analysis. Membranes were prehybridized for 4 h in a mixture containing 120 mM Tris, 600 mM NaCl, 0.1% Na4P2O7, 8 mM EDTA, 0.2% SDS, 625 μg/ml heparin, and 10% dextran sulfate at pH 7.4. Hybridization was performed overnight at 68°C in the same buffer. The human γ-GCS probe was obtained from ATCC (GenBank/EMBL M90656), and the multidrug resistance protein 1 (MRP1) probe was a gift from Dr. Susan Cole of Queen’s University (Kingston, ON, Canada). The CFTR pcDNA3 was a gift from Dr. Gergely Lukacs of the Hospital for Sick Children (Toronto, ON, Canada). The probe was prepared by using a 1.1-kb fragment after EcoRI digestion. Each probe was labeled with the multiprime DNA labeling system (Amersham) using [α-32P]dCTP (specific activity >3,000 Ci/mM). The membrane was then washed once at room temperature for 20 min in 2× SSC and for 1 h at 68°C in 0.1% SDS, 0.1× SSC and was rinsed at room temperature in 0.1× SSC. The membrane was exposed to X-OMAT film (Kodak, Rochester, NY) with intensifying screens at −80°C. As a control for RNA integrity, the blot was hybridized with a 1-kb PstI cDNA probe (ATCC) of the housekeeping gene GAPDH. Signal intensity was quantitated densitometrically with a PowerLook II scanner (UMAX Technologies, Dallas, TX). Densitometric values are expressed as the ratio of γ-GCShs to GAPDH, MRP1 to GAPDH, or CFTR to GAPDH densitometric quantifications.
mRNA stability and nuclear run-on assays.
To determine mRNA stability in the presence and absence of BHQ, T84 cells were seeded at a density of 6 × 106 cells/well in six-well plates and cultured to confluence in Dulbecco’s modified Eagle’s medium with Ham’s F-12 medium (1:1) supplemented with 5% FBS under 5% CO2 at 37°C. The cells were then washed, and fresh medium containing 100 μM BHQ or medium alone was added to the cells for 3 h. The supernatant was then replaced by medium free of BHQ and containing 8 μg/ml actinomycin D. Northern blot analysis was performed on cell extracts 3, 6, and 9 h after the addition of actinomycin D. We also determined the transcriptional effect of BHQ on cystic fibrosis gene expression with nuclear run-on assays as previously described (1). Confluent T84 cells were incubated with 300 μM BHQ for 0, 1, or 3 h. The cells were washed in PBS and lysed in 1 ml of lysis solution [in mM: 10 Tris·HCl (pH 7.4), 5 MgCl2, and 10 KCl with 0.5% NP-40], and the nuclei were isolated. The nuclei were resuspended in nuclei storage buffer [in mM: 50 Tris·HCl (pH 7.4), 5 MgCl2, and 0.5 DTT with 40% glycerol] containing 20 U/ml RNAguard (Pharmacia). Nascent transcripts were elongated in vitro for 30 min at 30°C for run-on analysis as previously described. Nuclear [α-32P]UTP-labeled RNAs were hybridized to linearized DNA of CFTR and GAPDH.
Western blot analysis.
T84 cells were cultured in 100-mm tissue culture dishes to confluence in medium alone or with 100 or 300 μM BHQ for 24 h. The cells were then washed twice with cold PBS, harvested by scraping, and resuspended in 0.5 ml of RIPA buffer composed of 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 0.08% sodium deoxycholate, 20 mM Tris·HCl (pH 8.0), and 1 protease inhibitor cocktail tablet/50 ml (Roche, Mannheim, Germany) before being placed on ice for 10 min to lyse the cells. The lysate was centrifuged at 9,000 g for 10 min at 4°C, and the supernatant was collected. The protein concentration of the lysate was estimated with the Bio-Rad Protein Assay Kit. Samples containing 50 μg of protein were electrophoresed on a 8% SDS-polyacrylamide gel. The separated proteins were electrotransferred to a nitrocellulose membrane (Bio-Rad Laboratories, Mississauga, ON, Canada) for Western blot analysis.
cAMP-dependent iodide efflux assay.
cAMP-dependent anion efflux was determined in T84 cells. Cells were cultured to 90% confluence in six-well plates and labeled with 15 μCi/well 125I for 1 h at room temperature. The cells were then washed six times in efflux buffer (in mM: 119 Na gluconate, 1.2 K2HPO4, 0.6 KH2PO4, 25 NaHCO3) and incubated in efflux buffer. Supernatants were collected every minute for 4 min and replaced with fresh efflux buffer. After 4 min, efflux buffer containing 0.5 mM 2′-O-dibutyryl cAMP, 10 μM IBMX, and 10 μM forskolin (cAMP buffer) was added, and supernatants were collected every minute and replaced with fresh cAMP-containing medium. The radioactivity of the supernatants was determined using a gamma counter. Results are expressed as the percentage of counts per minute measured before the addition of IBMX, forskolin, and dibutyryl cAMP.
Mucin and BHQ cytotoxicity.
Secretory mucins are the major glycoproteins present in mucosal secretions at epithelial surfaces. One of the major roles of CFTR is to regulate the hydration and viscosity of mucins by modulating transepithelial chloride, sodium, bicarbonate, and glutathione flux (15, 45). It has been suggested that mucins have antioxidant properties (13, 17). A potential advantage of CFTR suppression during sustained oxidant exposure may be to increase the concentration and viscosity of mucins at epithelial surfaces. We therefore hypothesized that mucins protect epithelial T84 cells against cytotoxicity induced by high concentrations of BHQ. To test this hypothesis, T84 cells were labeled with 51Cr as described above and washed, and 0–10 mg/ml mucin (Sigma-Aldrich) was added to 500 μl of EBSS. At these concentrations, mucin was freely soluble and did not form layers in the medium. Subsequently, 1 mM BHQ was added to each well, and supernatants were collected after 7 h to calculate the cytotoxicity.
Statistical analysis was performed with Prism software (GraphPad, San Diego, CA) version 4.0 for Macintosh. The results are presented as means ± SE. The data from experiments with only one variable were analyzed using one-way ANOVA, and two-way ANOVA was applied to data from experiments with more than one variable. A Bonferroni multiple comparison test was applied after ANOVA. A value of P < 0.05 was considered significant.
HBQ oxidative stress.
Exposure of the T84 cell line to BHQ resulted in a significant oxidative stress that at high concentrations induced measurable cytotoxicity, as determined by the release of chromium into the cell supernatants. Exposure of cells to ≤400 μM BHQ did not induce significant cytotoxicity, whereas a concentration of 800 μM induced a cytotoxicity index of 10.3 ± 0.8% (n = 12; P < 0.05 compared with 0 μM BHQ). Exposure of T84 and Calu-3 cells to 300 μM BHQ for 24 h did not decrease the cell number [T84 controls = 37.0 ± 3.0 × 106 cells, 300 μM BHQ = 38.0 ± 3.6 × 106 cells (P > 0.05); Calu-3 controls = 13.8 ± 1.0 × 106 cells, 300 BHQ = 12.2 ± 0.9 × 106 cells (P > 0.05)], cell protein content [T84 controls = 46.8 ± 3.7 mg/ml, 300 μM BHQ = 43.3 ± 1.8 mg/ml (P > 0.05); Calu-3 controls = 20.5 ± 3.4 mg/ml, 300 BHQ = 20.1 ± 1.0 mg/ml (P > 0.05)], or viability as determined by Trypan blue dye exclusion. Increasing concentrations of BHQ resulted in progressively higher cytotoxicity (Fig. 1).
BHQ exposure and cellular glutathione.
Exposure of confluent monolayers of T84 cells to BHQ for 6–24 h resulted in time-dependent increases in cellular glutathione (Fig. 2A). At 6 h, the glutathione content of cells exposed to 100 μM BHQ was significantly increased above control (44.8 ± 0.6 vs. 32.3 ± 0.3 nmol/106 cells; P < 0.001, n = 12), and the increase persisted at all time points up to 24 h (24 h = 94.9 ± 2.5 vs. 45.5 ± 0.9 nmol/106 cells; P < 0.001 for all times compared with controls, n = 12). In contrast, extracellular glutathione from the same experiments (Fig. 2B) was slightly increased only at 12 h (BHQ 1.80 ± 0.05 vs. control 1.30 ± 0.04 nmol/106 cells; P < 0.001, n = 12) and did not differ at any time subsequent to 12 h (P > 0.05 at all other times).
Effect of oxidant stress on gene expression.
Exposure of T84 cells to BHQ was associated with a marked increase in γ-GCShs mRNA expression (Fig. 3, A and B). At 4 and 6 h, the increase in γ-GCShs mRNA reached maximal levels, returning to control levels by 18–24 h (γ-GCShs-to-GAPDH ratio at 6 h: control = 5.3 ± 1.1 vs. 25.3 ± 7.2 ratio units; P < 0.01, n = 6). In striking contrast, CFTR mRNA showed changes synchronous to those of γ-GCShs but in the opposite direction, with a marked decrease in CFTR mRNA detected after 6-, 18-, and 24-h exposure to 100 μM BHQ (Fig. 3, A and C; CFTR/GAPDH at 6 h: control = 8.2 ± 0.8 vs. BHQ = 1.8 ± 0.3 ratio units, P < 0.001; 18 h, P < 0.001; 24 h, P < 0.01; n = 6 independent experiments). The oxidant stress did not alter mRNA levels of the gene encoding the ATP-binding cassette (ABC) protein MRP1 (Fig. 3D; P > 0.05 for all comparisons).
CFTR nuclear run-on assay and mRNA stability.
To determine whether oxidant stress affected CFTR expression at the level of transcriptional initiation, cells were treated with 300 μM BHQ for 0, 1, and 3 h, and the cell nuclei were prepared for nuclear run-on assays. Equal counts of radioactively labeled transcripts were hybridized to DNA fragments immobilized on nitrocellulose filters. Signals were compared with the transcriptional levels of stable GAPDH. No difference in signal was observed at any time point, suggesting that oxidant stress did not affect CFTR transcription (Fig. 4A). In contrast, the rate of CFTR mRNA degradation was clearly accelerated in cells exposed to 100 μM BHQ (Fig. 4B). The mRNA degradation curves of BHQ-treated and control cells were significantly different from each other as determined by repeated-measures two-way ANOVA (P < 0.01, n = 4).
CFTR protein and oxidant stress.
Exposure of T84 cells to 100 and 300 μM BHQ for 24 h resulted in decreased levels of CFTR protein as determined by Western blot analysis (Fig. 5). A concentration-dependent decrease in CFTR protein could be observed after 24 h with 100 and 300 μM BHQ (control = 101.3 ± 4.6, BHQ 100 μM = 84.7 ± 4.1, 300 μM = 61.2 ± 5.2 CFTR/actin protein density as %control; P < 0.05 and P < 0.01 for BHQ 100 and 300 μM vs. control, respectively, n = 9). The level of the reference protein actin was not affected by either concentration of BHQ, regardless of exposure time.
cAMP-dependent 125I efflux.
A concentration-dependent decrease in cAMP-dependent 125I efflux was observed when cells were exposed for 24 h to 0–400 μM BHQ (Fig. 6A). The peak cAMP-stimulated 125I effluxes at 6, 7, and 8 min were decreased in cells exposed to 400 μM BHQ (125I efflux at 7 min: control = 188.5 ± 30.8% vs. 400 μM BHQ = 123.3 ± 14.2%; P < 0.001, n = 6), as well as in cells exposed to 300 and 200 μM BHQ. An analysis of the area under the curve for each concentration of BHQ showed a substantial decrease in 125I efflux that reached statistical significance at 300 and 400 μM BHQ (P < 0.05; Fig. 6B).
Effect of oxidant stress on gene expression in Calu-3 cell line.
Exposure of Calu-3 cells to BHQ was also associated with a marked increase in γ-GCShs and a decrease in CFTR mRNA expression (Fig. 7). At 12, 18, and 24 h, γ-GCShs mRNA expression was significantly higher than in cells not exposed to 300 μM BHQ (γ-GCShs-to-GAPDH ratio at 12, 18, and 24 h: P < 0.001 compared with controls at either 6 or 24 h; n = 6). In striking contrast, CFTR mRNA showed changes synchronous to those of γ-GCShs but in the opposite direction, with a marked decrease in CFTR mRNA detected after 12-, 18-, and 24-h exposure to 300 μM BHQ (CFTR-to-GAPDH ratio at 12, 18 and 24 h: P < 0.001 compared with controls at either 6 or 24 h; n = 6).
TauNHCl, glutathione, γ-GCShs, and CFTR mRNA in T84 cells.
Glutathione levels of T84 cells exposed to 500 μM TauNHCl for 24 h were markedly increased (control: glutathione = 49.2 ± 2.0 nmol/106 cells; 500 μM TauNHCl: glutathione = 151.0 ± 3.9 nmol/106 cells; P < 0.0001, n = 16; Fig. 8A). Exposure of T84 cells to 0–500 μM TauNHCl induced a concentration-dependent increase in γ-GCShs-to-GAPDH ratio (P < 0.01 for 250 and 500 μM vs. control, n = 5; Fig. 8B) and a synchronous concentration-dependent decrease in the CFTR-to-GAPDH ratio (P < 0.05 for 62.5 μM vs. control; P < 0.01 for 125, 250, and 500 μM vs. control, n = 5; Fig. 8C).
Protection of T84 cells against BHQ-induced cytotoxicity by mucin.
Mucin from porcine stomach provided concentration-dependent protection of T84 cells against cytotoxicity induced by 1 mM BHQ (Fig. 9). The cytotoxicity index decreased from 51.8 ± 3.0% in the absence of mucin to 22.0 ± 2.7% and 12.3 ± 4.8% in the presence of 5 and 10 mg/ml mucin, respectively (P < 0.01 and P < 0.001 compared with no mucin; n = 3).
Glutathione levels of airway epithelial cells are regulated, at least in part, by CFTR expression (15, 24). In the present study, we report that CFTR is suppressed by oxidant stress at the mRNA, protein, and functional levels. The oxidant-mediated actions on CFTR are associated with a marked increase in cellular glutathione, the major soluble antioxidant present in eukaryotic cells. Although cellular glutathione was markedly increased, extracellular levels of glutathione did not increase proportionately, suggesting that suppression of CFTR function may represent an adaptive mechanism of epithelial cells that contributes to the preservation of cellular glutathione during an oxidant stress. Interestingly, the decrease in CFTR gene expression is synchronous with a marked increase in expression of the gene encoding γ-GCShs, the heavy subunit of the rate-limiting enzyme in glutathione synthesis. The decrease in CFTR mRNA is specific, because the oxidant BHQ did not decrease the amount of mRNA of either GAPDH or another member of the ABC protein superfamily, MRP1. Similar effects of BHQ on γ-GCS and CFTR mRNA were also observed in Calu-3 cells, although some differences in the capacity to recover normal γ-GCS and CFTR mRNA expression after 24 h of oxidant exposure were observed between the cell lines, with partial recovery in T84 cells but no evidence of recovery in Calu-3 cells. In addition, higher concentrations of BHQ were needed to induce changes in Calu-3 cells similar to those observed in T84 cells. Because Calu-3 cells are derived from the human airways and T84 cells from the human intestine (37, 53), the effects of oxidant stress on CFTR expression are not cell or tissue specific. The oxidant-mediated decrease of CFTR in T84 cells was also associated with an accelerated degradation of CFTR mRNA as well as decreases in CFTR protein and function.
The effects of BHQ on glutathione, γ-GCShs, and CFTR mRNA expression were not specific to BHQ. TauNHCl, a physiologically relevant oxidant produced by activated neutrophils, is thought to play a role in inflammatory bowel disease and possibly in lung disease associated with cystic fibrosis (34, 59). Chloramine concentrations of 30–100 μM have been measured in the supernatants of activated neutrophils at a density of 0.2–1.6 × 107/ml (16, 38, 41). In the present study, TauNHCl increased cellular glutathione and γ-GCShs mRNA expression in T84 cells, whereas as little as 62.5 μM TauNHCl decreased CFTR mRNA expression. The TauNHCl concentrations at which CFTR mRNA suppression was observed are within the range reported to be present in the supernatants of activated neutrophils and in cystic fibrosis sputum (16, 38, 59). Further evidence supporting the concept that oxidants other than BHQ can suppress CFTR gene expression was presented by Baudouin-Legros et al. (3), who showed that ouabain, which induces the generation of reactive oxygen species (30), can suppress CFTR mRNA expression in Calu-3 cells and that this effect is blocked by the antioxidants N-acetyl-l-cysteine and diphenyleneiodonium.
Oxidative stress results in epithelial cell responses that can be characterized as either immediate or adaptive (51). The biphasic nature of such responses is illustrated in lung airway epithelium exposed to cigarette smoke. Acute exposure results in depletion of the epithelium’s surface fluid glutathione, whereas chronic smoke exposure results in an increase of glutathione (8, 28, 48). The immediate decrease in cytoplasmic glutathione during an acute oxidant stress results from the rapid scavenging of radical oxygen species as a first line of defense. The subsequent adaptive response involves a complex network of transcriptional and posttranscriptional events coordinated to maximize the effectiveness of cellular antioxidant defenses by increasing cellular glutathione (10, 46). Several genes encoding peptides and proteins related to the synthesis and metabolism of glutathione are targets of these adaptive responses. The rate-limiting step in glutathione synthesis is the formation of the peptide γ-glutamylcysteine catalyzed by the enzyme complex γ-GCS (21, 35). The γ-GCS complex is the product of two genes, one encoding a light subunit and the other a heavy subunit (20, 21). Transcription of the γ-GCShs gene has been reported to markedly increase in cells exposed to oxidative stress (48, 54). Similar results were observed in the present study, with epithelial cell γ-GCShs gene expression increasing after 4 and 6 h of exposure to BHQ. The increase in γ-GCShs was associated with a significant increase in cellular glutathione in both intestine- and airway-derived epithelial cell lines, suggesting a common adaptive response to the oxidant burden.
In addition to γ-GCShs, another key factor that regulates epithelial glutathione is transepithelial glutathione transport (7). Recently, at least three transmembrane proteins from the ABC family have been shown to facilitate the export of glutathione S-conjugates and glutathione across epithelial cell membranes: the multidrug resistance protein MRP1 (ABCC1), CFTR (ABCC7), and MRP4 (ABCC4) (15, 50). Patients lacking functional CFTR have low levels of extracellular glutathione as determined in their lung epithelial lining fluid and in their serum (52). Glutathione efflux from CFTR-deficient epithelial cells is defective and can be restored by CFTR repletion. Cells expressing CFTR have lower glutathione levels and are more sensitive to oxidative stress-induced apoptosis than cells not expressing CFTR (24). These observations are consistent with the present study, in which the oxidative stress-mediated decrease in CFTR gene, protein, and function was associated with an increase in cellular glutathione. Interestingly, a decrease in CFTR expression occurred at the same time as expression of the γ-GCShs gene was increased, suggesting that the cell has a coordinated adaptive response that increases synthesis and decreases export of cellular glutathione during oxidant stress. The mRNA encoding the structurally related MRP1 did not decrease on exposure to oxidant stress.
The effects of oxidant stress on chloride efflux and CFTR function have been studied by several investigators (11, 23, 40, 55). Most of these studies have focused on the immediate response of CFTR function within minutes of exposure to an oxidant stress. Using a wide range of oxidants, including tert-butylhydroperoxide, monochloramine, and hydrogen peroxide, these studies have consistently demonstrated that chloride efflux is rapidly increased. In contrast, we have observed that a prolonged exposure to oxidant stress over several hours has the opposite effect. Together these observations suggest that oxidant-mediated CFTR activation is an immediate host response, perhaps representing an attempt to flush the oxidant source away from the cell surface, whereas CFTR suppression represents a long-term adaptive response to favor preservation of intracellular glutathione and increase the viscosity of the mucous barrier to prevent oxidation of vital cellular components. Consistent with this concept, recent work reported by Kreindler et al. (27) indicates that cigarette smoke exposure can induce changes in human bronchial epithelial cells that mimic ion transport defects observed in cystic fibrosis epithelia.
The results of this study and the work of others suggest that oxidant-mediated CFTR suppression may occur through several mechanisms. Using nuclear run-on assays, we were unable to observe an oxidant-dependent decrease in CFTR gene transcription. However, CFTR mRNA stability was clearly decreased after oxidant exposure. Enhanced degradation of CFTR mRNA has been reported previously for cells exposed to TNF-α and γ-interferon, suggesting that posttranscriptional regulation may play a key role in defining CFTR mRNA levels generally (5, 39). In addition to decreasing CFTR mRNA levels, oxidant stress also decreased steady-state CFTR protein level and channel function. A decrease in CFTR mRNA may contribute to lower CFTR protein and function, but it is unlikely to account for all of the loss of CFTR function. It was recently shown that reactive nitrogen species can directly accelerate CFTR protein degradation (4). Furthermore, oxidants could act directly through interactions with cysteine residues in the nucleotide binding domains of CFTR, which results in the slowing of channel gating (18, 19), although this would be unlikely to persist for 24 h in the absence of oxidant stress. Severe oxidant stress may lead to reversible glutathionylation of CFTR and inhibition of its channel activity (58).
BHQ is a redox cycling quinone that produces hydrogen peroxide and hydroxyl radical, reactive oxygen species that can injure epithelial cells (31, 44). Mucin, a glycoprotein concentrated at the surface of mucosal epithelia, can scavenge hydroxyl radicals and is thought to protect epithelial cells against oxidant-mediated damage (17). One of the major functions of CFTR is to regulate chloride and bicarbonate secretion, thus modulating the water content and pH of mucus, the key parameters defining the viscoelastic properties of mucin (33, 56). Our data indicate that increasing concentrations of mucin protect epithelial cells against BHQ-mediated injury. We therefore suggest that in addition to modulating glutathione transport, a potential benefit of decreased CFTR expression during an oxidant stress may be the increased antioxidant protection provided by viscous mucin at the epithelial surface.
In summary, we have shown that oxidant stress can suppress CFTR gene expression, protein expression, and function while increasing the cellular antioxidant glutathione. We propose that CFTR suppression during an oxidant stress is an antioxidant glutathione-sparing response that may also increase mucous barrier viscosity to help protect the epithelium and occurs despite the risk of impaired mucus clearance. In contrast to the sustained deficiency of CFTR observed in cystic fibrosis that leads to substantial respiratory, gastrointestinal, and reproductive disease from mucus obstruction, the oxidant-mediated effects on CFTR expression are transient. These results may be highly relevant to situations in which mucosal epithelial tissues are chronically exposed to a significant oxidant burden such as occurs in the airways of cigarette smokers and in the intestinal tract of patients with inflammatory bowel diseases.
This study was funded by a grant from the Canadian Cystic Fibrosis Foundation. J. W. Hanrahan was a senior scientist of the Canadian Institutes of Health Research. A. M. Cantin was a chercheur national of the Fonds de la Recherche en Santé du Québec.
The authors thank Dr. Susan Cole for providing the MRP1 cDNA, Dr. Gergely Lukacs for the CFTR cDNA, and Tim Jensen and Dr. John Riordan for the CFTR antibody.
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.
- Copyright © 2006 the American Physiological Society