Nuclear erythroid-related factor 2 (Nrf2), a redox-sensitive transcription factor, is involved in transcriptional regulation of many antioxidant genes, including glutamate-cysteine ligase (GCL). Cigarette smoke (CS) is known to cause oxidative stress and deplete glutathione (GSH) levels in alveolar epithelial cells. We hypothesized that resveratrol, a polyphenolic phytoalexin, has antioxidant signaling properties by inducing GSH biosynthesis via the activation of Nrf2 and protects lung epithelial cells against CS-mediated oxidative stress. Treatment of human primary small airway epithelial and human alveolar epithelial (A549) cells with CS extract (CSE) dose dependently decreased GSH levels and GCL activity, effects that were associated with enhanced production of reactive oxygen species. Resveratrol restored CSE-depleted GSH levels by upregulation of GCL via activation of Nrf2 and also quenched CSE-induced release of reactive oxygen species. Interestingly, CSE failed to induce nuclear translocation of Nrf2 in A549 and small airway epithelial cells. On the contrary, Nrf2 was localized in the cytosol of alveolar and airway epithelial cells due to CSE-mediated posttranslational modifications such as aldehyde/carbonyl adduct formation and nitration. On the other hand, resveratrol attenuated CSE-mediated Nrf2 modifications, thereby inducing its nuclear translocation associated with GCL gene transcription, as demonstrated by GCL-promoter reporter and Nrf2 small interfering RNA approaches. Thus resveratrol attenuates CSE-mediated GSH depletion by inducing GSH synthesis and protects epithelial cells by reversing CSE-induced posttranslational modifications of Nrf2. These data may have implications in dietary modulation of antioxidants in treatment of chronic obstructive pulmonary disease.
- glutamate-cysteine ligase
- chronic obstructive pulmonary disease
- reactive oxygen species
oxygen-centered free radicals generated from cigarette smoke (CS) have been known to trigger lung inflammation and, thereby, progression of airway disease (8, 42). The airway epithelium is the primary target for inhaled oxidants. Epithelial lining fluid is the first point of contact between the lung and inhaled environmental oxidants, such as CS and ozone. Oxidant challenge to the airway and alveolar epithelium is normally neutralized by the antioxidants in the epithelial lining fluid. Oxidative stress occurs if antioxidant levels in the epithelial lining fluid are inadequate to neutralize inhaled oxidants/free radicals. Reduced glutathione (GSH), the most abundant cellular thiol antioxidant, plays a critical role in the maintenance of intracellular redox balance in epithelial lining fluid and is involved in the detoxification reaction through direct conjugation or by enzyme-catalyzed reactions (45). This essential antioxidant has been reported to be depleted in the airways in several pulmonary disorders, such as chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome, and cystic fibrosis, suggesting a role for oxidative stress in the pathogenesis of these chronic inflammatory lung diseases (45, 46).
Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the de novo synthesis of GSH (45, 49). The de novo synthesis of GSH depends on the rate of synthesis of the GCL catalytic (GCLC) subunit through increased transcription and mRNA stability (18, 49). GSH plays a key role in cellular antioxidant defense against oxidant injury, especially in lung epithelial cells, and depletion of GSH leads to lung injury and permeability (45, 46). Because of the central role of GSH in cellular protective mechanisms, the induction of regulatory enzymes involved in its synthesis plays a key role as a defense mechanism.
Nuclear erythroid-related factor 2 (Nrf2), a member of the cap-N-collar family, is the principal transcription factor that regulates antioxidant response element-mediated expression of phase II detoxifying antioxidant enzymes (27, 28). Under normal conditions, Nrf2 is sequestered in the cytoplasm by an actin-binding (Kelch-like) protein (Keap1); on exposure of cells to inducers such as oxidative stress or electrophilic compounds (aldehydes) with the ability to oxidize or covalently modify thiol groups (11, 24), Nrf2 dissociates from its repressor protein (Keap1), translocates into the nucleus, binds to antioxidant response elements, and transactivates phase II detoxifying and antioxidant genes (28, 52). Among the spectrum of antioxidant genes controlled by Nrf2, the gene encoding GCL is of particular interest, especially in CS-induced oxidative stress (45). In this context, Harju and co-workers (19) showed downregulation of GCL in the airways of smokers and patients with COPD compared with nonsmokers, suggesting that GCL plays a role in CS-induced oxidative stress in the progression of lung injury. Recently, the importance of Nrf2 in susceptibility to CS has been shown by Rangasamy et al. (48), who reported that genetic ablation of Nrf2 leads to enhanced susceptibility to CS-induced pulmonary emphysema in mice. Agents that modulate Nrf2 would therefore be expected to have significant beneficial health effects in CS-mediated oxidative stress by upregulation of phase II genes.
Recently, much attention has been focused on natural antioxidants in foods. Diets rich in fruits and vegetables have been found to reduce cardiovascular diseases, cancer, neurological disorders, and inflammation. It is thought that the high polyphenol content of these foods is responsible for their biological activity (2). These compounds may also have a beneficial role in preventing the cytotoxic effects of oxidative stress generated by CS in lung tissue. The mechanism may involve scavenging of free radicals and modification of gene transcription via induction or inhibition of specific transcription factors. Among these, resveratrol (3,4',5-trihydroxystilbene), a phytoalexin that is found in the skin and seeds of grapes and produced by some spermatophytes such as grapevines in response to injury or fungal attack, has been reported to have antioxidant (23), anti-inflammatory (12, 32), and anticarcinogenic (14) properties. Studies have shown that resveratrol more effectively inhibits oxidative damage than do the conventional antioxidants (40, 51) and has been shown to scavenge free radicals such as lipid hydroperoxyl, hydroxyl (·OH), and superoxide anion (O2•−) radicals (36). Interestingly, Birrell et al. (1) showed that resveratrol inhibits lipopolysaccharide-induced airway neutrophilia and inflammation through an NF-κB-independent and unidentified mechanism. In light of the findings described above, we hypothesize that resveratrol induces GSH synthesis via Nrf2-dependent mechanisms and attenuates CS-mediated oxidative stress and depletion of GSH in lung epithelial cells. Therefore, we investigated the protective role of resveratrol against CS extract (CSE)-mediated oxidative stress, Nrf2 modifications, and impairment of GSH biosynthesis in human lung epithelial cells.
MATERIALS AND METHODS
Resveratrol (trans-3,4',5-trihydroxystilbene) was purchased from BioMol Research Laboratories (Plymouth Meeting, PA); polyclonal anti-Nrf2 and anti-Keap1 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); GCLC primers for RT-PCR from Integrated DNA Technologies (Coralville, IA); dual-luciferase assay kit from Promega (Madison, WI); penicillin, streptomycin, and DMEM from Life Technologies (Gaithersburg, MD); and FBS from HyClone Laboratories (Logan, UT). All biochemicals were of analytic grade and purchased from Sigma Chemical (St. Louis, MO), unless otherwise stated.
The human type II alveolar epithelial cell line (A549) was purchased from American Type Culture Collection and maintained in continuous culture at 37°C in a 5% CO2 atmosphere in DMEM containing l-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% FBS.
Human primary small airway epithelial cells (SAEC) derived from a single healthy nonsmoker and small airway epithelial cell growth medium (SAGM), including all the growth supplements, were purchased from Clonetics (San Diego, CA). Cells were cultured according to the supplier's instructions. Passage number was kept to <7 from original stocks. SAEC were maintained in SAGM supplemented with bovine pituitary extract (52 μg/ml), human recombinant epidermal growth factor (0.5 ng/ml), epinephrine (0.5 μg/ml), transferrin (10 μg/ml), insulin (5 μg/ml), retinoic acid (0.1 ng/ml), triiodothyronine (6.5 ng/ml), gentamicin and amphotericin B (GA-1000; 50 μg/ml), and fatty acid-free BSA (50 μg/ml).
Preparation of CSE.
Research-grade (1R3F) cigarettes were obtained from the Kentucky Tobacco Research Council (Lexington, KY). Each cigarette contained 17.1 mg of total particulate matter, 15 mg of tar, and 1.16 mg of nicotine. CSE was prepared by bubbling smoke from one cigarette in 10 ml of culture medium supplemented with 1% FBS at a rate of one cigarette every 2 min, as described previously (4, 26, 57). The pH of the CSE was adjusted to 7.4, and CSE was sterile filtered through a 0.45-μm filter (25-mm Acrodisc, Pall, Ann Arbor, MI). CSE preparation was standardized by measurement of absorbance (0.74 ± 0.05 optical density at 320 nm). The pattern of absorbance (spectrogram) at 320 nm showed very little variation between different preparations of CSE. CSE was freshly prepared immediately before use for each experiment and diluted with culture medium supplemented with 1% FBS as required. Control medium was prepared by bubbling air through 10 ml of culture medium supplemented with 1% FBS, adjusting pH to 7.4, and sterile filtering as described for 10% CSE.
A549 cells were seeded at a density of 5 × 106 cells in 100-mm dishes containing culture medium supplemented with 10% FBS in a total final volume of 10 ml. After the cells were grown to ∼80–90% confluency, the cells were maintained in culture medium supplemented with 1% FBS. All treatments were performed in duplicate. Concentration-effect studies were performed with resveratrol to determine the effective dose. Resveratrol at 10 μM was found to effectively induce antioxidant (GSH, glutathione peroxidase, and GCL) levels and quench reactive oxygen species (ROS; data not shown). The cells were treated with 1.0–5.0% CSE with or without 10 μM resveratrol for 24 h at 37°C in a humidified atmosphere containing 5% CO2. Cells were also treated with 100 μM H2O2, a well-known oxidant, as positive control in selective experiments. After 24 h, cells were washed with cold sterile Ca2+- and Mg2+-free PBS, lysed, and then used in various assays.
Human SAEC were seeded in eight-well chamber slides containing SAGM and treated with 100 μM H2O2 or 1.0% CSE, inasmuch as >1.0% CSE was cytotoxic to these cells (26). After treatment, cells were fixed with 4% paraformaldehyde for detection of Nrf2 localization.
Electron paramagnetic resonance measurement.
Electron paramagnetic resonance (EPR; X-band miniscope MS-100, Magnettech, Berlin, Germany) was used to establish the antioxidant effect of resveratrol with respect to O2•− and ·OH generated from xanthine/xanthine oxidase, FeCl3 + H2O2, and menadione, respectively. Xanthine (100 μM)/xanthine oxidase (100 mU/ml), FeCl3 (50 μM) + H2O2 (10 μM), and menadione (500 μM) were incubated in PBS (pH 7.4, 37°C) containing the spin trap tempone-H (1 mM) (9), the oxidation of which generates 4-oxo-tempo, which exhibits a characteristic three-line EPR signal centered at 3,365 g. Development of this signal was monitored for 24 h from the addition of oxidizing species and compared with parallel incubations containing 10 μM resveratrol (n = 6). The amplitude of the first line of the spectrum was measured, and data were expressed in arbitrary units. To establish whether oxidation of resveratrol generates a stable radical species or whether the product of this reaction is spin silent, separate experiments (n = 6) were conducted with xanthine/xanthine oxidase, FeCl3 + H2O2, or menadione in the presence of resveratrol without the spin trap. The EPR parameters for these experiments were as follows: microwave frequency = 9.4 GHz, microwave power = 20 mW, modulation frequency = 100 kHz, modulation amplitude = 1,500 mg, center field = 3,365 g, sweep width = 50 g, sweep time = 20 s, number of passes = 1, and receiver gain = 3 × 101.
Measurement of ROS by flow cytometry.
ROS release was determined using 5- (and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), an oxidant-sensitive fluorescent probe. Inside the cell, H2DCFDA is deacetylated by esterases and forms H2DCF, which is oxidized by ROS to DCF, a highly fluorescent compound. The cells were seeded at a density of 5 × 105 cells in six-well plates in a total final volume of 2 ml and treated with 1.0–5.0% CSE with or without 10 μM resveratrol for 24 h. After the cells were treated, the culture medium was removed, and the cells were washed with PBS and incubated with 10 μM H2DCFDA at 37°C for 30 min. Cells were then trypsinized, washed, and resuspended in PBS. Flow cytometric analysis was performed using a flow cytometer (ELITE, Coulter, Hialeah, FL). For each sample, ≥10,000 events were acquired. Cells were gated out, and analysis was performed only on the live population.
Measurement of intracellular GSH levels.
Intracellular GSH levels in the cell extracts were measured by the 5,5'-dithiobis-2-nitrobenzoic acid-glutathione disulfide reductase recycling method described by Rahman et al. (43), and the amount of GSH in the sample was expressed as nanomoles of GSH per milligram of protein.
Determination of GCL activity.
GCL activity was assayed as described by Seelig and Meister (49) using a coupled assay with pyruvate kinase and lactate dehydrogenase, where NADH oxidation was monitored at 340 nm in a spectrometer (model DU 520, Beckman Coulter). The enzyme-specific activity was defined as micromoles of NADH oxidized per minute per milligram of protein, which is equal to 1 IU.
Preparation of nuclear and whole cell protein extracts.
For nuclear proteins, cells were washed twice with ice-cold PBS and resuspended in 400 μl of buffer A [in mM: 10 HEPES (pH 7.9), 10 KCl, 0.1 EDTA, 0.1 EGTA, 1 DTT, and 0.5 phenylmethylsulfonyl fluoride]. After 15 min, Nonidet P-40 was added to a final concentration of 0.6%. Samples were centrifuged for collection of the supernatants containing cytosolic proteins. The pelleted nuclei were resuspended in 50 μl of buffer B [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF]. After 30 min at 4°C, lysates were centrifuged, and supernatants containing the nuclear proteins were stored at −80°C. Whole cell protein extracts were prepared as described previously (34).
Western blot analysis for Nrf2 and GCLC proteins.
Western blot analysis for Nrf2 and GCLC proteins were performed as described previously (48, 56) using 20 μg of isolated soluble proteins. Lamin B and β-actin were used as loading controls for nuclear and whole cell extracts, respectively.
Immunocytochemistry for detection of Nrf2 localization.
Immunocytochemistry was performed for Nrf2 localization in response to CSE in SAEC. SAEC were seeded at 5 × 103 cells/well in eight-well glass chamber slides and cultured overnight in SAGM at 37°C. Cells were then treated with 1.0% CSE and 100 μM H2O2 for different time periods. At the end of incubation, cells were washed twice in PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. Cells were permeabilized with 0.1% Triton X-100, washed, and blocked with 10% normal goat serum for 1 h. Cells were then incubated overnight with rabbit polyclonal anti-Nrf2 antibody (1:200 dilution in 1% goat serum in PBS) in a humidified chamber at 4°C. Cells were washed with PBS and incubated with FITC-labeled anti-rabbit IgG diluted 1:200 in 1% goat serum for 1 h at room temperature in the dark and, finally, counterstained with Hoechst dye to show the nuclear morphology. After the slides were rinsed with PBS, coverslips were applied and cells were viewed under a fluorescence microscope.
Transient transfection and luciferase assay.
To study the transcriptional regulation of GCLC, we grew human alveolar epithelial (A549) cells to 60–70% confluency and then transiently transfected the cells with 2 μg of GCLC recombinant plasmid-3802/GCSh-5'-Luc (Ref. 37, kindly provided by Dr. Jerry Gipp) or pGL-3 basic vector (Promega) as a negative control using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 12 h, the cells were treated with 10 μM resveratrol with or without CSE for different time periods. Then cells were harvested and lysed in 200 μl of reporter lysis buffer (Promega) and assayed for firefly and Renilla luciferase activities using the Promega Biotech dual-luciferase reporter assay system.
After treatments, total RNA was isolated from A549 cells using the RNeasy kit (Qiagen, Valencia, CA). RT-PCR was performed using oligo(dT) primers and Superscript reverse transcriptase (Invitrogen Life Sciences) following the manufacturer's recommendations. The PCR conditions for the housekeeping gene GAPDH were 20 thermal cycles at 94°C for 45 s, 60°C for 45 s, and 72°C for 90 s followed by final extension for 10 min at 72°C. GCLC was subjected to 32 thermal cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min followed by an extension at 72°C for 10 min. The primer pairs were as follows: 5'-GCCAAGGTCATCCATGACAAC-3' (forward) and 5'-AGTGTAGCCCAGGATGCCCTT-3' (reverse) for GCLC and 5'-AGTGTAGCCCAGGATGCCCTT-3' (forward) and 5'-GCCAAGGTCATCCATGACAAC-3' (reverse) for GAPDH. Amplified products were resolved by 1.5% agarose gel electrophoresis, stained with ethidium bromide, visualized and scanned by a white/UV transilluminator, and quantified by densitometry.
Transient transfection with Nrf2 small interfering RNA.
The Nrf2 small interfering RNA (siRNA) duplex [5'-GUAAGAAGCCAGAUGUUAAdUdU-3' (sense) and 3'-dUdUCAUUCUUCGGUCUACAATT-5' (antisense)] was used to knock down Nrf2. The siControl nontargeting scrambled siRNA (5'-UAGCGACUAAACACAUCAAUU-3') was used as a negative control. A549 cells in the exponential growth phase were plated at a density of 0.2 × 106 cells/ml, grown for 12 h, and transfected with 100 nM siRNA using DharmaFECT1 according to the manufacturer's recommendations (Dharmacon Research, Lafayette, CO). Transfection efficiency was tested after 48 h by measurement of the respective level of mRNA in control vs. transcription factor-specific siRNA-transfected cells. Preliminary experiments were performed in these cells to choose the optimum time points for measurement of the effects of siRNA (data not given). After 48 h of transfection with Nrf2 siRNA, A549 cells were treated with 10 μM resveratrol and/or 1% CSE for 24 h, and whole cell lysates were prepared for the estimation of Nrf2 and GCLC protein levels by Western blotting. Total RNA was isolated using an RNeasy kit as described above for the detection of GCLC mRNA expression.
Immunoprecipitation and posttranslational modifications of Nrf2 and Keap1.
After the treatment period, the cells were harvested and the nuclear fraction was isolated. Nrf2 and Keap1 proteins were immunoprecipitated. Briefly, Nrf2/Keap1 antibody (Santa Cruz Biotechnology; 1:1,000 dilution) was added to 100 μg of nuclear protein in a final volume of 100 μl and incubated for 1 h. Protein A/G agarose beads (40 μl; Santa Cruz Biotechnology) were added to each sample and left for overnight at 4°C on a rocker. The samples were then centrifuged at 1,000 g at 4°C for 5 min. The supernatant was discarded, and the beads were washed twice and then resuspended in 150 μl of lysis buffer. The assay was allowed to continue at 37°C for 90 min with continuous mixing. Then the agarose beads were pelleted by centrifugation. For Western blots, 20 μg of the immunoprecipitated Nrf2/Keap1-agarose bead suspension was added to 20 μl of 5× sample buffer, boiled, and resolved by SDS-PAGE as described above. Agarose beads alone were used as negative controls. For determination of the posttranslational modification of Nrf2/Keap1, blots were probed with mouse monoclonal anti-4-hydroxy-2-nonenal (4-HNE; Oxis International) and monoclonal 3-nitrotyrosine (Cell Signaling Technology, Danvers, MA) antibodies.
Nrf2/Keap1 protein carbonyl assay.
Protein carbonyls were analyzed as described by Levine et al. (31) with slight modifications. A549 cells were treated with CSE with or without resveratrol for 24 h, and cytosolic protein extracts were immunoprecipitated with Nrf2 and Keap1 antibodies as described above. Approximately 100 μg of protein from each sample were precipitated with ice-cold trichloroacetic acid [10% (vol/vol) final concentration] and centrifuged at 11,000 g for 3 min. The protein pellet was resuspended in 0.5 ml of 10 mM 2,4-dinitrophenylhydrazine in 2 M HCl. Samples were vortexed continuously at room temperature for 1 h, precipitated with 0.5 ml of 20% trichloroacetic acid, and centrifuged at 11,000 g for 3 min. The resulting pellet was washed with 1 ml of 1:1 (vol/vol) ethanol-ethyl acetate for removal of free 2,4-dinitrophenylhydrazine reagent and allowed to stand for 10 min. The sample was centrifuged for 5 min at 11,000 g, and the supernatant was discarded. The resulting protein pellet was resuspended in 0.9 ml of 6 M guanidine (prepared in PBS, pH 6.5). The samples were incubated at 37°C for 15 min and centrifuged at 10,000 g for 3 min for removal of any insoluble material remaining in suspension. The carbonyl content was measured at 360 nm spectrophotometrically against a blank containing 1 ml of 6 M guanidine solution (prepared in PBS). The molar absorption coefficient of 22,000 M−1·cm−1 was used to quantify the levels of protein carbonyls. Protein carbonyl content was expressed as nanomoles per milligram of protein.
Total protein assay.
Total protein was measured from the cell lysate supernatants in all samples using the bicinchoninic acid kit (Pierce, Rockford, IL). Protein standards were obtained by dilution of a stock solution of BSA. Linear regression was used to determine the actual protein concentration of the samples.
Statistical analysis of significance was calculated by one-way ANOVA followed by Tukey's post hoc test for multigroup comparisons using STATVIEW and Sigma plot statistical packages. Values are means ± SE of 6 independent experiments. P < 0.05 was considered significant.
Resveratrol exerts antioxidant properties by scavenging CSE-induced ROS.
To elucidate the ROS-quenching ability of resveratrol, A549 cells were exposed to 1%, 2.5%, and 5% CSE in the presence or absence of 10 μM resveratrol for 24 h. ROS levels in A549 cells were measured by flow cytometry using the fluorescent probe H2DCFDA. CSE caused significant accumulation of ROS (Fig. 1). Resveratrol attenuated CSE-induced ROS production, suggesting free radical-quenching ability of resveratrol. Electron paramagnetic studies with resveratrol in the presence of O2•− and ·OH generators also revealed free radical-quenching ability of resveratrol (Fig. 2).
Resveratrol restores CSE-depleted GSH levels by inducing expression of GCLC in A549 cells.
CSE (1%, 2.5%, and 5%) depleted GSH levels in A549 cells at 24 h in a concentration-dependent manner (Fig. 3A) that was associated with decreased GCL activity (Fig. 3B). Cotreatment with 10 μM resveratrol significantly restored CSE-depleted GSH levels (Fig. 3A) and GCL activity (Fig. 3B). We also studied GCLC mRNA expression in response to CSE with or without resveratrol (Fig. 4). CSE (1%)-mediated inhibition of GCLC mRNA expression was significantly reversed by 10 μM resveratrol. Resveratrol alone significantly induced the basal levels of GSH and GCLC expression in untreated A549 cells, suggesting the antioxidant-inducing properties of resveratrol.
Resveratrol induces GCLC gene transcription by inducing nuclear translocation of Nrf2 in CSE-treated A549 cells.
It has been known that the redox-sensitive transcription factor Nrf2 plays an important role in cellular defense against oxidative stress by inducing the expression of phase II genes. Western blot analysis was performed to determine the nuclear translocation of Nrf2 in response to 1% CSE and 10 μM resveratrol. CSE did not result in nuclear translocation of Nrf2 at 24 h, in contrast to 100 μM H2O2, which induced marked translocation of Nrf2 from the cytoplasm into the nucleus in A549 cells (Fig. 5). However, nuclear translocation of Nrf2 was observed at 20 min–1 h in response to CSE exposure in A549 cells (data not shown). To confirm the findings in A549 cells, we used human primary airway epithelial cells (SAEC) (26) to determine the effect of CSE on Nrf2 translocation. Similar to the results of Nrf2 nuclear translocation by CSE in transformed cells, primary SAEC showed nuclear translocation of Nrf2 at 1 h and inhibition of nuclear translocation at 4–24 h in response to CSE, confirming the phenomenon in transformed and primary epithelial cells (Figs. 6 and 7 ). CSE + resveratrol caused significant nuclear translocation of Nrf2 compared with CSE alone in A549 cells at 24 h (Fig. 5), suggesting in part that resveratrol induces GCLC gene expression through an Nrf2-dependent mechanism.
siRNA-mediated knockdown of Nrf2 resulted in downregulation of GCL protein levels and mRNA expression.
To determine the role of Nrf2 in transcriptional regulation of GCL, we used Nrf2 siRNA to knock down the expression of Nrf2 in A549 cells. Nrf2 siRNA-transfected cells were treated with 1% CSE with or without 10 μM resveratrol for 24 h, and the expression of GCL was determined. The 70–80% decrease of Nrf2 protein levels in Nrf2 siRNA-transfected cells was associated with the increase of Keap1 protein (Fig. 8). In cells transfected with nontargeting siRNA, there was no evidence of silencing and Nrf2 levels were similar to Nrf2 in controls. In Nrf2 siRNA-transfected cells, the reduction of GCLC and modulatory GCL protein levels (Fig. 9, A and B) was associated with a concomitant decrease in GCLC mRNA expression (Fig. 9, C and D), suggesting that the decrease in Nrf2 resulted in downregulation of GCL gene transcription. Similarly, resveratrol-mediated upregulation of GCLC gene expression and its ability to upregulate GCLC in CSE-treated cells were lost in Nrf2 siRNA-transfected cells (Fig. 9).
Resveratrol mediates transcriptional regulation of GCLC.
To determine whether resveratrol regulates GCLC expression at the transcriptional level, we transiently transfected A549 cells with GCLC recombinant plasmid. After transfection, cells were treated with resveratrol with or without 1% CSE for 12 and 24 h. Resveratrol (10 μM) induced a time-dependent increase in the GCLC reporter activity (Fig. 10). Furthermore, GCLC reporter activity was significantly increased in the cells treated with resveratrol + CSE compared with those treated with CSE alone (Fig. 10).
Resveratrol attenuates CSE-induced posttranslational modification of Nrf2 and Keap1.
We hypothesized that CSE sequesters Nrf2 in the cytosol by causing posttranslational modifications of Nrf2 and Keap1. To investigate this possibility, we subjected Nrf2 and Keap1 proteins to immunoprecipitation followed by Western blotting for 4-HNE and 3-nitrotyrosine using specific antibodies. Our results revealed that 1% CSE resulted in modification of Nrf2 and Keap1 proteins by 4-HNE and increased tyrosine nitration in A549 cells; this effect was reversed when the cells were treated with 10 μM resveratrol + CSE, suggesting a protective role of resveratrol against CSE-induced modifications of Nrf2 and Keap1 at the posttranslational level (Fig. 11).
CSE causes posttranslational modification of Nrf2 and Keap1 by forming reactive protein carbonyls.
To further confirm that CSE-mediated localization of Nrf2 in the cytosol is due to posttranslational modifications of Nrf2 and Keap1, we measured reactive carbonyls in immunoprecipitated cytosolic Nrf2/Keap1 proteins. Protein carbonyl derivatives are the most widely studied products of protein oxidation. Our results showed a significant increase of Nrf2/Keap1 protein carbonyl adducts in cells treated with 1% CSE, suggesting that CSE leads to localization of Nrf2 in the cytoplasm as a result of the formation of Schiff's base (adduct formation by the Michael reaction) with aldehydes present in CSE or formed by lipid peroxidation, thereby inhibiting nuclear translocation of Nrf2 (Fig. 12). Our results also showed that Nrf2/Keap1 posttranslational modification was reversed when the cells were treated with 10 μM resveratrol + CSE, suggesting a protective role of resveratrol against CSE-induced modifications of Nrf2 and Keap1 at the posttranslational level (Fig. 12).
CS is a complex mixture of >4,700 chemical compounds; each puff of CS contains 1014–1016 free radicals, which include reactive aldehydes, quinones, and benzo(a)pyrene (8, 38, 41). Cigarette smoking is the key etiological factor in the pathogenesis of airway disease, such as COPD. GSH is an important intra- and extracellular lung antioxidant involved in maintenance of epithelial integrity, and its deficiency leads to airway injury and epithelial damage (45, 46). The redox-sensitive transcription factor Nrf2 is important in the transcriptional regulation of phase II genes, including GCL, thereby regulating GSH levels (46). Recent studies have shown that Nrf2−/− mice were more susceptible to CS-induced emphysema, apoptosis, and oxidative stress (48), suggesting a protective role of Nrf2 in lung injury. In the light of this finding, we hypothesized that CS impairs Nrf2-mediated transcriptional regulation of GCL and biosynthesis of GSH, whereas resveratrol attenuates CS-induced oxidative stress and restores CS-depleted GSH via an Nrf2-dependent mechanism. In the present study, we tested our hypothesis by investigating the mechanism of CSE-mediated downregulation of antioxidant genes and the protective role of resveratrol in human alveolar and airway epithelial cells. CSE exposure resulted in increased production of ROS in a concentration-dependent manner, which was associated with decreased levels of GSH and GCL activity in lung epithelial cells. A similar trend was observed in SAEC (26). Owing to the presence of highly reactive electrophilic compounds (aldehydes and quinones) and free radicals, such as O2•−, NO2, ·OH, OONO−, and H2O2, in CS (8, 38, 41), CSE exposure leads to increased production of ROS in A549 cells. Our results are consistent with earlier studies suggesting that CS induces oxidative stress by generating ROS in various epithelial cells (20). The decrease in the levels of GSH and GCL could be due to the formation of GSH conjugates with electrophilic β-carbonyl compounds present in CS and inhibitory action of CS on GCL activity (44). Indeed, our data showing decreased GCL activity support the notion of electrophilic inactivation of GCL activity by CS. In this context, other investigators have shown differential expression of various phase II enzymes/genes after chronic CS exposure (17). However, CSE-induced GCLC promoter reporter activity in epithelial cells is consistent with our previous data showing CSE-mediated induction of GCLC in A549 cells (47). Continuous treatment with CSE reduced GCL activity presumably due to CSE-mediated inactivation of GCL activity by interaction of electrophilic components of CSE with the cysteine group in the GCL active site.
Induction of antioxidant genes is an effective approach for protection against environmental factor-induced oxidative stress. Resveratrol has been well known for its possible antioxidant role and protective effects against oxidant damage (23). In the present study, we tested the ability of resveratrol to attenuate the CSE-mediated ROS production and GSH depletion in lung epithelial cells. Treatment with resveratrol + CSE significantly decreased CSE-induced ROS production, which was associated with increased levels of GSH and GCL activity, compared with treatment with CSE alone. It is possible that resveratrol attenuates CSE-mediated depletion of GSH levels by increasing the biosynthesis of GSH and also by scavenging CSE-induced ROS. Polyphenolic compounds with hydroxyl groups at 4' and 5 positions, as seen in resveratrol, have the potential to scavenge free radicals (16, 53). Hence, it is likely that resveratrol may be involved in direct interaction with free radicals generated by CSE under our experimental conditions. Our data corroborate previous observations of the ability of resveratrol to scavenge free radicals such as ·OH and O2•− (16, 30) and have been shown to possess the antioxidant property (3).
We further investigated the mechanism of CSE-mediated GCL depletion by studying the status of the key transcription factor Nrf2, which is involved in GCL transcription. We show that the CSE-mediated decrease in GSH levels and GCL activity is associated with localization of Nrf2 in the cytoplasm or, alternatively, failure of Nrf2 translocation into the nucleus of epithelial cells at 24 h. In contrast, treatment with H2O2 resulted in marked nuclear translocation of Nrf2. H2O2 is an oxidant and, therefore, elicits conformational changes by oxidation of thiol groups on Nrf2/Keap1, which in turn causes dissociation of Nrf2 from Keap1 and allows nuclear translocation of Nrf2, thereby increasing nuclear accumulation of Nrf2. Oxidative stress or electrophilic compounds, which have the ability to modify thiol groups of Keap1 or Nrf2 (24) and/or activate upstream signaling kinases, can cause dissociation of Nrf2, allowing it to translocate into the nucleus, and, thereby, results in increased transcription of phase II detoxifying and antioxidant genes. Surprisingly, CSE was unable to induce nuclear translocation of Nrf2 at 4–24 h in human alveolar and airway epithelial cells in vitro, perhaps because of posttranslational modifications of Nrf2/Keap1 proteins. However, nuclear translocation of Nrf2 was observed in response to CSE exposure at 20 min–1 h in A549 cells (data not shown) and SAEC. At this earlier time point, the oxidants in CSE oxidize the thiol group, resulting in dissociation of Nrf2 from Keap1 and translocation of Nrf2 into the nucleus and, thereby, increase in nuclear accumulation of Nrf2. At 24 h of CSE exposure, aldehydes present in CSE or formed by lipid peroxidation form protein carbonyl adducts with sulfhydryl groups of Nrf2/Keap1, thereby leading to modulation of these sulfhydryl groups. This could be the reason for failure of Nrf2 translocation into the nucleus or retention of Nrf2 in the cytoplasm of epithelial cells at a later time point.
Immunohistochemistry studies of human peripheral lung tissue in our laboratory also showed localization of Nrf2 predominantly in the cytoplasm of airway epithelium, alveolar type II cells, and macrophages in smokers and patients with COPD compared with nonsmokers (unpublished observation). We have further established that inhibition of nuclear translocation of Nrf2 was associated with decreased mRNA expression of GCLC. To confirm the possibility that the decrease in GCLC mRNA expression was due to the inhibition of nuclear translocation of Nrf2, we used transient transfection of Nrf2 siRNA in A549 cells. Using this approach, we showed that siRNA-mediated knockdown of Nrf2 resulted in decreased expression of GCL mRNA as well as GCL protein levels. The finding that resveratrol-mediated upregulation of GCLC gene expression and its ability to upregulate GCLC in CSE-treated cells were lost in Nrf2 siRNA-transfected cells consolidates our hypothesis that resveratrol induces GCLC by an Nrf2-dependent mechanism. Our observations are supported by previous reports showing lowered GSH levels and GCLC expression in Nrf2-knockout mice (5, 7) and increased GCLC promoter activity in mice where Nrf2 was induced (55, 56). Thimmulappa et al. (54) also showed lowered expression of GCLC in the lungs as well as in mouse embryonic fibroblasts of Nrf2-knockout compared with wild-type mice.
Furthermore, to test our hypothesis that CSE retains Nrf2 in the cytoplasm by causing posttranslational modifications of Nrf2 and Keap1, we studied the modifications of these proteins in response to CSE exposure in A549 cells. Our results revealed a significant increase in covalent modifications of Nrf2 and Keap1 by the aldehyde 4-HNE and increased tyrosine nitration in response to CSE in A549 cells. Tyrosine nitration of proteins has been known to reduce/disrupt protein function, leading to its proteasomal degradation (22, 29). Reactive 4-HNE, a major end product of lipid peroxidation formed during oxidative stress, has been known to react with cysteine, histidine, serine, and lysine residues (15). CSE has been shown to cause posttranslational modifications of various signaling molecules, such as peroxisome proliferator-activated receptor-γ, inhibitory κB kinase-α, and histone deacetylases (13, 33, 35, 57), thereby decreasing their functional/enzymatic activity. Since Keap1 contains a cysteine residue in the binding domain with Nrf2 (10, 24) and Nrf2 contains serine residues at the active site, the 4-HNE modifications and increased tyrosine nitration in response to CSE may cause modifications on cysteine groups or alter the phosphorylation site on Nrf2/Keap1 proteins. This hypothesis is confirmed by our data showing increased protein carbonyl formation on Nrf2/Keap1 in response to CSE in A549 cells. Thus our data suggest that the amino acid residues, particularly cysteine, on Nrf2/Keap1 proteins are the potential targets for electrophilic conjugation by CSE-derived aldehydes. It has also been shown that sulfhydryl groups of Nrf2/Keap1 modulate the dissociation, leading to proteasomal degradation of Nrf2 (10), whereas mutation or modification of these sulfhydryl groups does not cause dissociation of Nrf2 from Keap1. Our results show that treatment of epithelial cells with CSE leads to modification of sulfhydryl groups of Nrf2/Keap1 as a result of covalent formation of protein carbonyl adducts with aldehydes present in CSE or formed by lipid peroxidation. This, in turn, interferes with dissociation of the Nrf2-Keap1 complex and, thereby, inhibits translocation of Nrf2 into the nucleus. This modified Nrf2 then undergoes rapid proteasomal degradation, which could be the reason for the CSE-induced decrease of nuclear Nrf2 levels at 24 h in epithelial cells.
We also hypothesized that resveratrol, which induces GCL mRNA expression, would attenuate CSE-induced modifications in alveolar epithelial cells. We have reported increased nuclear accumulation of Nrf2 in A549 cells treated with resveratrol + CSE, along with a concomitant increase in GCLC mRNA expression, suggesting that resveratrol indeed attenuated posttranslationally modified Nrf2. Furthermore, the transcriptional activity of GCLC, which was measured by transient transfection of A549 cells with GCLC-luciferase construct, was also significantly increased with resveratrol treatment, suggesting that resveratrol-mediated nuclear translocation of Nrf2 was associated with GCLC expression at the transcriptional level.
Our data also show that CSE-induced posttranslational modifications of Nrf2 and Keap1 were attenuated by cotreatment with resveratrol in A549 human epithelial cells. There are several possibilities to explain this phenomenon. One possibility is the ability of resveratrol to reverse the protein carbonylation by inducting aldehyde reductases (39), quench ROS, and, thereby, attenuate CSE-induced posttranslational modifications of Nrf2 and Keap1. Alternatively, resveratrol might react with thiols in Keap1, increasing the levels of Keap1 or activating upstream signaling kinases, such as protein kinase C, phosphatidylinositol 3-kinase, and mitogen-activated kinases to activate Nrf2, despite the posttranslational modification of Nrf2/Keap1. In this context, recent studies have shown Nrf2-inducing capability of resveratrol via the activation of MAP kinases (6, 21).
In conclusion, CSE induces oxidative stress and causes decreased GSH levels, GCL activity, and GCLC expression and inhibition of nuclear translocation of Nrf2 as a result of its posttranslational modifications by reactive carbonyls. Resveratrol attenuated CS-induced oxidative stress by quenching ROS and also by upregulating GCLC via an Nrf2-dependent mechanism. Resveratrol also attenuated CSE-induced posttranslational modification of Nrf2 and upregulated GCLC expression in human lung epithelial cells. Our data also suggest that Nrf2 is modified at the posttranslational level, and mere activation of Nrf2 in susceptible smokers may not be enough for upregulation of phase II detoxifying and antioxidant enzymes. Because they can be included in the diet, are inexpensive, and lack side effects, dietary polyphenols such as resveratrol may more efficiently halt the progression of chronic lung inflammatory diseases where oxidative stress plays a major role. Further studies are required to understand the molecular mechanism of CSE-induced posttranslational modifications of Nrf2/Keap1 and upregulation of phase II detoxifying and antioxidant genes via Nrf2 by resveratrol in human lung epithelial cells and whether a similar protective effect of resveratrol can be seen in lungs in vivo in response to CS or other proinflammatory agents (39, 50). Understanding the molecular mechanisms of action of dietary polyphenols in inflammation and injury could lead to the pharmacological development of novel therapeutic approaches for chronic lung inflammatory diseases such as COPD.
This study was supported by National Institute of Environmental Health Sciences Research Grant ES-01247 and Toxicology Training Grant ES-07026 and National Heart, Lung, and Blood Institute Grant R01-HL-085613.
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