Background
Cigarette smoke, the major risk factor for COPD and lung cancer, contains over 4,500 chemical compounds, including free radicals and oxidants. These compounds are present in both the gas and the tar phase [
1] and have been shown to cause epithelial lung injury [
2,
3]. Epithelial integrity is normally restored by a repair process, that may also result in squamous cell metaplasia and/or goblet cell hyperplasia, especially after repeated injury [
4]. This altered composition of the airway epithelium can be observed in smokers [
5]. Although these epithelial changes have been observed both for smokers with and without airflow obstruction, some of these epithelial features are more pronounced in COPD patients than in asymptomatic smokers [
6,
7]. Furthermore, analysis of bronchial biopsies from smokers with chronic bronchitis showed an increased epithelial cell proliferation [
8], and studies in both current and former smokers revealed epithelial cell proliferation at sites of metaplasia [
9]. These studies indicate that epithelial cell proliferation is a key feature of the epithelial changes observed in smoking-induced lung disease.
Oxidative stress is considered to play a main role in the pathogenesis of inflammatory lung disease, including chronic obstructive pulmonary disease (COPD) [
3]. In smokers, this oxidative stress may result both from cigarette smoke itself, and from oxidants released by inflammatory cells that are recruited as a result of smoke-induced injury. The potential importance of oxidative stress in COPD is supported by various studies such as those showing an increase in markers of oxidative stress in patients with COPD [
3]. The airway epithelium is a main target for exogenous oxidants such as those present in cigarette smoke. Oxidative stress not only induces cell injury, but also appears to play a central role in e.g. gene expression and cell proliferation. An efficient anti-oxidant system that is present in the lung provides protection against these oxidants, and glutathione (GSH) is considered to be a main antioxidant molecule [
10].
Epithelial cell proliferation, as well as various other cellular processes in epithelial cells, is regulated at least in part by Epidermal Growth Factor (EGF)-like factors and the EGF receptor (EGFR). Analysis of the expression of EGF-like growth factors and EGFR in human lung disease has provided evidence for a role of these factors in epithelial remodeling. Kurie
et al. observed that EGFR expression was increased in metaplastic bronchial epithelium, and reversal of bronchial metaplasia was associated with decreased EGFR expression [
11]. Furthermore, Vignola
et al. observed that EGF expression was significantly increased in chronic bronchitis patients in comparison with healthy non-smokers [
12]. Both these studies suggest a role for EGFR and its ligands in the epithelial pathological features observed in smokers with and without COPD.
Downstream signaling pathways that are activated via the EGFR and regulate cell survival and proliferation include phosphorylation of mitogen activated protein kinases (MAPK) and Akt/PI-3 kinase pathways. Activation of the MAPK extracellular-regulated kinase (ERK) 1/2 has been associated with cell survival and proliferation, whereas c-jun N-terminal kinases (JNK) and p38 MAPK are linked to induction of apoptosis [
13]. In addition to ligands of the EGFR, oxidants have been shown to cause activation of EGFR [
14]. Therefore, oxidants may not only cause direct killing of epithelial cells, but also activate specific signaling pathways. Anti-oxidants such as N-acetyl-L-cysteine (NAC) have been found to be an important tool to study the cellular consequences of oxidative stress. Such studies have shown that the increase in cellular GSH/thiol provided by NAC protects cells against oxidative stress.
The aim of the present study was to analyze the effect of cigarette smoke on cell proliferation and wound repair using an in vitro cell culture model. The underlying mechanisms were explored by analyzing the role of MAPK activation and the contribution of an oxidant/antioxidant imbalance in these cellular functions.
Materials and methods
Preparation of cigarette smoke condensate (CSC)
Commercial (Caballero, British American Tobacco Group) and standard research cigarettes (Research cigarettes produced for the University of Kentucky Research Foundation, Reference cigarette: code 1R3, date 3/74) were used in this study. CSC was prepared immediately before use essentially as described by Kim JK
et al. [
15]. Briefly, cigarette smoke derived from one cigarette was drawn slowly into a 50 ml glass syringe and bubbled into a tube containing 1 ml of phosphate-buffered saline (PBS), at room temperature. Each cigarette was completely burned after an average of 8 draws of the syringe, with each individual draw taking approximately 10 seconds to complete. The pH of the CSC solution was between pH 7.0 and 7.4. Subsequently, the CSC was filtered through a 0.22 μm pore filter (Schleicher & Schuell GmbH, Dassel, Germany). To prevent possible inactivation of compounds present in the CSC, the CSC was kept in the dark. The concentration of CSC in the solution was calculated by measuring the OD value of the 100-fold diluted solution at a wavelength at which the maximal absorbance (OD
max) was detected. In the CSC solution this OD
max was achieved between OD 270–280. The pattern of absorbance observed showed very little difference between different batches of CSC. The concentration, expressed as arbitrary units (AU) per ml, was calculated based on the following formula: OD
max × 2 × dilution factor. The CSC was further diluted to the required concentration in culture medium. Ten AU/ml was found to correspond to a mean of 5 % (vol/vol) CSC. Bronchial epithelial cells were exposed to various concentrations of CSC within 30 min after CSC preparation.
Cell culture
NCI-H292 cells, a human pulmonary muco-epidermoid carcinoma cell line, were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were routinely cultured in RPMI 1640 (Gibco, Grand Island, NY) medium containing 2 mM L-glutamine, 20 U/ml penicillin, 20 μg/ml streptomycin (all from Bio Whittaker, Walkersville, MD), and 10% heat-inactivated FCS (Gibco) at 37°C in a humidified 5% CO2 atmosphere. Cells were passaged weekly using Trypsin Versene (Bio Whittaker, Walkersville, MD), and starved for growth factors by overnight incubation in serum-free medium before exposure to CSC.
Subcultures from primary bronchial epithelial cells (PBEC) were derived from bronchial tissue that was obtained from resected lungs, derived from patients that underwent lung surgery for lung cancer at the Leiden University Medical Center (Leiden, The Netherlands). In this study, we used cells obtained from seven smokers: four without airflow limitation (FEV
1 > 81% of the predicted value) and three with airflow limitation (FEV
1 < 70% of the predicted value). PBEC were isolated from bronchial rings using enzymatic digestion of tissue as previously described [
16]. For the experiments, cells from passage two were cultured in DMEM/Ham F12 (1:1) medium (Gibco) supplemented with 10 ng/ml recombinant EGF (Sigma), 2% (v/v) Ultroser G (Gibco), 1 μM isoproterenol, 1 μM insulin (Sigma), 1 μM hydrocortisone (Sigma), 2 mM L-glutamine, 1 mM Hepes (Gibco), 20 U/ml penicillin and 20 μg/ml streptomycin. PBEC were cultured in tissue culture plates precoated with 10 μg/ml fibronectin (isolated from human plasma), 30 μg/ml Vitrogen (Cohesion technologies Inc., Palo Alto, CA) and 10 μg/ml bovine serum albumin (Sigma Chemical Co.).
Prior to the experiments, PBEC were starved for growth factors by overnight incubation in DMEM/HamF12 medium without UltroSer and EGF.
Cell proliferation
Cell proliferation was assessed using 5-bromo-2-deoxyuridine (BrdU) incorporation as previously described [
17]. Briefly, after stimulation, cells were incubated with BrdU (Sigma) for 20 or 24 hours in the presence of the stimulus in starvation medium. Cells were washed twice in PBS and fixed in ethanol 70% (v/v) for at least 1 hour. Cells were then permeabilized with 1 M hydrochloric acid followed by subsequent washes with 0.1 M sodium tetraborate and PBS. BrdU incorporation was demonstrated by incubation with a mouse anti-BrdU mAb followed by incubation with a peroxidase-labeled rabbit anti-mouse polyclonal antibody (both Dako, Glostrup, Denmark). BrdU incorporation was visualized using Nova RED (Vector Laboratories, Burlingame, CA) and the percentage BrdU-positive nuclei was calculated. The percentage BrdU positive nuclei was determined from images that were collected using a digital camera and Axiovision (Carl Zeiss Vision GmbH, München-Hallbermoos, Germany) and Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA) software.
To study the role of oxidants and GSH in the effects of CSC, cells were exposed to N-acetyl cysteine (NAC; Sigma) at 1 mM and CSC. In other experiments, cells were preincubated with DL-buthionine sulphoximine (BSO, Sigma) for 12 hours at a concentration of 10 μM before addition of CSC; BSO was also present during CSC exposure.
Wound closure model
Epithelial wound closure was studied essentially as described by Aarbiou
et al. [
18]. Both NCI-H292 and PBEC were cultured to confluence. After overnight starvation for growth factors, three circular wounds of 3 mm in diameter were prepared in each well using a Pasteur pipette with sharpened silicone tube. After washing with PBS to eliminate debris, cells were allowed to recover for one hour in starvation medium and subsequently incubated in starvation medium in presence or absence of CSC or TGF-α. In experiments using BSO, cells were pretreated for 12 hours prior to stimulation. NAC was replaced every 12 hours. Images of wounded areas were collected using a digital camera and Axiovision software (Carl Zeiss Vision GmbH, Munchen-Hallbermoos, Germany) at the start of the experiments and at various time points as indicated. Images were used to determine the percentage remaining wound area as compared to the start of the experiment (t = 0) using the Axiovision interactive measurement module (Carl Zeiss Vision).
Immunoblotting for ERK1/2
PBEC and NCI-H292 cells were cultured to confluence, starved overnight and subsequently stimulated with transforming growth factor (TGF)-α (20 ng/ml) or various concentrations of CSC for 15 minutes, 1, 6 or 24 hours. After washing with washing buffer (5 mM Tris, pH 6.4, 100 mM NaCl, 1 mM CaCl2, 1 mM MgCl2), cells were lysed in ice-cold lysis buffer (0.5% [v/v] Triton X-100, 0.1 M Tris-HCl pH 7.4, 100 mM NaCl, 1 mM MgCl2, 1 mM CaCl2 1 mM Na3VO4, mini complete protease inhibitor cocktail [Roche, Basel, Switzerland]). Following incubation for 10 minutes on ice, cell lysates were centrifuged at 13,000 rpm for 5 minutes at 4°C to remove insoluble debris. Aliquots of the samples containing equal amounts of protein were suspended in reducing SDS-PAGE sample buffer and boiled for 5 minutes. Proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes using the Mini-transblot system (both Biorad, Hercules, CA). These membranes were incubated in blocking buffer (0.05% Tween-20 in PBS containing 0.5% (w/v) casein) for one hour, followed by overnight incubation with rabbit antibodies directed against total (t) ERK1/2 and phosphorylated (p) ERK1/2 at 4°C (New England Biolabs, Beverly, MA). After incubation with a secondary horseradish peroxidase (HRP) conjugated goat anti-rabbit polyclonal antibody (BD Transduction Laboratories, Franklin Lake, NJ), immunoreactivity was detected by electrochemiluminescent (ECL) detection system (Amersham Pharmacia Biotech, Uppsala, Sweden). In selected experiments, cells were preincubated with the inhibitor of EGFR tyrosine kinase activity AG1478 (Calbiochem, La Jolla, CA).
Immunoblotting for p38 and JNK
Subconfluent cell cultures were starved overnight and stimulated with TGF-α (20 ng/ml) and various concentrations of CSC for 15 minutes, 1, 6 and 24 hours in RPMI 1640 medium containing glutamine, penicillin and streptomycin. After washing with ice-cold PBS, stimulated cells were lysed with reducing sample buffer and incubated for 10 minutes on ice. Proteins were separated by SDS-PAGE using 10% acrylamide gels and proteins were then transferred to nitrocellulose membrane (Schleicher & Schuell GmbH, Dassel, Germany). These were incubated with 0.05% Tween-20 in Tris Buffered Saline (TBST) containing 5% (w/v) skimmed milk (ELK, Campina, Zoetermeer, The Netherlands) for at least one hour, followed by incubation with antibodies directed against total and phosphorylated p38 and JNK at 4°C (New England Biolabs, Beverly, MA), diluted in TBST. After incubation with horseradish peroxidase (HRP) conjugated donkey anti-rabbit polyclonal antibody (Amersham Pharmacia Biotech, UK), immunoreactivity was visualized as described above.
Measurement of cellular GSH content
GSH content of epithelial cells was assessed in cellular lysates that were prepared after washing the cells with ice-cold PBS [
19]. Briefly, washed cells were lysed by adding ice-cold lysis buffer (0.6 % [w/v] sulfosalicylic acid, 0.1 % [v/v] Triton X-100, 5 mM EDTA in 0.1 M potassium phosphate buffer, pH 7.5) and incubation for 10 min on ice. Lysates were harvested and cell pellets, obtained after centrifugation, were disrupted using a Teflon pestle followed by vortexing. This solution was cleared by centrifugation, and the GSH content of the supernatant was assessed using the method of Tietze [
20]. GSH content was calculated using a standard curve, and expressed as nmol/mg protein. The protein content of the lysates was determined using the bicinchonic acid (BCA) method (Pierce Chemical Co, Rockford, IL). In the experiments where the effect of NAC was assessed, cells were preincubated for 16 hours with NAC.
Statistical analysis
The data are expressed as mean ± SEM. Statistical analysis was performed with Student's t test for paired samples following analysis of variance. Differences were considered statistically significant when p < 0.05.
Discussion
The results from the present study show a dose-dependent and dual effect of cigarette smoke on bronchial epithelial cell proliferation and wound repair. In cultures of the bronchial epithelial cell line NCI-H292, proliferation was inhibited at high and stimulated at low concentrations of CSC. Similar effects of CSC on epithelial wound closure in NCI-H292 or PBEC supported these results. Experiments using NAC, GSH and BSO to modify the intracellular thiol status, revealed a critical role of oxidants/free radicals in mediating these effects of CSC. Activation of ERK1/2, a MAPK involved in cell proliferation and survival, was increased by various concentrations of CSC, and sustained up to 24 h only at mitogenic concentrations of CSC (1 AU/ml). Higher, cytotoxic concentrations of CSC resulted in activation of the pro-apoptotic MAPK p38 and JNK. These results suggest an involvement of different MAP kinases in CSC-induced cell proliferation and cytotoxicity.
Various studies have demonstrated marked effects of cigarette smoke and its aqueous extracts on epithelial cell behavior, including proliferation and wound repair. However, most of these studies focused on high, cytotoxic concentrations that were found to inhibit proliferation and wound repair in bronchial epithelial cells [
22]. In contrast to our observations, Lannan
et al. did not observe any increase in proliferation of alveolar A549 epithelial lung adenocarcinoma cells by CSC using 1–10% of CSC [
2], which may be a specific feature of these cells or the CSC concentration used. Our results are however in line with the observation that short term exposure of rats to cigarette smoke condensate results in an increase in cell proliferation in the bronchiolar epithelium and the pulmonary vasculature [
23]. Furthermore, our conclusions are also supported by studies showing that a broad range of oxidants, other than those present in cigarette smoke, can stimulate epithelial cell proliferation [
24‐
26].
The importance of an oxidant/antioxidant imbalance in regulating both CSC-induced cell death, inhibition of wound repair and mitogenesis was demonstrated in studies using NAC, GSH and BSO. Previous to our study, the importance of GSH in cellular defense against CSC was demonstrated by the ability of both GSH and NAC to protect cells from CSC-induced cell death [
2,
27]. The inhibitory action of NAC on the effects of CSC may have been the result of both the extracellular scavenging action of NAC or its ability to increase cellular GSH since NAC was present in the culture medium during exposure to CSC. NAC prevented the CSC-induced decrease in GSH, but did not increase GSH in the absence of CSC. This finding is in line with previous observations, showing that NAC does increase total non-protein thiols but does not increase GSH [
28,
29]. More direct evidence for the involvement of cellular GSH came from our observation that pharmacological inhibition of GCL by BSO increases the sensitivity of epithelial cells to oxidative stress resulting from CSC exposure. This observed effect of BSO may also be relevant for our insight into the way TGFβ expression may alter the response to CSC in the (susceptible) smoker. TGFβ is a pleiotropic cytokine and its expression is higher in smokers with COPD when compared to those without COPD [
30]. For the present study it is interesting to note that TGFβ blocks GCL synthesis in cultured epithelial cells [
31], and thereby – like BSO, a chemical inhibitor of GCL – may decrease intracellular GSH levels. It needs to be noted that GSH and glutathione
S-transferases (GSTs) not only protect cells from the action of oxidants, but also play a more general role in the detoxification of electrophilic components that are present in CSC [
32]. Therefore the observed depletion of GSH and modulatory effects of NAC and BSO do not provide definitive proof for an exclusive role of oxidants in the observed effects of CSC. GSH can also directly scavenge the electrophilic compounds present in CSC [
19].
What is the mechanism involved in the increase in epithelial wound repair and the proliferative response following exposure to low concentrations of CSC? It is known that oxidants and CSC are able to cause ligand independent transactivation of the epidermal growth factor receptor (EGFR) [
14]. Reactive oxygen species may employ EGFR phosphorylation to activate MAPK such as extracellular-signal regulated kinase (ERK) 1/2, that in turn may induce shedding of the EGF receptor ligands such as TGFα and thereby lead to further activation of the EGFR [
33]. Our finding that the EGFR tyrosine kinase inhibitor AG1478 blocks CSC-induced ERK1/2 activation is in accordance with these observations. In line with this, reports demonstrated the involvement of ADAM 17 (tumor necrosis factor α converting enzyme; TACE) in shedding of TGFα and amphiregulin from bronchial epithelial cells exposed to suspended smoke particles [
34,
35]. The present study confirms the stimulatory effect of CSC on epithelial cell proliferation, and links this effect to an imbalance between oxidants and antioxidants and to MAPK activation. Our observation that CSC at mitogenic concentrations induces phosphorylation of ERK1/2 suggests a potential role of this MAPK pathway in the development of both epithelial hyperplasia and metaplasia in smokers, features that may predispose to the development of lung cancer [
36,
37]. Furthermore, Richter
et al. demonstrated that CSC not only induces release but also increases expression of selected EGFR ligands from airway epithelial cells [
38]. Taken together these data and our observation on prolonged activation of ERK1/2 following exposure to subtoxic concentrations of CSC may provide a mechanistic basis for the observed stimulatory effect of CSC on cell proliferation and epithelial wound repair
in vitro. Future studies are needed to define a functional involvement of ERK1/2 activation in the observed effects of CSC on proliferation and wound closure. Our results are in agreement with a recent study showing that two compounds of cigarette smoke, nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, activate the serine/threonine kinase Akt leading to increased cell survival and tumorigenesis in human airway epithelial cells [
39], and a study showing that nicotine induces cell proliferation in neoplastic epithelial cells [
40]. The relative contribution of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and nicotine or other reactive aldehydes to the effects of CSC observed in the present study is not known, also because it is not clear whether intracellular thiols block the stimulatory effect of these components. Furthermore, we have not explored the role of the serine/threonine kinase Akt in the mitogenic effects of CSC observed in the present study. In addition to the MAPK pathway, the Akt/PI-3 kinase pathway may play an important role in CSC-induced epithelial cell proliferation. Finally, a stimulatory effect of aged suspended smoke particles on cultured human bronchial epithelial cells was recently described [
34].
At higher concentrations, CSC has been shown to cause cell death, which seems to be due to high concentrations of oxidants and other radicals [
27] and this study). Moderate oxidative stress induces apoptosis, whereas necrosis occurs when cells are exposed to a higher dose of oxidants [
41,
42]. It has been demonstrated that p38 and JNK are involved in oxidant-induced cell death [
43], and cell fate is regulated by a balance between all three MAP kinase pathways [
44]. Interestingly, at the higher concentrations tested (10 and 5 AU/ml) we observed activation of both pro-apoptotic (p38 and JNK) and pro-survival/proliferation (ERK1/2) MAPK pathways. Based on the observation that these high concentrations of CSC induce cell death, it appears that activation of pro-apoptotic signals predominates. Studies using inhibition of these separate MAPK signaling pathways are needed to delineate their role in the cellular effects of CSC observed in the present study.
In our epithelial wound repair model, we observed differences between NCI-H292 cells and PBEC that are relevant to the interpretation of the effects of CSC on repair. Following injury in NCI-H292 cells, we observed marked proliferation in the cells that covered the original wound area, indicating a contribution of proliferation to the repair process. In contrast, in PBEC cultures very few proliferating cells were present in the wound area, suggesting that in PBEC repair of wounds of the size used in the present study is mainly mediated by migration or cell spreading. Therefore, a stimulatory effect of CSC on proliferation of PBEC could not be observed. Nevertheless, a small but significant effect of low concentrations of CSC on wound closure of both NCI-H292 and PBEC was observed. This may indicate an effect of CSC on cell migration that is dependent on the concentration of CSC used and the cell type investigated. Previously, an inhibitory effect of toxic concentrations of CSC on epithelial migration was reported [
21]. We also observed that PBEC appear more resistant to the cytotoxic effects of CSC than NCI-H292. Because of the role of intracellular GSH in mediating cellular defense against oxidative stress resulting from CSC exposure, we hypothesized that GSH levels may differ between PBEC and NCI-H292. Furthermore, since all PBEC cultures used in the present study were derived from smokers, the possibility that bronchial epithelial cells from smokers may display increased GSH levels needs to be considered [
3]. In addition, further studies are required to delineate differences between the effects of CSC on NCI-H292 and PBEC. In our study, we have used an aqueous extract of cigarette smoke to gain insight into the effect of cigarette smoke on epithelial cells. Much of our knowledge on the cellular effects of smoke is based on studies using such smoke extracts instead of smoke. Notwithstanding the inherent limitations of this model, it can be argued that epithelial cells in the lung – like those in our cultures – are also exposed to smoke components that have been extracted into a fluid,
i.e. the epithelial lining fluid [
45]. The use of the terms "low" and "high" to describe the CSC concentration does not imply any comparison with actual levels of cigarette smoke compounds in lungs. Since the pulmonary levels of individual compounds of cigarette smoke are unknown, comparisons between the
in vivo and
in vitro situation are difficult to make. However, the CSC concentrations used in this study are comparable to the concentrations used in other
in vitro studies.
In our study we have focussed on the effects of cigarette smoke on proliferation and wound closure in cultures of bronchial epithelial cells. These
in vitro results may add new elements to our insight into the pathogenesis of smoking-induced lung injury, and more specifically to the epithelial changes that may accompany COPD and chronic bronchitis. Our observation on CSC-induced epithelial cell proliferation suggests that cigarette smoke alone may partly explain the increased amount of proliferating cells observed in bronchial biopsies obtained from smokers [
8], and may be relevant for our understanding of mechanisms involved in the epithelial hyperplasia and metaplasia that is frequently observed in smokers with and without airflow limitation [
7]. Our findings may also be relevant for our insight in the development of smoking-induced lung cancer, since epithelial metaplasia and hyperplasia induced by cigarette smoke is considered a precancerous lesion [
46]. In this respect it is interesting to note that constitutive activation of ERK1/2 may suffice to cause transformation [
47], and that carcinoma cells often demonstrate high basal levels of ERK1/2 activation [
48]. Therefore, a better understanding of the mechanisms by which cigarette smoke in particular by redox signaling affects wound repair may lead to improved therapeutic interventions for the prevention and treatment of smoking-induced lung disease.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
FL carried out the cell culture experiments, analysis of MAP kinase activation and wrote the manuscript. JA, SW and IR introduced techniques used in the present study. WIB, KFR and PSH were involved in the design, supervision and writing of the manuscript. All authors have participated in the study design and evaluation, and have read, contributed and approved the manuscript.