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Cigarette smoke exposure causes changes in Scavenger Receptor B1 level and distribution in lung cells
The International Journal of Biochemistry & Cell Biology 43 (2011) 1065–1070
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Cigarette smoke exposure causes changes in Scavenger Receptor B1 level and distribution in lung cells Giuseppe Valacchi a,b,∗ , Paul A. Davis c , Elaine M. Khan d , Roni Lanir d , Emanuela Maioli e , Alessandra Pecorelli e , Carroll E. Cross d , Tzipora Goldkorn d a
Department of Biomedical Sciences, University of Siena, Siena 53100, Italy Department of Food and Nutrition, Kyung Hee University, Seoul, South Korea c Department of Nutrition, University of California Davis, Davis, 95616 CA, United States d CCRBM, University of University of California Davis, Davis, 95616 CA, United States e Department of Physiology, University of Siena, Siena 53100, Italy b
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Article history: Available online 2 June 2009 Keywords: Cigarette smoke Scavenger Receptor B1 Lung Vitamin E Ubiquitination
a b s t r a c t Scavenger Receptor B1 has been shown to play a prominent role in the uptake and delivery of vitamin E from HDL and is likely involved in regulating vitamin E in the lung. We have previously demonstrated that lung Scavenger Receptor B1 levels (protein and mRNA) are modulated by cigarette smoke in mice and this was accompanied by changes in lung vitamin E. To further characterize the molecular mechanism(s) involved in this process, human alveolar epitheliall cells were exposed to cigarette smoke and Scavenger Receptor B1 cellular levels and distribution were assessed. Results demonstrated that Scavenger Receptor B1 localizes in patches on the cellular membrane and in the perinuclear area of control cells. Upon cigarette smoke exposure, Scavenger Receptor B1 first translocated to the cell surface (within the first 12 h of exposure) and then cell levels (protein and mRNA levels) decreased significantly at 24 h. This decline was accompanied by increased Scavenger Receptor B1 ubiquitination which may explain the decrease in the protein levels. Cigarette smoke induced changes in both sub-cellular redistribution and ubiquitination of Scavenger Receptor B1 together with our previous in vivo data provides evidence that cigarette smoke exposure may alter lung’s ability to control its tocopherol levels. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Cigarette smoke (CS)- and CS-related activation of inflammatory processes are known to induce oxidative stress and thus conceivably play a pathobiologic role in the CS induced lung diseases such as chronic obstructive pulmonary disease (COPD) and lung cancer (Rahman and Kilty, 2006; MacNee, 2005). A number of in vitro studies have shown that CS and its constituents induce oxidative stress and apoptosis in both bronchial epithelial cells and alveolar cells (A549 and L2 cells) (Jiao et al., 2006; Ramage et al., 2006). In particular, Hoshino has identified volatile phase CS extract able to induce apoptosis but this effect was attenuated when oxidant
Abbreviations: CS, cigarette smoke; SR-B1, Scavenger Receptor B1; ALF, alveolar lining fluid; SR-B1 KO, Scavenger Receptor B1 knock out mice; COPD, chronic obstructive pulmonary disease; HDL, high density lipoprotein; eNOS, endothelial nitric oxide synthase; MAPK, mitogen-activated protein kinase; FACS, fluorescence activated cell sorting; BSA, bovine serum albumin; DAPI, 4 ,6-diamidino-2phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ∗ Corresponding author at: Department of Biomedical Sciences, University of Siena, Via Aldo Moro, 7, 53100 Siena (SI), Italy. Tel.: +39 0577 234 107; fax: +39 0577 234 219. E-mail address: [email protected] (G. Valacchi). 1357-2725/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2009.05.014
scavengers were added to the cells (Hoshino et al., 2001). Additionally, a diet high in fruits and vegetables presumably containing “antioxidant substances” has been reported to be associated with a decreased lung diseases risk (Dow et al., 1996). Alveolar lining fluid (ALF) contains antioxidant substances that protect alveolar surfactant and cells from exogenous cytotoxic oxidative stress such as CS (Müller et al., 1998). In CS-related lung diseases these defenses may be compromised (Carp and Janoff, 1978). As studies have shown that oxidant-related lung damage is enhanced in lungs deficient of vitamin E (Hybertson et al., 1995), efforts have been made to augment ALF’s antioxidant levels by increasing vitamin E (␣-tocopherol) via dietary supplementation (Romieu, 2005). However, these efforts, in general, did not show to be effective (Lonn et al., 2005). This lack of efficacy is not well understood and may in part be derived from overall micronutrient antioxidant kinetics in alveolar tissues. Specifically, with respect to vitamin E, the role of the Scavenger Receptor B1 (SR-B1) in lung tocopherol transport remains incompletely understood despite the strong evidence that SR-B1 plays a major role arising from knockout (KO) studies where lung ␣-tocopherol levels are 64% lower in SR-BI KO mice lungs compared to control animals (Rigotti et al., 2003). We recently reported that acute CS exposure reduced lung SR-B1 expression (protein and mRNA) in mice (Valacchi et al.,
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2007). Given that SR-B1 likely plays a major role in the delivery of tocopherol to alveolar tissues, the current study sought to further characterize the effect of acute CS on alveolar cells and the possible mechanism by which CS is able to modulate SR-B1 levels. For our studies we used A549 cells which are a cell line with type II cell characteristics, e.g. production of surfactant proteins and has been widely used to study the responses of alvelolar epithelial type II cells to oxidants (Mairbaurl et al., 1997; Lin et al., 2001; Burvall et al., 2002; Geiser et al., 2004; Kwong et al., 2004). In the present study, A549 cells were exposed to CS and probed either immediately or at timed intervals up to 24 h post CS exposure for changes in the levels of SR-B1 mRNA, protein and cellular localization. The results demonstrate that CS not only produces a decline in SR-B1 mRNA and protein levels, but also induces changes in the cellular localization of SR-B1 protein and increases SR-B1 ubiquitinylation. 2. Materials and methods 2.1. Cell culture A549 cells (ATCC; Manassas, VA) were grown in F-12K nutrient mixture (Invitrogen, Carlsbad, CA), supplemented with 10% FCS and 1% penicillin/streptomycin on Vitrogen-coated (Collagen Corporation, Palo Alto, CA) Costar clear Transwells (0.4-mm pore size; Costar Corporation, Cambridge, MA) until a confluent monolayer was established (approximately 4 × 106 cells/well). For experiments with the proteasome inhibitor MG132 (Calbiochem, USA) the cells were either treated with or without MG132 (final concentration in culture 10 M) dissolved in DMSO (final concentration in culture 0.01%), and incubated for 2 h at 37 ◦ C. At the end of the incubation period, the cells were washed three times and exposed to CS as described below. 2.2. CS exposure Just prior to exposure, the cells’ apical media was aspirated, whereas the media in the basolateral compartment remained in the well to supply cells with nutrients during the exposure. Control cells were exposed to filtered air for the same duration (45 min). The time and the way of exposure were chosen based on our recently published results (Khan et al., 2008) with no difference in the cell viability as measured by Trypan blue exclusion was detected between control (air) and CS treatment (data not shown). A549 cultures were exposed to fresh CS in an exposure system that generated CS by burning one UK research cigarette (12 mg tar, 1.1 mg nicotine) using a vacuum pump to draw air through the burning cigarette and leading the smoke stream over the A549 cultures as described previously by our group (Khan et al., 2008). Briefly, overnight serum-starved A549 cells in culture were placed with lids removed in a 37 ◦ C chamber having a volume of 0.45 cu. ft., smoke from one cigarette was introduced into the chamber by readmitting air (a decrease of ∼5 in. Hg over ∼30 s) in the chamber after achieving a partial vacuum (25 in. Hg) via a short piece of tubing holding a lit cigarette (UK research cigarette; 12 mg tar, 1.1 mg nicotine). After the exposure (air or CS) fresh media was added to the cells. 2.3. Immunohistochemistry and confocal microscopy A549 cells grown on coverslips at the same density of that of the cells cultured in transwells, were treated as indicated above and fixed in 4% paraformaldehyde in phosphate buffered saline (PBS). Cells were permeabilized for 15 min at room temperature with PBS containing 1% BSA, 0.2% Triton X-100, and 0.02% sodium azide, then the coverslips were blocked in PBS containing 1% BSA, 0.2% Nonidet
P-40, 5% goat serum, and 0.02% sodium azide at room temperature for 1 h. Coverslips were then incubated for 1 h with primary antibody, followed by 1 h with secondary antibodies (Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, Eugene, OR)). Nuclei were stained with 1 g/ml DAPI or 1 g/ml propidium iodide (Molecular Probes) for 3 min after removal of secondary antibodies. Coverslips were mounted onto glass slides using the ProLong Antifade Kit (Molecular Probes). Confocal microscopy at 20 and 60× magnification was carried out using an Olympus FV1000 Fluoview confocal laser scanning microscope. 2.4. Western blot analysis Total cell lysates were extracted in solubilization buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM EGTA, 0.1% SDS, 5 mM N-ethylmaleamide (Sigma–Aldrich Corp.), protease and phosphatase inhibitor cocktails (Sigma–Aldrich Corp.). Membrane extracts were prepared using the ProteoExtract TM Native Membrane Protein Extraction Kit (Calbiochem, Milan, Italy), following the manufacturer’s instructions. Lysates were cleared by centrifugation (16,000 × g) for 45 min and protein concentration was measured using the Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA). Equivalent amounts of proteins were subjected to 10% SDSPAGE, transferred onto a nitrocellulose membrane which was then blocked for 1 h in Tris-buffered saline, pH 7.5, containing 0.5% Tween 20 and 5% milk. Membranes were then incubated overnight at 4 ◦ C with the appropriate primary antibody (SR-B1) (Abcam, Cambridge, MA) for 24 h. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h, and the bound antibodies were detected using chemiluminescence (Pierce Chemical Co., Rockford, IL). Images of the bands were digitized and the densitometry of the bands were performed using NIH-Image software. 2.5. Immunoprecipitation and immunoblotting for ubiquitination After treatments, cells were extracted in solubilization buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM EGTA, protease and phosphatase inhibitor cocktails (Sigma). Lysates were cleared by centrifugation and 400 g of protein in the supernatant were immunoprecipitated by overnight incubation with 2.5 g anti-SR-B1 (Abcam, Cambridge, MA) at 4 ◦ C, followed by protein A (Repligen Corp., Needham, MA) precipitation for 1–2 h. Immunoprecipitates were washed three times with HNTG buffer containing 20 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol, resolved by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were blocked for 1 h with Tris-buffered saline, pH 7.5, containing 0.5% Tween 20, 1% BSA, and 5% non-fat milk, incubated overnight at 4 ◦ C with monoclonal primary antibody anti-ubiquitin (Covance Research Products, Inc., Berkeley, CA), followed by 1 h incubation at room temperature with a 1:10,000 dilution of horseradish peroxidaseconjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). Immunoreactive protein bands were detected with the SuperSignal West Pico Substrate (Pierce, Rockford, IL). 2.6. SR-B1 messenger RNA levels and stability SR-B1 mRNA was evaluated by real-time quantitative PCR on total RNA isolated from A549 cells using SYBER Green (Perkin Elmer Applied Biosystems; Foster City, CA) and ABI Prism 7700 Sequence Detector (Perkin Elmer Applied Biosystems). Primers were synthe-
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Fig. 1. Cigarette Smoke exposure induces changes in SR-B1 levels and localization in lung epithelial cells. Immunocytochemistry of A549 cells showing localization of SR-B1 (green) before and after CS exposure for different time points. Images are merged and are representative of at least 100 cells viewed in each experiments (n = 5). Nuclei (blue) were stained with DAPI. A) Cells overview at different time points 20×; B) Individual cell at different time points 60×; C) Representative Western blot of proteins extracted from the membranes of cells exposed to Cigarette Smoke at different time points. The signals of SR-B1 protein levels were determined by densitometric analysis of the scanned images (bottom panel). Data are expressed in arbitrary units and are averages of the values for five different experiments. * p < 0.05.
sized using Primer Express v. 1.0 software (Perkin Elmer Applied Biosystems) and according to the published cDNA sequences for SR-B1 and GAPDH the internal control. No-template (water) reaction mixture was prepared as a negative control. Quantitative analysis of PCR products during the PCR amplification was measured via fluorescence emission and then analyzed by the Sequence Detector v1.6.3 program (Perkin Elmer Applied Biosystems) of the ABI Prism 7700 detection system. mRNA expression of the gene of interested was normalized by using GAPDH mRNA expression levels. The mRNA level analysis was done in triplicate and repeated. Assessment of SR-B1 mRNA stability upon exposure to CS was done by adding actinomycin (5 g/ml) at time zero to halt new RNA synthesis. Cells were harvested and cellular mRNA was isolated immediately or at 1, 2, 3 h post CS exposure. 3. Results 3.1. CS exposure affects SR-B1 cellular localization and membrane protein levels The results from confocal microscopy (Fig. 1) demonstrate that in the control (Fig. 1A and B), air-exposed A549 cells, SR-B1 immunofluorescence was distributed in the perinuclear area and in the cell membrane. After CS exposure, there was an increase in SR-B1 membrane location and a decreased perinuclear staining. This pattern changed at 6 and 12 h post CS exposure as the perinuclear staining was almost lost and the membrane staining was less evident. At 24 h SR-B1 levels were dramatically decreased in
all the cellular compartments. These changes were not present in the control, air-exposed cells at the same time points (0–24 h) (data not shown). The changes in SR-B1 membrane levels were confirmed by Western blot analysis of cell membrane extracts. Fig. 1C showed the increase in SR-B1 membrane levels and the following significant decrease. 3.2. CS exposure decreased SR-B1 protein levels The level of SR-B1 protein from whole A549 cell lysates as assessed by Western blot analysis decreased markedly upon CS exposure in a time dependent manner as depicted in Fig. 2. A reduction of approximately 50% occurred after 45 min of CS exposure and was further reduced after 24 h (approximately 75% decline). Although the total SR-B1 (membrane and cytosol) decreased after CS exposure (Fig. 2), the shift of the receptor from the perinuclear area to the membrane leads to an increased membrane level (Fig. 1C). No changes were detected during the 24 h time course in the air treated cells. Furthermore, no differences in cell viability as measured by Trypan blue exclusion were apparent between control air and CS exposed cells (data not shown). 3.3. CS exposure decreased SR-B1 mRNA levels without affecting its stability The results showed in Fig. 3A demonstrated that CS exposure caused a rapid drop in SR-B1 mRNA levels immediately
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Fig. 2. Cigarette Smoke exposure decreases SR-B1 protein levels in A549 cells. Cells were exposed to Cigarette Smoke for 45 min and were harvested at different time points. SR-B1 proteins levels were detected by Western blot analysis. A representative immunoblot is shown at the top panel. The signals of SR-B1 protein levels were determined by densitometric analysis of the scanned images. Quantification of the SR-B1 bands is shown at the bottom panel. Data are expressed in arbitrary units and are the average of five different experiments. * p < 0.05.
Fig. 4. Cigarette Smoke exposure induced SR-B1 ubiquitination. A549 cells were exposed to Cigarette Smoke and cell lysates were immunoprecipitated using anti SR-B1. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with anti-ubiquitin (A). Re-probing the membrane with anti-SR-B1 antibody showed the presence of equal amounts of SR-B1 immunoprecipitates (bottom panel). B) Cells were pre-treated (+) or not (−) with the proteosome inhibitor MG132 at the final concentration of 10 M and after 2 h incubation the cells were exposed to CS for 45 min. Quantification of the SRB1 bands is shown at the bottom panel. The signals of SR-B1 protein levels were determined by densitometric analysis of the scanned images. All samples were normalized per protein per sample. Data are expressed in arbitrary units. A representative Western blot from five experiments is shown at the top. * p < 0.05 for Air vs CS; # p < 0.05 for +MG132 vs −MG132.
after exposure and the reduced mRNA level persisted at 6 and 12 h post exposure. mRNA stability studies revealed that SR-B1 mRNA decline in the presence of actinomycin paralleled each other between the air and the CS treated cells, indicating that CS exposure did not affect mRNA stability (Fig. 3B). 3.4. CS exposure induced SR-B1 ubiquitination
Fig. 3. Cigarette Smoke exposure decreased SR-B1 mRNA levels. A) SR-B1 gene expression in A549 cells exposed to air or to Cigarette Smoke was evaluated by real-time PCR. The isolated mRNA samples were analyzed using the specific primer for SR-B1 and compared to GADPH (housekeeping gene) levels. B) For mRNA stability studies the cells were pre-treated with 5 M of actonimycin D as described in Methods. Data are expressed as mean ± S.D. of three independent experiments. p < 0.05.
Immunoprecipitation and Western blot analysis for SR-B1 ubiquitination (Fig. 4) revealed that CS exposure increased ubiquitinated SR-B1 in A549 cells, with the increase apparent after 45 min of CS exposure and a further increase at 6 h post CS exposure (Fig. 4A). Re-probing of the membrane with anti-SR-B1 antibodies indicated that equivalent amounts of SR-B1 were immunoprecipitated from each sample (Fig. 4A, bottom panel). These data were confirmed using an inhibitor of the proteosome (MG132). CS exposed cells in the presence of MG132 exhibited higher levels of ubiquitinated SR-B1 while air-exposed cells did not
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show any differences in SR-B1 levels ubiquitination in the presence of MG132 (Fig. 4B). Although MG132 is not a fully selective inhibitor, the results point to both protein degradation (MG132/air control is higher than MG132/CS treated cells), as well as decreased synthesis as shown in Fig. 3A as being responsible for the declines noted.
4. Discussion Alveolar surfactant represents one of the first targets of airborne oxidants such as CS (Rahman and Kilty, 2006) and therefore likely plays a major role in the pathobiology of several respiratory tract diseases including COPD and lung cancer. CS exposure interacts with tocopherol as the effects of CS on lung surfactant synthesis and cell adherence constituents of type II pneumocytes are restored upon vitamin E supplementation, suggesting that vitamin E likely plays an important role in the lung related events in response to CS exposure (Xue et al., 2005; Guthmann et al., 2003). The present study examined the effect of CS exposure on SRB1, a receptor that has been shown to play a prominent role in cellular uptake of vitamin E in a variety of tissues including the lung (Kolleck et al., 2002). The studies were performed in A549 cell, a cell line that has many of the structural and biochemical features characteristics of type II alveolar pneumocytes, the alveolar cells most responsible for surfactant and vitamin E secretion into alveolar spaces (Mason, 2006). SR-B1, in addition to its role in tocopherol transport is known to play a vital role in the bidirectional cholesterol exchange between cells and HDL particles (Kolleck et al., 2002). Of particular relevance to the present study is the report of Hrzenjak and colleagues demonstrating that SR-B1 is the predominant mediator of tocopherol uptake in A549 cells and does so via its interaction with HDL (Hrzenjak et al., 2004). Critically, lung vitamin E levels are 64% lower in SR-BI KO mice compared to control animals (Rigotti et al., 2003). Our recent work demonstrated that mice exposed to CS had a significant decline in lung SR-B1 receptor alongside a decline in lung Vitamin E contents. Based on the known relationships between SR-B1 and tocopherol, the results of the current study showed that CS impairs tocopherol delivery to lung alveolar tissues and does so via its effects on SR-B1 (i.e. decreased SR-B1 levels in A549 lung alveolar type II cells upon exposure to CS). The fact that the decline in SR-B1 upon CS exposure in vitro replicates the in vivo results strongly suggests that SR-B1 represents a major connection between CS and vitamin E in the lung. Given its importance in lung tocopherol, the mechanisms responsible for the decline in SR-B1 protein upon CS exposure are of clear interest. The results of the current study suggest that exposure of lung alveolar type II cells to CS decreases SR-B1 levels and this, based on previous reports (Rigotti et al., 2003) potentially impairs the tocopherol delivery to lung alveolar tissues mediated by SR-B1. The decline in SR-B1 protein appears to be driven in part by increased SR-B1 ubiquitinylation and consequent degradation and does not appear to be caused by decreased mRNA stability. Interestingly, confocal microscopy analysis of the cells showed CS exposure induced changes in the localization of SR-B1. The SRB1 in air-exposed cells was found in a diffuse perinuclear pattern which then switched to a membrane bound patch like pattern upon CS exposure. The shift may via re-localization of already present SR-B1 receptors to the surface promote the destruction of the receptor upon internalization of the patches and ubiquitinylation. This would suggest that SR-B1 receptors have limited recycling given the decline in its level. This shift in SR-B1 localization and its impact on SR-B1 levels and tocopherol cellular uptake remain to be fully characterized as the functionality of receptor
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proteins such as SR-B1 depends directly upon their plasma membrane levels and the effect of this level on the uptake of their ligands is pivotal to many cellular events. The internalization and recycling pathway plays a major role in the regulation of protein surface level (Gong et al., 2008) and suggests that control of SRB1 localization may be of significance as SR-B1 has also recently been associated with the regulation of apoptosis suppressible by HDL (Rigotti et al., 2003). Li et al. have shown that SR-B1 contains a conserved putative redox motif at 323CXXC326 of SR-B1 that mediates the apoptotic activity of SR-B1 the induction of which occurs in a ligand-independent manner via the caspase-8 pathway with both HDL and eNOS suppressing the SR-B1-induced apoptosis (Li et al., 2005). The complexity associated with the SR-B1 induction of apoptosis makes it difficult to assess the possible role of SR-B1 in the induction of apoptosis within the cell culture system employed here. It is clear from the findings presented that SR-B1 levels and especially localization respond to CS in concert with increased apoptosis, which suggests that control of SR-B1 levels and by extension, tocopherol uptake, is involved in apoptosis, though whether the changes noted here are required or merely permissive for appropriate apoptosis to proceed remains to be determined. The mechanisms responsible for the declines in SR-B1 mRNA remain to be fully characterized. Murao et al. have shown that hepatic SR-B1 expression is mediated via the p38 MAPK/Sp-1 signaling cascade (Murao et al., 2008). In this elegant study, Murao’s group showed that p38 MAPK suppressed SR-B1 promoter activity using both constitutively active and dominant-negative forms of p38 MAPK effects on SR-B1 promoter activity. There are several reports showing that CS is able to activate p38 and also AKT (Low et al., 2007; Kuo et al., 2005; Mercer and D’Armiento, 2006; Cao et al., 2004). These suggest a possible pathway wherein CS activates p38 that then modulates the activity of Sp-1 promoter decreasing then the level of SR-B1 mRNA. Clearly the system is likely more complicated since it has been reported that AKT activity regulates SR-B1 levels with increased Akt phosphorylation inhibiting SR-B1 promoter activity (Cao et al., 2004). The implications of the results merit comment as CS induced decline in tocopherol content alongside reductions in tocopherol delivery systems seems counterintuitive. One might expect that the delivery of antioxidants to the site of oxidative stress might be a priority. However, these results reemphasize the fact that oxidative stress is biologically controlled as might be expected given oxidative stress’ central role in a variety of signaling and metabolic systems (Finkel, 2003). This means that the cell, organ or organism must balance the level of oxidants relative to that of antioxidants and that the effects of high levels of antioxidants are not going to be uniformly positive. Adding to the complexity is that vitamin E is only one of an increasing number of compounds that are thought to be involved in protecting cells from oxidative stress and the antioxidant system present likely relies on multiple overlapping or complementary antioxidant species to optimally control oxidant species and levels (Valacchi and Davis, 2008). In conclusion, the data herein presented suggest that CS exposure in the lung with its attendant oxidative stress, induces alterations in lung SR-B1that decrease lung vitamin E availability. This in turn suggests that CS interactions with tocopherol represent an initial event and that subsequent lung responses to continued CS exposure shift to or rely on other antioxidants or systems. This behavior may help explain the lack of a beneficial effect of vitamin E, delivered alone as a supplement on lung health compared to reports of increased lung health upon consuming a vegetable rich diet. More experiments using freshly isolated Type II cells together with vitamin E supplemented diets and tocopherol uptake studies will be needed to better understand the in vivo physiological consequences of the results presented.
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The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Cigarette smoke exposure causes changes in Scavenger Receptor B1 level and distribution in lung cells Giuseppe Valacchi a,b,∗ , Paul A. Davis c , Elaine M. Khan d , Roni Lanir d , Emanuela Maioli e , Alessandra Pecorelli e , Carroll E. Cross d , Tzipora Goldkorn d a
Department of Biomedical Sciences, University of Siena, Siena 53100, Italy Department of Food and Nutrition, Kyung Hee University, Seoul, South Korea c Department of Nutrition, University of California Davis, Davis, 95616 CA, United States d CCRBM, University of University of California Davis, Davis, 95616 CA, United States e Department of Physiology, University of Siena, Siena 53100, Italy b
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Article history: Available online 2 June 2009 Keywords: Cigarette smoke Scavenger Receptor B1 Lung Vitamin E Ubiquitination
a b s t r a c t Scavenger Receptor B1 has been shown to play a prominent role in the uptake and delivery of vitamin E from HDL and is likely involved in regulating vitamin E in the lung. We have previously demonstrated that lung Scavenger Receptor B1 levels (protein and mRNA) are modulated by cigarette smoke in mice and this was accompanied by changes in lung vitamin E. To further characterize the molecular mechanism(s) involved in this process, human alveolar epitheliall cells were exposed to cigarette smoke and Scavenger Receptor B1 cellular levels and distribution were assessed. Results demonstrated that Scavenger Receptor B1 localizes in patches on the cellular membrane and in the perinuclear area of control cells. Upon cigarette smoke exposure, Scavenger Receptor B1 first translocated to the cell surface (within the first 12 h of exposure) and then cell levels (protein and mRNA levels) decreased significantly at 24 h. This decline was accompanied by increased Scavenger Receptor B1 ubiquitination which may explain the decrease in the protein levels. Cigarette smoke induced changes in both sub-cellular redistribution and ubiquitination of Scavenger Receptor B1 together with our previous in vivo data provides evidence that cigarette smoke exposure may alter lung’s ability to control its tocopherol levels. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Cigarette smoke (CS)- and CS-related activation of inflammatory processes are known to induce oxidative stress and thus conceivably play a pathobiologic role in the CS induced lung diseases such as chronic obstructive pulmonary disease (COPD) and lung cancer (Rahman and Kilty, 2006; MacNee, 2005). A number of in vitro studies have shown that CS and its constituents induce oxidative stress and apoptosis in both bronchial epithelial cells and alveolar cells (A549 and L2 cells) (Jiao et al., 2006; Ramage et al., 2006). In particular, Hoshino has identified volatile phase CS extract able to induce apoptosis but this effect was attenuated when oxidant
Abbreviations: CS, cigarette smoke; SR-B1, Scavenger Receptor B1; ALF, alveolar lining fluid; SR-B1 KO, Scavenger Receptor B1 knock out mice; COPD, chronic obstructive pulmonary disease; HDL, high density lipoprotein; eNOS, endothelial nitric oxide synthase; MAPK, mitogen-activated protein kinase; FACS, fluorescence activated cell sorting; BSA, bovine serum albumin; DAPI, 4 ,6-diamidino-2phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ∗ Corresponding author at: Department of Biomedical Sciences, University of Siena, Via Aldo Moro, 7, 53100 Siena (SI), Italy. Tel.: +39 0577 234 107; fax: +39 0577 234 219. E-mail address: [email protected] (G. Valacchi). 1357-2725/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2009.05.014
scavengers were added to the cells (Hoshino et al., 2001). Additionally, a diet high in fruits and vegetables presumably containing “antioxidant substances” has been reported to be associated with a decreased lung diseases risk (Dow et al., 1996). Alveolar lining fluid (ALF) contains antioxidant substances that protect alveolar surfactant and cells from exogenous cytotoxic oxidative stress such as CS (Müller et al., 1998). In CS-related lung diseases these defenses may be compromised (Carp and Janoff, 1978). As studies have shown that oxidant-related lung damage is enhanced in lungs deficient of vitamin E (Hybertson et al., 1995), efforts have been made to augment ALF’s antioxidant levels by increasing vitamin E (␣-tocopherol) via dietary supplementation (Romieu, 2005). However, these efforts, in general, did not show to be effective (Lonn et al., 2005). This lack of efficacy is not well understood and may in part be derived from overall micronutrient antioxidant kinetics in alveolar tissues. Specifically, with respect to vitamin E, the role of the Scavenger Receptor B1 (SR-B1) in lung tocopherol transport remains incompletely understood despite the strong evidence that SR-B1 plays a major role arising from knockout (KO) studies where lung ␣-tocopherol levels are 64% lower in SR-BI KO mice lungs compared to control animals (Rigotti et al., 2003). We recently reported that acute CS exposure reduced lung SR-B1 expression (protein and mRNA) in mice (Valacchi et al.,
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2007). Given that SR-B1 likely plays a major role in the delivery of tocopherol to alveolar tissues, the current study sought to further characterize the effect of acute CS on alveolar cells and the possible mechanism by which CS is able to modulate SR-B1 levels. For our studies we used A549 cells which are a cell line with type II cell characteristics, e.g. production of surfactant proteins and has been widely used to study the responses of alvelolar epithelial type II cells to oxidants (Mairbaurl et al., 1997; Lin et al., 2001; Burvall et al., 2002; Geiser et al., 2004; Kwong et al., 2004). In the present study, A549 cells were exposed to CS and probed either immediately or at timed intervals up to 24 h post CS exposure for changes in the levels of SR-B1 mRNA, protein and cellular localization. The results demonstrate that CS not only produces a decline in SR-B1 mRNA and protein levels, but also induces changes in the cellular localization of SR-B1 protein and increases SR-B1 ubiquitinylation. 2. Materials and methods 2.1. Cell culture A549 cells (ATCC; Manassas, VA) were grown in F-12K nutrient mixture (Invitrogen, Carlsbad, CA), supplemented with 10% FCS and 1% penicillin/streptomycin on Vitrogen-coated (Collagen Corporation, Palo Alto, CA) Costar clear Transwells (0.4-mm pore size; Costar Corporation, Cambridge, MA) until a confluent monolayer was established (approximately 4 × 106 cells/well). For experiments with the proteasome inhibitor MG132 (Calbiochem, USA) the cells were either treated with or without MG132 (final concentration in culture 10 M) dissolved in DMSO (final concentration in culture 0.01%), and incubated for 2 h at 37 ◦ C. At the end of the incubation period, the cells were washed three times and exposed to CS as described below. 2.2. CS exposure Just prior to exposure, the cells’ apical media was aspirated, whereas the media in the basolateral compartment remained in the well to supply cells with nutrients during the exposure. Control cells were exposed to filtered air for the same duration (45 min). The time and the way of exposure were chosen based on our recently published results (Khan et al., 2008) with no difference in the cell viability as measured by Trypan blue exclusion was detected between control (air) and CS treatment (data not shown). A549 cultures were exposed to fresh CS in an exposure system that generated CS by burning one UK research cigarette (12 mg tar, 1.1 mg nicotine) using a vacuum pump to draw air through the burning cigarette and leading the smoke stream over the A549 cultures as described previously by our group (Khan et al., 2008). Briefly, overnight serum-starved A549 cells in culture were placed with lids removed in a 37 ◦ C chamber having a volume of 0.45 cu. ft., smoke from one cigarette was introduced into the chamber by readmitting air (a decrease of ∼5 in. Hg over ∼30 s) in the chamber after achieving a partial vacuum (25 in. Hg) via a short piece of tubing holding a lit cigarette (UK research cigarette; 12 mg tar, 1.1 mg nicotine). After the exposure (air or CS) fresh media was added to the cells. 2.3. Immunohistochemistry and confocal microscopy A549 cells grown on coverslips at the same density of that of the cells cultured in transwells, were treated as indicated above and fixed in 4% paraformaldehyde in phosphate buffered saline (PBS). Cells were permeabilized for 15 min at room temperature with PBS containing 1% BSA, 0.2% Triton X-100, and 0.02% sodium azide, then the coverslips were blocked in PBS containing 1% BSA, 0.2% Nonidet
P-40, 5% goat serum, and 0.02% sodium azide at room temperature for 1 h. Coverslips were then incubated for 1 h with primary antibody, followed by 1 h with secondary antibodies (Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, Eugene, OR)). Nuclei were stained with 1 g/ml DAPI or 1 g/ml propidium iodide (Molecular Probes) for 3 min after removal of secondary antibodies. Coverslips were mounted onto glass slides using the ProLong Antifade Kit (Molecular Probes). Confocal microscopy at 20 and 60× magnification was carried out using an Olympus FV1000 Fluoview confocal laser scanning microscope. 2.4. Western blot analysis Total cell lysates were extracted in solubilization buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM EGTA, 0.1% SDS, 5 mM N-ethylmaleamide (Sigma–Aldrich Corp.), protease and phosphatase inhibitor cocktails (Sigma–Aldrich Corp.). Membrane extracts were prepared using the ProteoExtract TM Native Membrane Protein Extraction Kit (Calbiochem, Milan, Italy), following the manufacturer’s instructions. Lysates were cleared by centrifugation (16,000 × g) for 45 min and protein concentration was measured using the Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA). Equivalent amounts of proteins were subjected to 10% SDSPAGE, transferred onto a nitrocellulose membrane which was then blocked for 1 h in Tris-buffered saline, pH 7.5, containing 0.5% Tween 20 and 5% milk. Membranes were then incubated overnight at 4 ◦ C with the appropriate primary antibody (SR-B1) (Abcam, Cambridge, MA) for 24 h. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h, and the bound antibodies were detected using chemiluminescence (Pierce Chemical Co., Rockford, IL). Images of the bands were digitized and the densitometry of the bands were performed using NIH-Image software. 2.5. Immunoprecipitation and immunoblotting for ubiquitination After treatments, cells were extracted in solubilization buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM EGTA, protease and phosphatase inhibitor cocktails (Sigma). Lysates were cleared by centrifugation and 400 g of protein in the supernatant were immunoprecipitated by overnight incubation with 2.5 g anti-SR-B1 (Abcam, Cambridge, MA) at 4 ◦ C, followed by protein A (Repligen Corp., Needham, MA) precipitation for 1–2 h. Immunoprecipitates were washed three times with HNTG buffer containing 20 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol, resolved by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were blocked for 1 h with Tris-buffered saline, pH 7.5, containing 0.5% Tween 20, 1% BSA, and 5% non-fat milk, incubated overnight at 4 ◦ C with monoclonal primary antibody anti-ubiquitin (Covance Research Products, Inc., Berkeley, CA), followed by 1 h incubation at room temperature with a 1:10,000 dilution of horseradish peroxidaseconjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). Immunoreactive protein bands were detected with the SuperSignal West Pico Substrate (Pierce, Rockford, IL). 2.6. SR-B1 messenger RNA levels and stability SR-B1 mRNA was evaluated by real-time quantitative PCR on total RNA isolated from A549 cells using SYBER Green (Perkin Elmer Applied Biosystems; Foster City, CA) and ABI Prism 7700 Sequence Detector (Perkin Elmer Applied Biosystems). Primers were synthe-
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Fig. 1. Cigarette Smoke exposure induces changes in SR-B1 levels and localization in lung epithelial cells. Immunocytochemistry of A549 cells showing localization of SR-B1 (green) before and after CS exposure for different time points. Images are merged and are representative of at least 100 cells viewed in each experiments (n = 5). Nuclei (blue) were stained with DAPI. A) Cells overview at different time points 20×; B) Individual cell at different time points 60×; C) Representative Western blot of proteins extracted from the membranes of cells exposed to Cigarette Smoke at different time points. The signals of SR-B1 protein levels were determined by densitometric analysis of the scanned images (bottom panel). Data are expressed in arbitrary units and are averages of the values for five different experiments. * p < 0.05.
sized using Primer Express v. 1.0 software (Perkin Elmer Applied Biosystems) and according to the published cDNA sequences for SR-B1 and GAPDH the internal control. No-template (water) reaction mixture was prepared as a negative control. Quantitative analysis of PCR products during the PCR amplification was measured via fluorescence emission and then analyzed by the Sequence Detector v1.6.3 program (Perkin Elmer Applied Biosystems) of the ABI Prism 7700 detection system. mRNA expression of the gene of interested was normalized by using GAPDH mRNA expression levels. The mRNA level analysis was done in triplicate and repeated. Assessment of SR-B1 mRNA stability upon exposure to CS was done by adding actinomycin (5 g/ml) at time zero to halt new RNA synthesis. Cells were harvested and cellular mRNA was isolated immediately or at 1, 2, 3 h post CS exposure. 3. Results 3.1. CS exposure affects SR-B1 cellular localization and membrane protein levels The results from confocal microscopy (Fig. 1) demonstrate that in the control (Fig. 1A and B), air-exposed A549 cells, SR-B1 immunofluorescence was distributed in the perinuclear area and in the cell membrane. After CS exposure, there was an increase in SR-B1 membrane location and a decreased perinuclear staining. This pattern changed at 6 and 12 h post CS exposure as the perinuclear staining was almost lost and the membrane staining was less evident. At 24 h SR-B1 levels were dramatically decreased in
all the cellular compartments. These changes were not present in the control, air-exposed cells at the same time points (0–24 h) (data not shown). The changes in SR-B1 membrane levels were confirmed by Western blot analysis of cell membrane extracts. Fig. 1C showed the increase in SR-B1 membrane levels and the following significant decrease. 3.2. CS exposure decreased SR-B1 protein levels The level of SR-B1 protein from whole A549 cell lysates as assessed by Western blot analysis decreased markedly upon CS exposure in a time dependent manner as depicted in Fig. 2. A reduction of approximately 50% occurred after 45 min of CS exposure and was further reduced after 24 h (approximately 75% decline). Although the total SR-B1 (membrane and cytosol) decreased after CS exposure (Fig. 2), the shift of the receptor from the perinuclear area to the membrane leads to an increased membrane level (Fig. 1C). No changes were detected during the 24 h time course in the air treated cells. Furthermore, no differences in cell viability as measured by Trypan blue exclusion were apparent between control air and CS exposed cells (data not shown). 3.3. CS exposure decreased SR-B1 mRNA levels without affecting its stability The results showed in Fig. 3A demonstrated that CS exposure caused a rapid drop in SR-B1 mRNA levels immediately
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Fig. 2. Cigarette Smoke exposure decreases SR-B1 protein levels in A549 cells. Cells were exposed to Cigarette Smoke for 45 min and were harvested at different time points. SR-B1 proteins levels were detected by Western blot analysis. A representative immunoblot is shown at the top panel. The signals of SR-B1 protein levels were determined by densitometric analysis of the scanned images. Quantification of the SR-B1 bands is shown at the bottom panel. Data are expressed in arbitrary units and are the average of five different experiments. * p < 0.05.
Fig. 4. Cigarette Smoke exposure induced SR-B1 ubiquitination. A549 cells were exposed to Cigarette Smoke and cell lysates were immunoprecipitated using anti SR-B1. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with anti-ubiquitin (A). Re-probing the membrane with anti-SR-B1 antibody showed the presence of equal amounts of SR-B1 immunoprecipitates (bottom panel). B) Cells were pre-treated (+) or not (−) with the proteosome inhibitor MG132 at the final concentration of 10 M and after 2 h incubation the cells were exposed to CS for 45 min. Quantification of the SRB1 bands is shown at the bottom panel. The signals of SR-B1 protein levels were determined by densitometric analysis of the scanned images. All samples were normalized per protein per sample. Data are expressed in arbitrary units. A representative Western blot from five experiments is shown at the top. * p < 0.05 for Air vs CS; # p < 0.05 for +MG132 vs −MG132.
after exposure and the reduced mRNA level persisted at 6 and 12 h post exposure. mRNA stability studies revealed that SR-B1 mRNA decline in the presence of actinomycin paralleled each other between the air and the CS treated cells, indicating that CS exposure did not affect mRNA stability (Fig. 3B). 3.4. CS exposure induced SR-B1 ubiquitination
Fig. 3. Cigarette Smoke exposure decreased SR-B1 mRNA levels. A) SR-B1 gene expression in A549 cells exposed to air or to Cigarette Smoke was evaluated by real-time PCR. The isolated mRNA samples were analyzed using the specific primer for SR-B1 and compared to GADPH (housekeeping gene) levels. B) For mRNA stability studies the cells were pre-treated with 5 M of actonimycin D as described in Methods. Data are expressed as mean ± S.D. of three independent experiments. p < 0.05.
Immunoprecipitation and Western blot analysis for SR-B1 ubiquitination (Fig. 4) revealed that CS exposure increased ubiquitinated SR-B1 in A549 cells, with the increase apparent after 45 min of CS exposure and a further increase at 6 h post CS exposure (Fig. 4A). Re-probing of the membrane with anti-SR-B1 antibodies indicated that equivalent amounts of SR-B1 were immunoprecipitated from each sample (Fig. 4A, bottom panel). These data were confirmed using an inhibitor of the proteosome (MG132). CS exposed cells in the presence of MG132 exhibited higher levels of ubiquitinated SR-B1 while air-exposed cells did not
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show any differences in SR-B1 levels ubiquitination in the presence of MG132 (Fig. 4B). Although MG132 is not a fully selective inhibitor, the results point to both protein degradation (MG132/air control is higher than MG132/CS treated cells), as well as decreased synthesis as shown in Fig. 3A as being responsible for the declines noted.
4. Discussion Alveolar surfactant represents one of the first targets of airborne oxidants such as CS (Rahman and Kilty, 2006) and therefore likely plays a major role in the pathobiology of several respiratory tract diseases including COPD and lung cancer. CS exposure interacts with tocopherol as the effects of CS on lung surfactant synthesis and cell adherence constituents of type II pneumocytes are restored upon vitamin E supplementation, suggesting that vitamin E likely plays an important role in the lung related events in response to CS exposure (Xue et al., 2005; Guthmann et al., 2003). The present study examined the effect of CS exposure on SRB1, a receptor that has been shown to play a prominent role in cellular uptake of vitamin E in a variety of tissues including the lung (Kolleck et al., 2002). The studies were performed in A549 cell, a cell line that has many of the structural and biochemical features characteristics of type II alveolar pneumocytes, the alveolar cells most responsible for surfactant and vitamin E secretion into alveolar spaces (Mason, 2006). SR-B1, in addition to its role in tocopherol transport is known to play a vital role in the bidirectional cholesterol exchange between cells and HDL particles (Kolleck et al., 2002). Of particular relevance to the present study is the report of Hrzenjak and colleagues demonstrating that SR-B1 is the predominant mediator of tocopherol uptake in A549 cells and does so via its interaction with HDL (Hrzenjak et al., 2004). Critically, lung vitamin E levels are 64% lower in SR-BI KO mice compared to control animals (Rigotti et al., 2003). Our recent work demonstrated that mice exposed to CS had a significant decline in lung SR-B1 receptor alongside a decline in lung Vitamin E contents. Based on the known relationships between SR-B1 and tocopherol, the results of the current study showed that CS impairs tocopherol delivery to lung alveolar tissues and does so via its effects on SR-B1 (i.e. decreased SR-B1 levels in A549 lung alveolar type II cells upon exposure to CS). The fact that the decline in SR-B1 upon CS exposure in vitro replicates the in vivo results strongly suggests that SR-B1 represents a major connection between CS and vitamin E in the lung. Given its importance in lung tocopherol, the mechanisms responsible for the decline in SR-B1 protein upon CS exposure are of clear interest. The results of the current study suggest that exposure of lung alveolar type II cells to CS decreases SR-B1 levels and this, based on previous reports (Rigotti et al., 2003) potentially impairs the tocopherol delivery to lung alveolar tissues mediated by SR-B1. The decline in SR-B1 protein appears to be driven in part by increased SR-B1 ubiquitinylation and consequent degradation and does not appear to be caused by decreased mRNA stability. Interestingly, confocal microscopy analysis of the cells showed CS exposure induced changes in the localization of SR-B1. The SRB1 in air-exposed cells was found in a diffuse perinuclear pattern which then switched to a membrane bound patch like pattern upon CS exposure. The shift may via re-localization of already present SR-B1 receptors to the surface promote the destruction of the receptor upon internalization of the patches and ubiquitinylation. This would suggest that SR-B1 receptors have limited recycling given the decline in its level. This shift in SR-B1 localization and its impact on SR-B1 levels and tocopherol cellular uptake remain to be fully characterized as the functionality of receptor
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proteins such as SR-B1 depends directly upon their plasma membrane levels and the effect of this level on the uptake of their ligands is pivotal to many cellular events. The internalization and recycling pathway plays a major role in the regulation of protein surface level (Gong et al., 2008) and suggests that control of SRB1 localization may be of significance as SR-B1 has also recently been associated with the regulation of apoptosis suppressible by HDL (Rigotti et al., 2003). Li et al. have shown that SR-B1 contains a conserved putative redox motif at 323CXXC326 of SR-B1 that mediates the apoptotic activity of SR-B1 the induction of which occurs in a ligand-independent manner via the caspase-8 pathway with both HDL and eNOS suppressing the SR-B1-induced apoptosis (Li et al., 2005). The complexity associated with the SR-B1 induction of apoptosis makes it difficult to assess the possible role of SR-B1 in the induction of apoptosis within the cell culture system employed here. It is clear from the findings presented that SR-B1 levels and especially localization respond to CS in concert with increased apoptosis, which suggests that control of SR-B1 levels and by extension, tocopherol uptake, is involved in apoptosis, though whether the changes noted here are required or merely permissive for appropriate apoptosis to proceed remains to be determined. The mechanisms responsible for the declines in SR-B1 mRNA remain to be fully characterized. Murao et al. have shown that hepatic SR-B1 expression is mediated via the p38 MAPK/Sp-1 signaling cascade (Murao et al., 2008). In this elegant study, Murao’s group showed that p38 MAPK suppressed SR-B1 promoter activity using both constitutively active and dominant-negative forms of p38 MAPK effects on SR-B1 promoter activity. There are several reports showing that CS is able to activate p38 and also AKT (Low et al., 2007; Kuo et al., 2005; Mercer and D’Armiento, 2006; Cao et al., 2004). These suggest a possible pathway wherein CS activates p38 that then modulates the activity of Sp-1 promoter decreasing then the level of SR-B1 mRNA. Clearly the system is likely more complicated since it has been reported that AKT activity regulates SR-B1 levels with increased Akt phosphorylation inhibiting SR-B1 promoter activity (Cao et al., 2004). The implications of the results merit comment as CS induced decline in tocopherol content alongside reductions in tocopherol delivery systems seems counterintuitive. One might expect that the delivery of antioxidants to the site of oxidative stress might be a priority. However, these results reemphasize the fact that oxidative stress is biologically controlled as might be expected given oxidative stress’ central role in a variety of signaling and metabolic systems (Finkel, 2003). This means that the cell, organ or organism must balance the level of oxidants relative to that of antioxidants and that the effects of high levels of antioxidants are not going to be uniformly positive. Adding to the complexity is that vitamin E is only one of an increasing number of compounds that are thought to be involved in protecting cells from oxidative stress and the antioxidant system present likely relies on multiple overlapping or complementary antioxidant species to optimally control oxidant species and levels (Valacchi and Davis, 2008). In conclusion, the data herein presented suggest that CS exposure in the lung with its attendant oxidative stress, induces alterations in lung SR-B1that decrease lung vitamin E availability. This in turn suggests that CS interactions with tocopherol represent an initial event and that subsequent lung responses to continued CS exposure shift to or rely on other antioxidants or systems. This behavior may help explain the lack of a beneficial effect of vitamin E, delivered alone as a supplement on lung health compared to reports of increased lung health upon consuming a vegetable rich diet. More experiments using freshly isolated Type II cells together with vitamin E supplemented diets and tocopherol uptake studies will be needed to better understand the in vivo physiological consequences of the results presented.
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