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Cigarette Smoke Induces an Unfolded Protein Response in the Human Lung

A Proteomic Approach

² Department of Biochemistry and ¹ Department of Medicine, Division of Pulmonary Critical Care and Pulmonary Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania

Correspondence and requests for reprints should be addressed to Steven G. Kelsen, M.D., 761 Parkinson Pavilion, Temple University Hospital, 3401 N. Broad Street, Philadelphia, PA 19140. E-mail: kelsen{at}temple.edu

	Abstract

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Cigarette smoking, which exposes the lung to high concentrations of reactive oxidant species (ROS) is the major risk factor for chronic obstructive pulmonary disease (COPD). Recent studies indicate that ROS interfere with protein folding in the endoplasmic reticulum and elicit a compensatory response termed the "unfolded protein response" (UPR). The importance of the UPR lies in its ability to alter expression of a variety of genes involved in antioxidant defense, inflammation, energy metabolism, protein synthesis, apoptosis, and cell cycle regulation. The present study used comparative proteomic technology to test the hypothesis that chronic cigarette smoking induces a UPR in the human lung. Studies were performed on lung tissue samples obtained from three groups of human subjects: nonsmokers, chronic cigarette smokers, and ex-smokers. Proteomes of lung samples from chronic cigarette smokers demonstrated 26 differentially expressed proteins (20 were up-regulated, 5 were down-regulated, and 1 was detected only in the smoking group) compared with nonsmokers. Several UPR proteins were up-regulated in smokers compared with nonsmokers and ex-smokers, including the chaperones, glucose-regulated protein 78 (GRP78) and calreticulin; a foldase, protein disulfide isomerase (PDI); and enzymes involved in antioxidant defense. In cultured human airway epithelial cells, GRP78 and the UPR-regulated basic leucine zipper, transcription factors, ATF4 and Nrf2, which enhance expression of important anti-oxidant genes, increased rapidly (< 24 h) with cigarette smoke extract. These data indicate that cigarette smoke induces a UPR response in the human lung that is rapid in onset, concentration dependent, and at least partially reversible with smoking cessation. We speculate that activation of a UPR by cigarette smoke may protect the lung from oxidant injury and the development of COPD.

Key Words: oxidant defense • lung injury • COPD

CLINICAL RELEVANCE Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References   Chronic cigarette smoking induces an endoplasmic reticulum stress response in the human lung termed the unfolded protein response. Failure of this compensatory system in some cigarette smokers may contribute to the development of chronic obstructive pulmonary disease.

 
Cigarette smoking is the major risk factor for several forms of lung disease, including chronic obstructive pulmonary disease (COPD) (1–3). In the United States alone, more than 45 million people or 20.9% of the population are chronic smokers (4, 5). The gaseous and particulate phases of cigarette smoke contain more than 4,500 separate compounds, many of which are highly toxic reactive oxygen/nitrogen species (RONS) and xenobiotic materials (2, 6–8). In fact, cigarette smoke contains more than 10¹⁴ free radicals in each puff of smoke (6, 7). Although the molecular mechanisms underlying development of COPD remain incompletely understood, these toxic substances are believed to induce an inflammatory response by adversely affecting oxidant/antioxidant and protease/anti-protease balance in the lung (1–3, 9–12).

Of considerable importance, the propensity to develop COPD varies widely across cigarette smokers and correlates only weakly with the smoking history as reflected in the number of cigarette pack-years (13, 14). In fact, it is estimated that only a minority (i.e., 15–35%) of chronic, continuous cigarette smokers develop COPD (14, 15). That the majority of long-term smokers do not develop COPD suggests that failure of compensatory mechanisms that protect the lung from ROS or xenobiotic materials contributes to development of the disease. In support of this concept, the expression of antioxidant genes believed to be important in protection of the lung from cigarette smoke–induced injury (e.g., thioredoxin, peroxiredoxin and glutathione-S-transferase, glutathione peroxidase) varies widely in airway epithelial cells harvested from chronic cigarette smokers (16, 17).

Recent studies indicate that a complex molecular cascade termed the "unfolded protein response" (UPR) plays an important role in the regulation of expression of a variety of antioxidant, xenobiotic metabolizing and pro- and anti-inflammatory genes (18–24). The UPR is activated in response to cellular stressors such as RONS, increases in cytosolic calcium, and hypoxia, which impair protein folding in the lumen of the endoplasmic reticulum (ER) (18, 19, 22). Activation of the UPR compensates for abnormalities in protein folding by increasing the expression of genes involved in protein chaperoning and folding, protein translation, and protein degradation (18, 19, 25). Moreover, since the processes involved in protein transport and folding consume ATP and generate ROS, the UPR response induces expression of genes involved in energy synthesis and ROS quenching (21). Of interest, many basic cellular processes which depend on an adequate supply of fully functional membrane and secreted proteins (e.g., cell cycle regulation, apoptosis, energy metabolism, inflammation, and acute phase reactants) are also regulated by the UPR (20, 26–28).

The effect of cigarette smoke exposure, which is likely to create conditions that foster development of a UPR by virtue of its effects on the cell RONS burden and cytosolic calcium, has not, however, been studied (29, 30). In the present study, we tested the following hypotheses: (1) chronic cigarette smoke exposure activates a UPR in the human lung; (2) the UPR is reversible with smoking cessation; and (3) UPR induction by cigarette smoke is dose-dependent and rapid in onset. Studies were performed on lung tissue samples obtained from three groups of human subjects: nonsmokers, chronic cigarette smokers, and ex-smokers. Comparative proteomic technology was used to examine the expression of several proteins that are hallmarks of a UPR and its downstream targets (18, 26–28). Specifically, activation of a UPR was tested by examining the expression of the chaperones, GRP78, calnexin and calreticulin, and a foldase, protein disulfide isomerase. Cultured human airway epithelial cells (i.e., the 16-HBE cell line), a cell which plays an important role in the pathogenesis of COPD, were used to examine the UPR response to a range of concentrations of cigarette smoke extract (CSE), and its time course (1–3). In addition, experiments in 16-HBE cells sought to determine if CSE activates one of several ER sensors that detect misfolded proteins (i.e., the protein kinase-R-like endoplasmic reticulum kinase [PERK]) (18, 19). The PERK pathway is of considerable importance in the cellular response to oxidant stress, since it augments nuclear expression of the basic leucine zipper transcription factors, nuclear factor, erythroid factor 2 p45 related factor 2 (Nrf2), and ATF4 (21, 26, 31–33). In particular, Nrf2 heterodimerizes with several different transcription factors, including ATF4, to regulate a large number of genes involved in protection of the lung against oxidant injury and metabolism of xenobiotic substances (12, 34–38).

The results of the present study indicate that the several chaperones and foldase, which are markers of a UPR, are up-regulated in the lungs of chronic cigarette smokers, and that several downstream targets of the UPR including the antioxidant gene, thioredoxin-dependent peroxidase reductase, are up-regulated. Moreover, ex-smokers demonstrate a UPR response intermediate between that of nonsmokers and that of active smokers, supporting the idea that the UPR induced by chronic cigarette smoking is at least partially reversible. Finally, in cultured airway epithelial cells, the UPR is dose-dependently activated by CSE, occurs in less than 24 hours, and specifically involves PERK activation and its downstream targets, the transcription factors Nrf2 and ATF4.

	MATERIALS AND METHODS

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Clinical Study Material
Lungs were obtained by the region's transplant network, Gift of Life Inc., under a protocol approved by the Temple University Institutional Review Board. The lungs were obtained from three chronic smokers; three lifelong, nonsmoking subjects; and three ex-smokers. Criteria for inclusion included a history negative for lung disease and normal lung radiography.

All lungs were obtained from beating heart donors, immediately placed in sterile, ice-cold preservation medium (39), and shipped to our laboratory on ice within 12 hours of collection. Radiologically and macroscopically normal-appearing regions were selected for this study. The region selected, usually an entire lobe, was slowly inflated using a cannula inserted into the supplying airway to instill a 1:1 mix of cryo-embedding medium (Cryomatrix; Thermo Electron Corp., Waltham, MA) and 50% sucrose in distilled water.

Portions of the inflated organ were then frozen by suspension in liquid nitrogen fumes for 20 to 25 minutes, as previously described (40), then stored at –80°C until used. Samples of frozen tissues were cut into 1 x 1 x 1 cm blocks and embedded in OCT (Tissue Tek, Electron Microscopy Sciences, Hatfield, PA). Sample sections (6 µm) were cut from each block using a cryostat at –20°C, then stained with hematoxylin and eosin for inspection. Only those blocks showing normal lung architecture microscopically were selected for subsequent proteomic analysis. Since whole lung tissue is composed of at least six major cell types, considerable effort was made to obtain approximately equal amounts of lung parenchyma, small airways, and pulmonary vessels. In this regard, the amount ( 1 cm³) and location (peripheral tissue extending to include the pleural surface) of the sample were standardized.

Human Airway Epithelial Cell Culture
Human airway epithelial cells, the 16-HBE cell line, were cultured in 100-mm tissue culture dishes coated with type VI human placental collagen (Sigma/Aldrich, St. Louis, MO) in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 mg/ml) in 5% CO₂ at 37°C. Cells were grown to 80% to 100% confluence before study. At the time of study, medium was replaced with medium containing CSE.

CSE was prepared as previously described (41, 42). Briefly, smoke from two unfiltered, research cigarettes (2R4; University of Kentucky, Lexington, KY) was bubbled through 25 ml of cell culture medium in a 50-ml plastic centrifuge tube at a flow rate of 50 ml/minute. Smoke was allowed to equilibrate in the medium for 10 to 15 minutes, and the resultant suspension was then passed through a 0.2-µm pore filter (Nalgene, Rochester, NY). CSE prepared in this manner was considered to have a concentration of 100%. From this stock, serial dilutions were made with cell culture medium to achieve final concentrations of 15, 30, and 45% CSE. CSE was applied to cell cultures within 30 minutes of preparation and maintained for 24 hours. Cells exposed to medium alone served as control.

Cultured cells were washed twice with cold PBS, scraped and lysed in 1% SDS, 4 µg/ml aprotinin, 4 µg/ml leupeptin, 4 µg/ml pepstatin, 2 mM phenylmethylsulfonyl fluoride, 6 mM Na₃VO₄, and 6 mM NaF. Protein concentrations were determined by DC protein assay (Bio-Rad, Hercules, CA).

Sample Preparation for Two-Dimensional Gel Analysis
Frozen lung tissue from three nonsmokers, three chronic smokers, and three ex-smokers was individually processed by grinding with a mortar and pestle cooled with liquid nitrogen. Frozen powders from each individual tissue block were thawed by adding 0.8 ml of cold lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, and 60 mM DTT) and sonicated in an ice bath for approximately 1 minute at 5 watts and 70% duty cycle (Sonic Dismembrator; Fisher Scientific, Pittsburgh, PA). Sonicates were centrifuged at 10,000 x g for 12 minutes at 4°C. Supernatants were collected and acetone added to precipitate proteins and remove cryo-embedding medium and sucrose solution. Precipitated proteins were resolubilized using the above lysis buffer. Protein concentrations in the extracts were measured in triplicate using a Bio-Rad Bradford-based protein assay with bovine serum albumin as the standard.

Two-Dimensional Gel Electrophoresis
Lung lysates were processed individually (nine in total; three from each group of subjects). For first-dimension separation, 40 µg of sample protein was diluted in 125 µl with rehydration buffer and loaded onto an immobilized pH gradient (IPG) strip by overnight passive in-gel rehydration. A global view of the proteome was obtained initially using IPG strips of pI 3-10. To enhance resolution and sensitivity, narrow range IPG strips (pI 4–7 and 6–10) were subsequently used. The rehydration buffer contained 8 M urea, 2% CHAPS, 0.2% carrier ampholytes, and 10 mM DTT for pH 4–7 linear and pH 3–10 nonlinear IPG strips. For pH 6–10 IPG strips, rehydration was with 15 mg/ml Destreak reagent. Isoelectric focusing (IEF) within the strips was performed at 20°C with a MultiPhor II system (Amersham Biosciences Corp., Piscataway, NJ) using a total of 12,000 V·h with a maximum of 5,000 V.

For second-dimensional separation, the IPG strips were removed from the MultiPhor II chamber, soaked for 15 minutes in 10 ml of an equilibration buffer (6 M urea, 30% glycerol, 2% SDS, 1% DTT, and 0.05 M pH 8.8 Tris), then for 15 minutes in 10 ml of a second equilibration buffer (with 2.5% iodoacetamide substituted for 1% DTT), and positioned against 10 to 14% SDS polyacrylamide gels in a BioRad Mini-PROTEAN 3 System at 200 V for 45 minutes. Polyacrylamide gels were then fixed twice using 50% methanol, 7% acetic acid, balance water. Protein spots in the gels were revealed by staining with either Sypro-Ruby fluorescent total protein stain or Pro-Q Diamond phosphoprotein stain.

Image Analysis of Two-Dimensional Gels
Fluorescence images of individual gels from the nine lung lysates were captured with a FLA-5000 Fluor Imager (Fuji Photo Film Co., Ltd., Tokyo, Japan) and analyzed using PDQuest Software (Version 8.0). After automatic detection of spots by PDQuest software, the files were inspected manually to assess accuracy of computer-generated images. The software calculated individual spot "volumes" in each gel by density/area integration. To control for slight differences in protein loading across gels, the spot volume density obtained from each individual lung lysate was calculated by image analysis software and normalized to total spot volume on that gel.

In-Gel Trypsin Digestion
Differentially expressed spots were excised using an Xcise automated robotic system (Shimadzu Biotech, Columbia, MD). Destaining of excised gel pieces was performed by two 30-minute washes with 50% acetonitrile containing 50 mM ammonium bicarbonate. After dehydration with 100% acetonitrile, 10 µl of 12.5 ng/µl sequencing grade trypsin (Promega, Madison, WI) was added to the gel pieces and incubated overnight at 37°C. Resulting tryptic peptides were extracted twice with 15 µl (5% formic acid, 50% acetonitrile, balance water) for 20 minutes and the pooled extracts were processed with desalting ZipTips.

MALDI-TOF-TOF Analysis
The desalted peptides from each spot were mixed 1:1 with matrix solution (1% -cyano-4-hydroxy cinnamic acid in 50% acetonitrile and 50% 0.1% trifluoroacetic acid) and 1.0 µl volumes were applied to wells of an AnchorChip sample target plate used for the Bruker Auto-flex MALDI-TOF-TOF instrument. Peptide mass fingerprints were obtained using the reflective and positive ion mode. Mass spectra were collected from the sum of 100 to 400 laser shots and mono-isotopic peaks were generated by FlexAnalysis software with signal-to-noise ratio of 2:1. Mass peak value calculations used two trypsin auto-digestion peptides with M+H values 842.509 and 2211.104 as internal standards. Proteins were identified by matching the calibrated peptide mass values within either Swiss-Prot or NCBInr protein databases for Homo sapiens using an in-house version of Mascot Server 2.1 imbedded in Bruker's Biotool software. Match variances allowed were a mass tolerance of 50 ppm, one missed trypsin cleavage, fixed modification of carbamidomethyl cysteine, and variable modification of methionine oxidation. For the samples that did not produce a "hit" with a confident score, peptide peaks with good signal were further fragmented using "Laser-induced decomposition" to obtain LIFT-TOF/TOF spectra, and these MS/MS data alone or combined with the previously produced MS data were used to search against the protein database through the Mascot.

Western Blot Analysis
Proteins (30 to 80 µg) from the same lung lysates as used for the above two-dimensional gels or from CSE-exposed 16 HBE cells was separated by 10 to 14% gradient SDS-PAGE. The separated proteins were transferred to a nitrocellulose membrane in a semi-dry blotting chamber according to the manufacturer's protocol (Bio-Rad) or in a transfer apparatus in CAPS buffer (10 mM 3-cyclohexylamino-1-propanosulfonic acid in 15% methanol, pH 10.6).

Blots were blocked with 5% milk in Tris-buffer saline solution (pH 7.6) containing 0.05% Tween-20 (TBS/T), and probed with the following rabbit anti-human antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) at a concentration of 0.4 µg/ml: GRP-78 (SC-13968), PDI (SC-20132), calreticulin (SC-11398), calnexin (SC-11397), GAPDH (SC-25778), Nrf2 (SC-13032), and ATF4 (SC-200). In addition, rabbit anti-human total eIF2 (cat# 9722) and anti-phospho eIF2 (cat# 9721) from Cell Signaling Technology (Beverly, CA) were used at 1:1,000 dilution. Blots were incubated with primary antibody overnight at 4°C at with gentle shaking, then incubated with a mouse anti-rabbit horseradish peroxidase–conjugated secondary Ab (1:10,000) (Biomeda Corp., Foster City, CA) for 1 hour at room temperature. Blots were exposed using a chemiluminescent detection method (Enhanced ECL Detection System, Amersham Biosciences).

In some 16-HBE cell experiments, cytoplasmic and nuclear fractions were prepared to examine expression, respectively, of the translational initiation factor, eIF2, a classical target of activated PERK, as well as the transcription factors, Nrf2 and ATF4. Cells were washed with cold PBS twice, scraped in 1 ml PBS/1 mM EDTA buffer, and then centrifuged. The pellet was resuspended in ice-cold harvest buffer (10 mM HEPES, pH 7.9, 50 mM NaCl, 0.5 M Sucrose, 0.1 mM EDTA, 0.5% Triton X-100, 1 mM dithiothreitol, and proteinase inhibitor [cat#78410; Pierce, Rockford, IL]) for 10 minutes. The lysate was centrifuged and the supernatant containing the cytoplasmic fraction was collected and cleared at 14,000 rpm. The pellet containing the nuclear fraction was washed in ice-cold buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and proteinase inhibitor), pelleted and then resuspended in ice-cold buffer C (10 mM HEPES, pH 7.9, 500 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% NP-40, with 1 mM dithiothreitol, and proteinase inhibitor). The suspension was vortexed, vigorously rocked, and then centrifuged. The supernatant containing the nuclear proteins was removed. Nuclear and cytoplasmic fractions were stored at –80°C before Western blotting.

Statistical Analysis
Group mean ± SD values of normalized spot volumes in two-dimensional gels of individual smokers, nonsmokers, and ex-smokers were determined. Statistical significance of differences in group mean values was determined by one-way ANOVA and Student's t test with statistical significance accepted at the P < 0.05 level. Spots on two-dimensional gels, which were statistically significantly different (P < 0.05), were considered differentially expressed.

Western blots for proteins of interest were scanned and differences in band density assessed statistically by one-way ANOVA and Students' t test. Statistical significance was accepted at the P < 0.05 level.

	RESULTS

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Subject Groups
Demographics, smoking history, co-morbidities, and cause of death are shown in Table 1. Smokers and ex-smokers had similar, heavy smoke exposure (36 ± 19 SE pack-years, range 12–75 yr, and 38 ± 13 SE pack-years, range 15–60 yr, for smokers and ex-smokers, respectively; P > 0.44). Ex-smokers had stopped smoking from 8 to 18 years before death.

The microscopic appearance of the lung in the region from which samples were obtained was normal (Figure 1).

View larger version (193K): [in this window] [in a new window]   Figure 1. Typical lung architecture in representative smoker and nonsmoker. The lungs were inflated with cryo-embedding medium and frozen over liquid nitrogen fumes. Frozen sections (6 µM) were stained with hematoxylin and eosin. Each field shows normal-appearing small intra-pulmonary airways (large arrows), pulmonary arterioles (small arrows), and adjacent parenchyma (x1,000 magnification).

View larger version (74K): [in this window] [in a new window]   Figure 2. Differential expression profiling of lung homogenates from smoker and nonsmoker using two-dimensional electrophoresis. Proteins were separated by isoelectric focusing (pI) and by molecular weight as described in MATERIALS AND METHODS. The sub-proteome from each sample was assessed using a pI range of 4 to 7 (A) or of 6 to 10 (B). The proteins were stained with sypro-ruby and images compared by PDQuest software. The red arrows indicate up-regulated proteins and the blue arrows down-regulated proteins in lungs of smokers.

Of the differentially expressed proteins observed in the lungs of smokers compared with those of the nonsmokers, 20 were up-regulated, 5 were down-regulated and 1 (major vault protein) was detected only in the smoking group. The differentially expressed proteins, their identity, and the magnitude of difference with the nonsmoker group are shown in Table 2.

View larger version (17K): [in this window] [in a new window]   Figure 3. Expression of the UPR proteins GRP78, calreticulin, and PDI in smokers, nonsmokers, and ex-smokers (group mean ± SEM). Expression levels of proteins visualized on the sypro-ruby stained two-dimensional gel were quantitated as described in MATERIALS AND METHODS. All three proteins were significantly up-regulated in the lungs of smokers compared with those of nonsmokers and ex-smokers (P < 0.05 for each comparison).

Up-regulation of the UPR chaperones and foldase in the lungs of smokers was confirmed by Western blotting for GRP78, calreticulin, and PDI (Figure 4). Another UPR marker not detected on two-dimensional gels, the membrane-bound chaperone calnexin, was also up-regulated as detected by Western blotting (Figure 4).

View larger version (74K): [in this window] [in a new window]   Figure 4. Western blot for GRP78, calreticulin, calnexin and PDI in lungs of individual smokers and nonsmokers. (A) Results for individual subjects are shown as separate lanes. (B) Group mean ± SE data of bands scanned and quantitated. *P < 0.05 compared with nonsmokers.

Phosphoproteome
Additional gels performed at pI 4 to 7 two-dimensional gels, which were stained for phosphoproteins, demonstrated four differentially expressed spots in the lungs of smokers (P < 0.05), one of which was not previously identified in the total protein stain (Figure 5). Of the four proteins, three were up-regulated and one was down-regulated. The three up-regulated phosphoproteins (elongation factor-1β, elongation factor-1, and 60S acidic ribosomal protein P2) were also identified in gels stained for total proteins (Table 2). In contrast, the one phosphoprotein not detected in the gels stained for total protein, calgranulin-B/S-100 A9, was down-regulated. Interestingly, all four proteins were annotated as phospho-serine proteins in Swiss-Prot protein knowledge database.

View larger version (16K): [in this window] [in a new window]   Figure 5. Differentially expressed phosphoproteins. The two-dimensional gels (pI 4–7) of smokers and nonsmokers were prepared as described in MATERIALS AND METHODS. Phosphoproteins were labeled using Pro-Q diamond staining. The three up-regulated proteins identified were: (1) 60S acidic ribosomal protein P2, (2) elongation factor 1-β, and (3) elongation factor 1-. The decreased phosphoprotein (4) was protein S100-A9.

View larger version (27K): [in this window] [in a new window]   Figure 6. Effect of cigarette smoke extract (CSE) on GRP78 protein expression in cultured 16 HBE cells. Cells were treated with a range of CSE concentrations for 24 hours. GRP78 levels were assessed by Western blotting. (Top) One experiment representative of five. (Bottom) Group mean ± SEM data of five experiments of bands scanned and quantitated. Note CSE concentration-dependent increase in GRP78 levels (*P = 0.002 by one-way ANOVA).

View larger version (78K): [in this window] [in a new window]   Figure 7. Effect of CSE on eIF2 phosphorylation and nuclear expression of ATF4 and Nrf2 in cultured 16 HBE cells. eIF2, ATF4, and Nrf2 were assessed by Western blotting. (A) CSE increased phosphorylated eIF2 in the cytoplasmic fraction without affecting total eIF2. One experiment representative of four. (B) CSE (30%) increased Nrf2 and ATF4 in the nuclear fraction. Lamin A/C was used as a loading control for nuclear protein. Cells were harvested at 6 hours of exposure to CSE, tBHQ (100 µM), or TG (0.3 µM). tBHQ = tert-butyl hydroquinone, an organic oxidant known to activate Nrf2 (44). TG = thapsigargin, a known activator of ATF4 (21). One experiment representative of two.

	DISCUSSION

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The lungs studied were obtained from donors who had been scrutinized by history and chest X-ray and felt to be free of chronic lung disease. The lung was preserved in the usual manner for transplanted organs and quick-frozen within 12 hours of removal from the body. The region of lung sampled was macroscopically and microscopically normal and standardized to minimize variability in cell composition across subjects. However, given the manner in which the lungs were obtained, lung function data (FEV₁, FVC, etc.) were not available in any of the subjects. Hence, we cannot rule out the possibility that some subjects may have had COPD. However, if present, COPD was almost certainly mild, since it was not associated with symptoms or abnormalities in chest X-ray or histology.

To our knowledge, the results of the present study are the first description of the proteome of the normal human lung and the effects of chronic cigarette smoking on that proteome.

The results indicate differential expression in the smoking group of at least 26 proteins in the two-dimensional gel electrophoresis approach stained for total proteins and 4 differentially expressed phosphoproteins.

The differentially expressed proteins detected by these methods included proteins classically involved in the unfolded protein response. These included major effectors that define the UPR and enhance the capacity for protein folding: GRP78, calreticulin, calnexin, and PDI (18–20, 26–28). In fact, GRP78, calreticulin, and calnexin are ER chaperones and calcium-binding proteins that stabilize nascent proteins until they are properly folded or targeted for degradation by ER-associated degradation mechanisms (18, 19, 26).

GRP78 is believed to be the key molecule in the regulatory system that initiates a UPR because it complexes with the three resident ER membrane proteins that sense misfolded proteins in the ER lumen and activate the UPR. These three sensors are: (1) PERK; (2) the proto-transcription factor, ATF6; and (3) the combined kinase/RNA ribonuclease, IRE-1 (18, 19, 26, 28). While the precise mechanism by which the three sensors are activated is not completely understood, it has been postulated that a higher affinity of GRP78 for unfolded proteins in the ER lumen causes dissociation of GRP78 from the luminal surface of the sensors, thereby allowing them to become activated (18, 19, 26, 28).

In contrast to GRP78, calreticulin and calnexin are ER lectins, which bind most monoglucosylated glycoproteins either separately or jointly, and present them to ER foldases of the PDI family (18, 19, 26). PDI in conjunction with the FAD-dependent oxygenase, ER oxidase 1, promotes protein folding by enhancing disulfide bond formation (18, 19, 26). Of interest, in the process of thiol oxidation, PDI ultimately produces the reactive oxygen specie, H₂O₂, perhaps explaining why antioxidant defense genes are downstream targets of the UPR (18, 22, 26, 43).

GRP78, calreticulin, and PDI levels were significantly less in the lungs of ex-smokers compared with active smokers, suggesting that the unfolded protein response induced by cigarette smoking is at least partially reversible with smoking cessation.

Follow-up experiments in cultured human airway epithelial cells (16-HBE cells), the lung cell type with the greatest smoke exposure, demonstrated that cigarette smoke extract up-regulated GRP78 at 24 hours. These results indicate that a UPR is induced in airway epithelial cells by the aqueous and particulate material present in cigarette smoke and that the UPR occurs rapidly and in a CSE concentration–dependent fashion.

Studies in 16-HBE cells also confirmed activation of the ER kinase, PERK, by demonstrating rapid phosphorylation of eIF2 and increased nuclear expression of ATF4 and Nrf2. Of interest, although phosphorylation of eIF2 inhibits protein translation globally, it augments expression of selected proteins like ATF4, GRP78, and possibly Nrf2 by enhancing translation of their transcripts (18, 19, 21, 31). PERK also directly phosphorylates Nrf2, thereby releasing it from its cytoplasmic inhibitor, Kelch-like ECH–associated protein 1 (Keap1) and allowing translocation to the nucleus (32, 33).

Of particular interest in the setting of cigarette smoke exposure, ATF4 and Nrf2 bind to antioxidant response elements in the promoter region of a variety of genes coding for important antioxidant enzymes (e.g., heme oxygenase-1, glutathione-S-transferase, glutathione peroxidase, superoxide dismutase, etc.) (12, 34–36). In fact, Nrf2 regulates genes involved in two major redox systems, the glutathione and thioredoxin systems, by promoting expression of enzymes involved in glutathione synthesis, transfer, and reduction and thiodoxin synthesis and reduction (12, 35). Several Nrf2-regulated enzymes, both glutathione dependent (e.g., UDP-glucuronosyl transferase) and glutathione independent (e.g., NAD(P)H:quinone oxidoreductase1), are also important in the detoxification of tobacco smoke products (6, 12, 24, 36, 44).

Direct evidence of the importance of Nrf2 in the pathogenesis of cigarette smoke–induced lung inflammation and emphysema has been provided in animal models and the protection of lung cells against oxidant injury (12, 37). For example, Nrf2 knockout mice demonstrate enhanced susceptibility to cigarette smoke–induced emphysema and lung inflammation compared with wild-type mice (12, 36, 37). Moreover, type II pneumocytes from Nrf2 knockout mice demonstrate impaired growth and increased sensitivity to oxidant-induced cell death (38).

In the present study, lungs from the chronic smokers demonstrated up-regulation of the Nrf2-regulated antioxidant enzyme, mitochondrial thioredoxin-dependent peroxide reductase (12). Thioredoxin-dependent peroxide reductase scavenges mitochondrial hydrogen peroxide by NAPDH-dependent oxidation of the two thiol groups in thioredoxin, the ubiquitous intracellular reducing agent (11). Increases in thioredoxin peroxide reductase would be expected, therefore, to prevent accumulation of oxidized thioredoxin, redox imbalance in the mitochondria, and cytochrome c–induced apoptosis (45–47).

As mentioned, the UPR response involves depression of overall protein translation while selectively enhancing translation of selected proteins (18, 19, 26). It is of interest, therefore, that four of the differentially expressed proteins in chronic smokers are involved in translation and ribosome formation (60S acidic ribosomal protein P2, heat shock protein 27, and elongation factors-1β and -1). In particular, heat shock protein 27 inhibits formation of the large and small ribosomal complex (48), and 60S acidic ribosomal protein P2 associates with elongation factor-2 to form the large and small ribosomal complex (38, 39). Of note, 60S acidic ribosomal protein P2 is increased when the UPR is induced by hypoxia, a condition associated with a global inhibition of protein synthesis (31, 49).

Protein folding and the correction of protein misfolding is an ATP-dependent process. It is of interest, therefore, that several enzymes involved in energy synthesis (glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, and ATP synthase subunit beta) were up-regulated in the lungs of chronic cigarette smokers. These may represent downstream targets of the UPR.

Finally, a protein with inflammatory activity, S100-A9/calgranulin C, a member of the S100 family of EF hand calcium-binding proteins, was down-regulated in the lungs of chronic smokers. Among other targets, S100-A9 binds the transcription factors NF-B and P53, and activates the P38 and ERK MAPK kinases and the receptor for advanced glycated end products (RAGE) (50). Down-regulation of S100-A9 suggests a tilt toward an anti-inflammatory response in the smokers. To our knowledge, the relationship of S100-A9 to the ER stress response is unstudied.

The present study did not identify the nature of the stimulus/stimuli that initiated the UPR in smokers. However, a variety of organic and inorganic oxidants induce a UPR by increasing cytosolic calcium and interfering with protein folding directly (21, 51–53). It seems possible, therefore, that ROS present in cigarette smoke may induce a UPR in the lung directly. In addition, nicotine per se induces a UPR response in several cell types, presumably by increasing cytosolic calcium (29, 30).

The overall consequences of the UPR in the lung of smokers were not elucidated in this study. However, the pattern of proteins affected—namely, up-regulation of antioxidant/xenobiotic defense molecules (i.e., ATF4, Nrf2, and mitochondrial thioredoxin-dependent peroxide reductase)—suggest that the UPR may increase the capacity of the lung to maintain redox homeostasis and degrade xenobiotic material contained in cigarette smoke. In fact, induction of a UPR in the setting of oxidant stress promotes cell survival, whereas knockdown or knockout of GRP78, calreticulin, or PDI increases ROS burden and promotes cell death (21, 52, 53). Moreover, activation of a UPR may decrease the inflammatory response to cigarette smoke. In fact, inhibition of the UPR in IRE-1B knockout mice promotes colonic inflammation in response to application of irritant chemicals to the colonic mucosa (54). Accordingly, cigarette smoke–induced activation of a UPR response may be a protective mechanism defending the lung against the deleterious effects of cigarette smoke. If so, differences in the magnitude of a UPR could contribute, in part at least, to observed individual differences in susceptibility to cigarette smoke–induced lung diseases like COPD. The sample size in this study was relatively small (three in each group), however. Accordingly, larger population studies will be required to test these concepts by characterizing the effects of cigarette smoke on the UPR in the lung and the role of the UPR in the susceptibility to cigarette smoke–induced lung disease.

In summary, the present study indicates that a number of proteins are differentially regulated in the lungs of cigarette smokers. These include proteins involved in the unfolded protein response (GRP78, calreticulin, calnexin, and PDI) and several downstream targets involved in defense against antioxidant/xenobiotic injury, protein synthesis and degradation, inflammation, and cell structure.

	Footnotes

 
^* These authors contributed equally to this work.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0221OC on December 13, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form June 13, 2007

Accepted in final form November 7, 2007

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