Jan 22, 2010 - ... formerly g-glutamylcysteine synthase heavy subunit) and glutamate cysteine ligase modifier (GCLM, formerly g-glutamylcysteine synthase, ...
Cellular Response to Cigarette Smoke and Oxidants Adapting to Survive Andre´ M. Cantin1 1
Pulmonary Division, Department of Medicine, Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke, Quebec, Canada
The gaseous and soluble phases of cigarette smoke are sources of oxidants that contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD). Chronic oxidative stress of cigarette smoking induces mucus secretion and inhibits cystic fibrosis transmembrane conductance regulator function. The increased mucus viscosity renders the airways susceptible to bacterial infections, a hallmark of chronic bronchitis. Furthermore, lungs chronically exposed to the toxic mixture of oxidants in cigarette smoke show signs of endoplasmic reticulum stress, unfolded protein response, altered ceramide metabolism, and apoptosis. Fortunately, the respiratory tract has developed effective adaptive cellular mechanisms to limit oxidant damage. Numerous antioxidant enzymes and glutathione-dependent detoxification systems are increased in healthy smokers. The regulation of the antioxidant response is largely dependent on the nuclear factor erythroid 2–related factor2 (Nrf2) pathway. However, patients with COPD have defective Nrf2 responses. Novel therapies such as 2-cyano-3,12-dioxooleana-1,9dien-28-oic acid (CDDO) to correct defective Nrf2-dependent cellular response may hold promise for patients with COPD. Keywords: oxidants; epithelium; glutathione; chronic obstructive pulmonary disease; Nrf2
Cigarette smoke exposure is the leading cause of chronic obstructive pulmonary disease (COPD) (1). Cigarette smoke is a strong oxidant. The airway, alveolar cell, and molecular biology changes induced by cigarette smoke reflect an elaborate, coordinated response allowing the lung to adapt and survive. Researchers have made great strides in defining the key pathways by which the respiratory tract adapts to oxidative stress. Their work has also uncovered some critical failures in adaptive responses that may increase the risks of developing COPD. The progress made in our understanding of cellular responses to cigarette smoke has opened exciting new areas for the development of therapeutics in COPD.
OXIDANTS AND ANTIOXIDANTS Oxidants can be simply defined as molecules with the potential to subtract electrons from other molecules. A molecule that gains an electron is reduced, and one that loses an electron is oxidized. Many oxidant species are in the form of free radicals, meaning that they have one or more unpaired electrons. Other oxidants do not have unpaired electrons but are highly susceptible to reduction, a process that oxidizes electron donor molecules such as thiols. Such oxidants include oxygen and hydrogen peroxide (Figure 1). The single-electron reduction of
(Received in original form January 22, 2010; accepted in final form August 2, 2010) Supported by a grant in aid of research from the Canadian Cystic Fibrosis Foundation. Correspondence and requests for reprints should be addressed to Andre´ M. Cantin, M.D., Pulmonary Division, Faculty of Medicine, University of Sherbrooke, 3001, 12th Avenue N, Sherbrooke, PQ, J1H 5N4 Canada. E-mail: Andre.Cantin@ USherbrooke.ca Proc Am Thorac Soc Vol 7. pp 368–375, 2010 DOI: 10.1513/pats.201001-014AW Internet address: www.atsjournals.org
oxygen forms the free radical superoxide, a reaction that is catalyzed by a variety of cellular oxidases, particularly NADPH oxidase in the plasma membranes of macrophages and polymorphonuclear cells (2). Cigarette smokers have increased numbers of alveolar macrophages, which release high levels of superoxide (3). Superoxide further undergoes reduction with one electron to form hydrogen peroxide, a process that occurs either spontaneously or is catalyzed by superoxide dismutase. The amounts of superoxide and hydrogen peroxide formed by phagocytes at the alveolar surface of the smoker’s lungs are sufficient to inactivate a1-antitrypsin, a major human leukocyte elastase inhibitor that plays a key role in preventing emphysema (4, 5). Surprisingly however, mice without functional NADPH oxidase are not protected against cigarette smoke–induced emphysema (6). Hydrogen peroxide in the presence of a reduced transition metal, particularly iron (Fe21), is rapidly converted through the Fenton reaction to the highly reactive and toxic hydroxyl radical (OH) (7). The formation of OH can be prevented by the ferroxidase activity of proteins such as ceruloplasmin that keep iron in its oxidized form (Fe31) (8). However, superoxide is a potent iron-reducing agent allowing the formation of Fe21, which catalyzes the formation of OH from hydrogen peroxide, a chain of events known as the HaberWeiss reaction. Not only do cigarette smokers have increased numbers of alveolar phagocytes that generate high levels of reduced oxygen species, but their alveoli also contain increased amounts of iron that facilitate the Fenton and Haber-Weiss reactions (9).
CIGARETTE SMOKE AND OXIDANTS Cigarette smoke is an exceptionally rich source of oxidants both in the gaseous and water-soluble phases (10, 11). The presence of unpaired electrons can be detected by electron spin resonance (ESR) (11). The spin trap N-t-butyl-a-phenylnitrone forms cigarette smoke radical adducts that produce a quantifiable ESR spectrum. It has been estimated that cigarette smoke contains 1015 radicals per puff and 1017 ESR-detectable radicals per gram of tar (12). Furthermore, tobacco leaf pyrolysis generates more than 3,000 aromatic compounds, semiquinones/ quinones, iron, and oxides of nitrogen derived from nitrates (10, 12). Among the most significant sources of oxidants are longlived semiquinone radicals present in the tar of cigarette combustion products. The quinone, hydroquinone, and semiquinone system reduces oxygen to superoxide that dismutates to H2O2, and in the presence of iron forms the OH radical (10). Soluble semiquinone can be extracted from the tar of cigarette smoke and is sufficient to oxidatively inactivate a1-antitrypsin (13). Some investigators have found oxidized a1-antitrypsin in the bronchoalveolar lavage fluid of cigarette smokers (4). High concentrations of 8-hydroxydeoxyguanosine, an indicator of oxidized DNA, have also been observed in the bronchoalveolar lavage fluid of cigarette smokers, and found to correlate with the number of cigarettes smoked per day (14). These results indicate that cigarette smoking induces measurable oxidative stress in the lung.
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Figure 1. Illustration of oxidant and antioxidant pathways. GSSG 5 oxidized glutathione.
ANTIOXIDANTS One of the more striking observations is that the lungs of many chronic cigarette smokers seem to function normally despite being heavily exposed to such potent oxidants. Although many factors likely protect the airways of smokers, one of the most important sources of protection is the antioxidant armamentarium of the respiratory epithelium. Among the major antioxidants are the enzymes superoxide dismutase, catalase, and the glutathione (GSH)-related systems. Superoxide dismutases are both cellular (SOD1, CuZnSOD in the cytosol; SOD2, MnSOD in the mitochondria) and extracellular (SOD3, a high molecular weight CuZnSOD, also known as EC-SOD). Superoxide dismutases catalyze the single-electron reduction of superoxide to H2O2 that then undergoes a two-electron reduction to oxygen and water by catalase. Interestingly, SOD may play a key role in preventing some of the key steps involved in the pathogenesis of emphysema associated with chronic smoke exposure. Cigarette smoke induces lung cell ceramide accumulation that in turn induces superoxide release and apoptosis (15). The cigarette smoke–induced apoptosis cascade can be prevented by SOD1, a gene target of nuclear factor erythroid 2–related factor-2 (Nrf2) (discussed below) (16). Another major adaptive antioxidant system involves GSH, the most abundant thiol in biological systems (17). Glutathione is a tripeptide synthesized by most living organisms. It occurs in reduced and disulfide (GSSG) forms, and as mixed disulfides with various proteins, leukotrienes, and other metabolites. Glutathione is composed of glutamate linked through its carboxylate side chain to the amine group of cysteine, which is in turn linked through a peptide bond to glycine. Although it is a rather simple peptide, GSH synthesis is highly regulated and involves three genes and two enzymes (18). Glutamate–cysteine ligase catalytic (GCLC, formerly g-glutamylcysteine synthase heavy subunit) and glutamate cysteine ligase modifier (GCLM, formerly g-glutamylcysteine synthase, light subunit) are modified post-translationally to form the rate-limiting enzyme in glutathione synthesis, glutamate–cysteine ligase (GCL) (19). The genes of both of these enzymes are under the transcriptional control of Nrf2 (20, 21). A third gene is needed to synthesize the enzyme GSH synthase (GS; formerly GSH synthetase), also induced by Nrf2 (22). Glutathione alone can scavenge free radicals; however, it is most efficient when acting
in concert with GSH peroxidase (GPX), GSH reductase (GSR), and the hexose monophosphate shunt system that regenerates NADPH, the electron donor needed to reduce GSSG. Glutathione is also an essential component of the Nrf2-inducible glutathione-S-transferase system that catalyzes the conjugation of GSH with endogenous and exogenous electrophilic compounds (23). Glutathione-conjugated electrophiles can be exported from cells through multidrug resistance–associated proteins (MRPs) (24). Genes that encode the MRPs also have an antioxidant response element in their 59-flanking region that is induced by binding to Nrf2 (25). Evidence that GSH plays a key role in human airway biology stems from numerous in vitro and in vivo observations (26–31). Acute exposure of airway epithelial cells to cigarette smoke depletes airway cell GSH (32). Airway cell GSH depletion is likely related to the formation of glutathione–aldehyde derivatives that cannot be reduced (33). However, a distinction probably needs to be made between the acute effects of cigarette smoke in vitro and the adaptive response of the respiratory epithelium to chronic cigarette smoke exposure in the human lung. Healthy nonsmokers have 100 times more GSH in their alveolar epithelial lining fluid than in plasma (34). Strikingly, the epithelial lining fluid GSH levels are significantly higher in healthy chronic smokers than in nonsmokers. Almost all GSH is in the reduced state. Furthermore, the expression of at least four genes related to GSH synthesis and metabolism (G6PD, GCLC, GPX, GSR) is increased between 1.5- and 5-fold in the small airway epithelial cells of cigarette smokers when compared with nonsmokers (35). These results strongly suggest that the modification of GSH synthesis and metabolism, in which Nrf2 plays a major role, is a key adaptive response to protect the respiratory epithelium against cigarette smoke exposure.
Nrf2 AND CIGARETTE SMOKE A critically important cellular response to cigarette smoke is the Nrf2 pathway (36). Nrf2 is normally bound to its inhibitor, Kelch-like ECH-associated protein-1 (KEAP1). Oxidants present in cigarette smoke will change the conformation of KEAP1 and prevent it from inhibiting Nrf2 (Figure 2). Once activated, Nrf2 is stabilized by DJ-1, a protein that prevents KEAP1mediated Nrf2 degradation in the proteasome (37). DJ-1 is
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Figure 2. Summary of the cellular responses to cigarette smoke in health and disease. KEAP1 changes conformation as cysteine residues are oxidized and Nrf2 is free. Nrf2 binds to the antioxidant response elements of several key antioxidant genes and increases their transcription. This response to cigarette smoke is observed in the lungs of healthy smokers and likely is protective. However, some smokers with COPD have a defective Nrf2 response with deficient DJ1 protein, a protein needed to stabilize Nrf2. Nrf2 insufficiency could lead to a decrease in the expression of several antioxidant genes, and may contribute to the development of COPD. ATF6 5 activating transcription factor-6; COPD 5 chronic obstructive pulmonary disease; G6PD 5 glucose-6-phosphate dehydrogenase; GCL 5 glutamate–cysteine ligase; GPX 5 GSH peroxidase; GSH 5 glutathione; KEAP1 5 Kelch-like ECH-associated protein-1; MUC5AC 5 mucin 5AC; NF-kB 5 nuclear factor-kB; Nrf2 5 nuclear factor erythroid 2–related factor2; PKC 5 protein kinase C; PMN 5 polymorphonuclear cells; SOD1 5 superoxide dismutase-1; UPR 5 unfolded protein response.
susceptible to oxidative inactivation, and can be affected by cigarette smoke exposure. Alterations in Nrf2–KEAP1 equilibrium have been reported in the lungs of patients with pulmonary emphysema (38, 39). Furthermore, patients with COPD demonstrate markedly decreased levels of DJ-1 (40). In the absence of functional DJ-1, the lungs of patients with COPD show decreased Nrf2 and Nrf2-dependent antioxidant gene expression as well as glutathione. Stabilizing Nrf2 by transfecting small interfering RNA against KEAP1 restores Nrf2 and Nrf2-dependent antioxidant gene expression in the DJ-1– disrupted cells (40). Furthermore, activation of Nrf2 in Clara cell–specific KEAP1 knockout mice markedly protects lung cells against oxidative stress in vivo (41). These results point to the importance of the Nrf2-dependent adaptive response in preventing smoke-induced COPD. Nrf2 is therefore a logical therapeutic target. Pharmacological agents aimed at increasing Nrf2 levels are available. Sulforaphane, an isothiocyanate present in high concentration in young broccoli sprouts, can restore defective Nrf2 in cultured airway cells (40). Another promising agent is 1-[2-cyano-3–12-dioxooleana-1,9(11)-dien28-oyl]imidazole (CDDO-Im). CDDO-Im is a synthetic triterpenoid with lipophilic properties and a key electrophilic carbon capable of activating Nrf2 without inducing significant nonspecific oxidation of other cellular components (42). This new class of compounds (bardoxolone, RTA 402) has been tested in patients with cancer and preliminary results indicate that it is well tolerated (43). Clinical trials are ongoing in patients with chronic kidney failure (see NCT00811889 at http://clinicaltrials. gov). Of particular interest, CDDO-Im protects Nrf21/1 but not Nrf22/2 mice against smoke-induced emphysema (44).
EFFECTS OF CIGARETTE SMOKE ON LUNG CELL SURVIVAL Increased 4-hydroxy-2-nonenal levels confirm the presence of oxidative stress in the lungs of patients with COPD (45). Chronic exposure of lung tissue to cigarette smoke initiates pathways that lead to inflammation, cell injury, protease– antiprotease imbalance, loss of interstitial matrix, and alveolar wall cell death. The increase in alveolar cell death observed in patients with COPD likely involves several mechanisms including necrosis, apoptosis, and anoikis (death by detachment from extracellular matrix) (46). Specifically, cigarette smoke has been shown to trigger both the death receptor (extrinsic, Fas
ligand dependent and independent) and mitochondrial (intrinsic) apoptosis pathways (47–49). One of the major mechanisms associated with increased epithelial and endothelial cell death in the COPD lung is a decrease in vascular endothelial growth factor (VEGF) and the VEGF receptor R2 protein (50). Tuder and colleagues have demonstrated that blockade of the VEGF receptor alone is sufficient to increase alveolar oxidative stress and apoptosis (51). Interestingly, oxidative stress, alveolar wall apoptosis, and emphysema are prevented by the superoxide dismutase mimetic M40419 (51). Furthermore, VEGF receptor blockade results in the accumulation of ceramide, a membrane sphingolipid increased in the lungs of patients with COPD that enhances oxidative stress and induces apoptosis (15). The emphysema associated with VEGF blockade can be prevented by sphingosine 1-phosphate, a prosurvival derivative of ceramide metabolism (52). Although apoptotic cells are increased in the COPD lung, cigarette smoke decreases their removal by phagocytic cells, or efferocytosis (53). Deficient efferocytosis can be restored in cigarette smoke–exposed mice by treatment with the antioxidant L-2-oxothiazolidine-4-carboxylate (procysteine) (54). A key pathway relating cigarette smoke, apoptosis, and oxidative stress likely passes through the triggering of RTP801 (also known as Redd1), a stress-related protein involved in apoptosis and oxidative stress (55, 56). RTP801 expression is increased in the lung tissues of healthy smokers and patients with COPD. RTP801 activates the TSC2 gene product, tuberin, which negatively regulates the mammalian target of rapamycin (mTOR) (57). Inhibition of mTOR decreases VEGF. Strikingly, RTP801 knockout mice are markedly protected against emphysema induced by chronic cigarette smoke exposure (57).
SUSCEPTIBILITY TO CIGARETTE SMOKE–INDUCED LUNG DISEASE The first suggestion of a molecular link between cigarette smoke, oxidative stress and emphysema was made by Janoff and colleagues, who observed that cigarette smoke inhalation inactivates a1-antitrypsin activity in the rat lung (58). Susceptibility to cigarette smoke–induced emphysema is markedly increased in individuals with an inherited deficiency of a1-antitrypsin. In addition to its capacity to inactivate a1-antitrypsin, cigarette smoke increases the lung expression of several metalloproteases
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(MMP2, 9, 12, 13, and 14) and cysteine proteases (59–61). Among these proteases, MMP12 seems to play a key role in driving emphysema, because mice lacking MMP12 are fully protected against smoke-induced emphysema (62). Alveolar macrophage MMP12 expression is increased up to ninefold in cigarette smokers (63). Interestingly, the minor allele of a singlenucleotide polymorphism in MMP12 (rs2276109 [282AG]) is associated with reduced MMP12 promoter activity through less efficient binding of the AP-1 transcription factor, and with a reduced risk of COPD (64). Investigators have also linked decreased lung function, increased risk of COPD-related mortality, and SOD3 polymorphisms (65). Several other COPD susceptibility molecular markers, some of which are clearly related to oxidant and antioxidant pathways, have been identified and include polymorphisms of genes encoding metalloproteinases, heme oxygenase-1, glutathione-S-transferases, enzymes that catalyze xenobiotic epoxides, the cytochrome P-450 superfamily, and the cystic fibrosis transmembrane conductance regulator (CFTR) (66).
CIGARETTE SMOKE AND MUCINS In addition to antioxidants the protective mechanisms of the airways include the epithelial mucus layer and the mucociliary escalator. When functioning normally this system provides protection in at least two distinct ways. First, the mucus layer acts as a barrier between the epithelial cell and toxic particles, microorganisms, gases, and allergens present in the 8,000 L of air we breathe in every day. One can add to this list the host proteases released during episodes of acute bronchitis, and hydrochloric acid associated with gastroesophageal reflux. Second, mucus facilitates the clearance of unwanted materials from the airways through the action of cilia at the airway epithelial surface. The barrier and clearance functions of airway mucus may require different rheological properties. Regulation of mucus rheology is therefore critically important. The key to the regulation of mucus rheology lies largely in the structure of secreted airway mucins and in the function of CFTR. Mucins represent the most abundant protein family within mucus lining epithelial airways (67). The mucin superfamily comprises many genes encoding glycoproteins that are characterized by the presence of several proline, threonine, and serine (or PTS) domains. The PTS domains form the sites to which abundant oligosaccharide side chains are attached through
Figure 3. Effects of cigarette smoke exposure on airway epithelial cells and mucus. Oxidants within cigarette smoke increase MUC5AC gene transcription, activate protein kinase C to induce mucin granule exocytosis, and stimulate goblet cell and submucosal gland hyperplasia, all of which increase the abundance of airway mucus. MUC5AC 5 mucin 5AC; PKC 5 protein kinase C.
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glycosidic bonds. The sugars present on the mucin proteins represent more than 70% of the glycoprotein mass, give mucins their hygroscopic (water-absorbing) properties, and are heavily sialylated and sulfated. The latter property provides mucins with a net negative charge at physiological pH. Secreted mucins are also rich in cysteine residues that form cysteine knots contributing to mucin polymerization. Inflammation, oxidative stress, and proteases have been linked to goblet cell hyperplasia and submucosal gland hypertrophy accompanied by hypersecretion of mucins (68–70). Oxidants induce goblet cell hyperplasia, increase polymeric mucin gene transcription, and stimulate protein kinase C (PKC)-dependent exocytosis of mucin proteins that are stored in specialized granules (Figure 3) (71, 72). PKC comprises an Nterminal regulatory domain and a C-terminal catalytic domain. The N-terminal domain has an autoinhibitory sequence known as the pseudo-substrate (73). Oxidative stress suppresses the autoinhibitory function and activates PKC. A major PKC substrate is myristoylated alanine-rich C kinase substrate (MARCKS), a key molecule in the regulation of mucus exocytosis by human airway epithelial cells (74, 75). The C-terminal catalytic domain of PKC contains free cysteines that can react with a variety of antioxidants such as polyphenols, seleno compounds, vitamin E succinate, and tocopheryl quinone to inactivate PKC (73). Mucins have been suggested to have some oxidant-scavenging properties, particularly with respect to hydroxyl radical and hypochlorous acid; however, the physiological importance of such a scavenging role is unknown (76–78). Another possible role of cysteine-rich mucins may be to provide amino acid precursors for airway epithelial GSH synthesis. Such a role has been described for albumin (79). Albumin is a cysteine-rich protein shown to be an essential component of serum in preserving GSH synthesis by several cell types. Members of the mucin superfamily, encoded at least 17 MUC genes, are mostly cell-tethered mucins (80). The genes
Figure 4. Prolonged cigarette smoke exposure suppresses CFTR. The UPR activates ATF6, which decreases cftr gene transcription. CFTR protein and function are also decreased by cigarette smoke exposure. Simultaneously, oxidative stress activates the Nrf2 transcription factor that binds to the antioxidant response element of several antioxidant genes such as GLC, the rate-limiting enzyme in glutathione synthesis. ATF6 5 activating transcription factor-6; CFTR/cftr 5 cystic fibrosis transmembrane conductance regulator protein/gene; GCL 5 glutamate– cysteine ligase; Nrf2 5 nuclear factor erythroid 2–related factor-2; UPR 5 unfolded protein response.
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encoding secreted polymeric mucins MUC2, MUC5AC, and MUC5B are located in a gene complex on chromosome 11p15.5. These mucins are packaged tightly within granules of epithelial secretory cells and kept in this compact configuration by high concentrations of calcium and a low pH. Concurrent bicarbonate secretion is essential to allow proper mucus release (81). Once mucus is secreted, the extracellular chloride and bicarbonate concentrations likely allow the mucin proteins to unfold from their highly compact state (82). The secreted mucins are hygroscopic and their physical properties will be directly defined by the water, salt, and bicarbonate present in or at the apical surface of mucous membranes (83). Each of these players is tightly controlled in the human airway by the CFTR (84, 85).
CIGARETTE SMOKE AND CFTR CFTR functions as an anion channel with highest specificity for chloride and bicarbonate. Cells expressing functional CFTR at their apical surface respond acutely within minutes to oxidative stress by markedly increasing CFTR anion permeability and chloride secretion (86–89). In contrast to the immediate effects of oxidants on CFTR function, prolonged (24 h) exposure of CFTR-expressing epithelial cells to sublethal oxidative stress induced by t-butyl hydroperoxide, taurine chloramines, or cigarette smoke results in marked suppression of CFTR gene and protein expression (Figure 4) (90, 91). Cadmium, an important component of cigarette smoke that induces oxidative stress, has also been shown to suppress CFTR, an effect that could be prevented by vitamin E (92). A decrease in airway CFTR may increase the ‘‘barrier’’ function of mucus. These changes in CFTR function occur simultaneously with an increase in GCLC transcription and GSH synthesis. GCLC transcription is increased by oxidant-mediated Nrf2 activation. The mechanism by which oxidants decrease cftr gene expression is related to the unfolded protein response (UPR)–activated ATF6 (activating transcription factor-6) (93). Furthermore, oxidants may directly interfere with protein function by altering key cysteine residues in CFTR to affect its open probability (94). Although in vitro studies provide many clues to mechanisms of oxidant-mediated cellular responses, confirmation of the relevance of such data must come from observations in humans. The nose of healthy cigarette smokers provides a window through which one can look to obtain such confirmation. The respiratory epithelium of the nose is a tissue that can be probed to study signatures of CFTR function. If one assumes that the nose of a cigarette smoker is chronically exposed to cigarette smoke, then measurements of the nasal potential difference (NPD) between the nasal mucosal surface and submucosal tissue should provide valuable information about the in vivo effects of smoking on CFTR function. The NPD signature of CF is an increased difference in the baseline NPD caused by an excessive absorption of sodium, followed by an absence of NPD changes in response to a chloride-free solution and b-agonist stimulation (95). The baseline NPD of healthy smokers is normal but CFTR stimulation in the presence of a chloridefree solution and b-agonist stimulation is greatly reduced, strongly suggesting that CFTR is dysfunctional in chronic cigarette smokers (91). The consequences of CFTR dysfunction in patients with CF include chronic cough; increased sputum production; chronic airway infection initially with Haemophilus influenzae and eventually with Staphylococcus aureus, Pseudomonas aeruginosa, and other gram-negative bacteria; chronic progressive airway obstruction; and repeated respiratory exacerbations, all features that can be found in a significant proportion of patients
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with advanced cigarette smoke–induced COPD (96). CFTR dysfunction leads to mucus dehydration, which has been clearly linked to increased susceptibility to bacterial infections and inflammation (97). The cellular changes in CFTR function may therefore place the cigarette smoker at risk of developing a condition akin to an ‘‘acquired’’ form of CF in their airways, although further work is needed to determine whether CFTR functional deficiency actually contributes to the clinical manifestations of COPD.
CIGARETTE SMOKE AND THE UNFOLDED PROTEIN RESPONSE Oxidative stress is a key link between cigarette smoke, respiratory epithelial cell responses, and COPD. Oxidative stress induces endoplasmic reticulum (ER) stress leading to the unfolded protein response (UPR) in several cell types, including the airway epithelial cell. Proteomic analysis has confirmed that cigarette smoke induces expression of the protein kinase R–like endoplasmic reticulum kinase (PERK)–dependent bZip transcription factors (98). The induction of UPR by oxidants leads to the dissociation of ER-resident signaling molecules (ATF6, Ire1 [inositol requiring1], and PERK) previously bound to the ER chaperone BiP (binding protein; also named GRP78 [78-kD glucose-regulated protein]) (99). BiP facilitates protein folding and increases markedly on initiation of UPR. BiP is a signature of the UPR. Activated ATF6 and Ire1 increase the transcription of ER chaperones involved in protein folding and in the targeting of misfolded proteins for degradation, both of which increase the probability of cell survival (100). PERK activates the phosphorylation of eukaryotic initiation factor (eIF)-2a, thus decreasing protein synthesis. The decrease in protein synthesis again plays a key role in allowing the cell to survive ER stress (101). Protein folding requires abundant disulfide binding and therefore is associated with increased oxidation in the ER environment. The proximity of the ER to mitochondria places the mitochondrial membrane proteins at increased risk of oxidative damage and opening of a mitochondrial transition pore that leads to cell death. One of the normal responses of the UPR is to adapt to this oxidative stress. This response can be initiated through Nrf2 (102). It is possible that in some cigarette smokers, the UPR initiated by ER stress is not successful in allowing the cell to survive, and Nrf2 activation is inadequate, thus contributing to the pathogenesis of cigarette smoke–induced emphysema.
CONCLUSIONS Cigarette smoke is a mix of highly concentrated soluble and gaseous electrophiles placing lungs at increased risk of protein and lipid oxidation, abnormal ceramide metabolism, ER stress, and cell death. Cells adapt to chronic cigarette smoke exposure by changing the conformation of KEAP1. Oxidant-dependent alteration of KEAP1 frees up Nrf2, a key transcription factor that increases important lung antioxidants such as GSH. Because of oxidative inactivation of the Nrf2-stabilizing protein DJ-1, some smokers may be at increased risk for COPD. Several other factors related to oxidative stress responses increase the susceptibility of some cigarette smokers to develop COPD. Airway cell adaptation to cigarette smoke also involves oxidant-dependent increases in mucin synthesis and mucus secretion and decreases in CFTR function, all changes known to favor bacterial infections as seen in patients with COPD. Novel therapies aimed at the Nrf2 pathway are now being developed and may hold promise for patients with COPD.
Cantin: Cigarette Smoke and Lung Oxidant Stress Author Disclosure: A.C. was on the Advisory Board for CSL-Behring ($1,001– $5,000) and received lecture fees from Boehringer Ingelheim International GmbH (up to $1,000). He received grant support from the Bayer Partnership Fund, the CIHR, and the Canadian CF Foundation ($50,001–$100,000).
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