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Protein Misfolding and Endoplasmic Reticulum Stress in Chronic Lung Disease
CHEST
Translating Basic Research Into Clinical Practice
Protein Misfolding and Endoplasmic Reticulum Stress in Chronic Lung Disease James Wei, BSc; Sadaf Rahman, BSc; Ehab A. Ayaub, BSc; Jeffrey G. Dickhout, PhD; and Kjetil Ask, PhD
The pathogenesis of chronic lung disorders is poorly understood but is often thought to arise because of repeated injuries derived from exposure to exogenous or endogenous stress factors. Protein-misfolding events have been observed in a variety of genetic and nongenetic chronic lung disorders and may contribute to both the initiation and the progression of lung disease through endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). Evidence indicates that exposure to common lung irritants such as cigarette smoke, environmental pollutants, and infectious viral or bacterial agents can induce ER stress and protein misfolding. Although the UPR is thought to be a molecular mechanism involved in the repair and restoration of protein homeostasis or “proteostasis,” prolonged activation of the UPR may lead to compromised cellular functions, cellular transformation, or cell death. Here, we review literature that associates protein-misfolding events with ER stress and UPR activation and discuss how this basic molecular repair mechanism may contribute to the initiation and progression of various genetic and nongenetic chronic lung diseases. CHEST 2013; 143(4):1098–1105 Abbreviations: 4-PBA 5 4-phenylbutyrate; AAT 5 a1-antitrypsin; ATF6 5 activating transcription factor 6; CF 5 cystic fibrosis; CFTR 5 cystic fibrosis transmembrane conductance regulator; CS 5 cigarette smoke; ER 5 endoplasmic reticulum; ERAD 5 endoplasmic reticulum-associated degradation; GRP78 5 glucose-regulated protein 78; IPF 5 idiopathic pulmonary fibrosis; IRE1 5 inositol-requiring kinase 1; NRF2 5 nuclear erythroid-2-related factor; PERK 5 protein kinase RNA-like endoplasmic reticulum kinase; SERCA 5 sarcoendoplasmic reticulum Ca21 pump; UPR 5 unfolded protein response; VCP 5 valosin-containing protein
endoplasmic reticulum (ER) is a membraneThebound organelle found in eukaryotic cells. It con-
sists of smooth and rough sections: The former is responsible for the synthesis of lipids, metabolism of carbohydrates, and detoxification of xenobiotics, whereas the latter synthesizes proteins for integration into the cell membrane, use by other organelles, or secretion.1 Approximately one-third of proteins assembled in the cell travel through the rough ER (Fig 1), where they undergo folding and posttranslational modifications.1,2 Because proteins are involved Manuscript received August 28, 2012; revision accepted October 10, 2012. Affiliations: From the Department of Medicine (Messrs Wei and Ayaub, Ms Rahman, and Drs Dickhout and Ask), Department of Biochemistry and Biomedical Sciences (Dr Ask), and McMaster Immunology Research Center (Mr Ayaub and Dr Ask), McMaster University; Firestone Institute for Respiratory Health (Messrs Wei and Ayaub, Ms Rahman, and Dr Ask), St. Joseph’s Healthcare; and Hamilton Centre for Kidney Research (Drs Dickhout and Ask), Hamilton, ON, Canada.
in a variety of biologic processes, their “proteostasis” (ie, their efficient synthesis, folding, and adequate degradation) is fundamental to ensuring optimal cellular function and maintaining tissue homeostasis. For appropriate protein production to occur, it is critical that (1) the physiochemical conditions within the ER lumen are adequate and (2) its molecular transport systems are operational. The former requires the maintenance of a high Ca21 concentration, an oxidizing redox state for disulfide bond formation, and sufficient amounts of subcellular sugars and molecular chaperones, all aspects that facilitate protein folding.3 Correspondence to: Kjetil Ask, PhD, Department of Medicine, McMaster University, Firestone Institute for Respiratory Health, 50 Charlton Ave E, Room T2112, Hamilton, ON, L8N 4A6, Canada; e-mail: [email protected] © 2013 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details. DOI: 10.1378/chest.12-2133
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tional. Any external or internal perturbations of these conditions can reduce the ability of the ER to perform its native physiologic roles, causing ER stress. ER Stress in Lungs The numerous inducers of ER stress are also common causes of a wide array of lung diseases, suggesting that protein-misfolding events may serve as a central unifying mechanism. Evidence indicates that ER stress can be induced by common pathogens to which the lungs are exposed, such as viral and bacterial infections,4 environmental pollution,5 chemical compounds,6 and cigarette smoke (CS)7 (Fig 2). ER stress may also be triggered by a default in the molecules associated with the secretory pathway and in the ERAD pathway8 or by the trapping of misfolded proteins in the ER as a result of the genetic mutations discussed later. Aging may also contribute to heightened ER stress because of a loss of chaperones and folding molecules localized in the ER.9 Unfolded Protein Response
Figure 1. The secretory pathway. mRNA is transcribed from DNA and posttranscriptionally modified and exported out of the nucleus (1). Proteins that are destined to be secreted, embedded in the plasma membrane or membranes of the Golgi complex, lysosomes, and endosomes have the mRNA targeted for translation in the rough endoplasmic reticulum (RER). In the RER, newly translated proteins are folded into their three-dimensional state (2) and transported to the Golgi complex in transporting vesicles for posttranslational modifications (3). Mature protein exits the Golgi in vesicles and is either sent to its final destination in a membrane or shipped via exocytosis to the extracellular space (4). Proteins that reach the Golgi and are recognized as not properly folded can be transported back to the endoplasmic reticulum for reprocessing (5) or targeted to the endoplasmic reticulumassociated degradation pathway and sent to the proteasome for degradation.
The latter requires the secretory pathway and the ER-associated degradation (ERAD) pathway (which recycles defective molecules) to be intact and func-
The unfolded protein response (UPR) is an evolutionarily conserved biochemical pathway that restores cellular homeostasis when the ER is stressed.10 The UPR acts to resolve the accumulation of misfolded proteins that compromise ER function. It acts when unfolded proteins lead to the activation of three known ER transmembrane transducers: activating transcription factor 6 (ATF6), inositol-requiring kinase 1 (IRE1), and protein kinase RNA-like endoplasmic reticulum kinase (PERK). PERK activation can reduce global protein production by inducing the phosphorylation of eukaryotic translation initiation factor 2a. ATF6 and IRE1 activation increases the expression of chaperones to increase protein-folding capacity, and IRE1 activation increases ERAD protein expression, allowing greater misfolded protein degradation, all of which assist in restoring ER homeostasis11 (Fig 3). The UPR can also stimulate programs aimed at increasing the surface area and volume of the ER, enhancing the cellular capacity for folding and processing12 and it has been shown to have a key role in cellular differentiation processes.13 If these initiatives do not restore cellular homeostasis, the UPR can ultimately promote cell death. Thus, it is not surprising that persistent deregulation of the UPR can contribute to a wide range of diseases, including various neurodegenerative, cardiovascular, and metabolic diseases. In this article, we discuss the current knowledge of the implications of prolonged ER stress and UPR in the development and progression of chronic lung diseases,
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diseases of uncertain causes, such as COPD, idiopathic pulmonary fibrosis, and asthma. Cystic Fibrosis
Figure 2. Contributors to ER stress. Mutations in DNA (1) or defaults in the transcription machinery (2) may lead to the production of incorrectly coded mRNA sequences that may have replaced, frame-shifted, or deleted nucleotide sequences (3). Ultimately, this may lead to the translation of mutant proteins that may not fold properly in the ER. Appropriate levels of molecules must be present in the ER to maintain an optimal folding environment. This includes high Ca21 concentrations, oxidizing redox state, and sufficient amounts of sugars and molecular chaperones (4). The production of unfolded or improperly folded proteins is a hallmark of ER stress (5). Improperly folded proteins may still proceed through the secretory pathway (6). However, the cell will attempt to refold the protein when possible. If this cannot be achieved, the mutant proteins are targeted to the proteasome for degradation and recycling (7). ER 5 endoplasmic reticulum.
including the genetic disorders cystic fibrosis (CF), a1-antitrypsin (AAT) deficiency, and pulmonary surfactant mutation-induced interstitial lung disease, and
Approximately 90% of CF cases are at least partially attributable to the DF508 mutation on the ion channel protein, cystic fibrosis transmembrane conductance regulator (CFTR).14 This mutation results in a misfolded CFTR protein that is primarily targeted for degradation through the proteasome. The fraction that reaches the apical surface of epithelial cells has reduced function, and the associated compromised transmembrane ion transport and deficient mucociliary clearance have severe implications in the airway, causing chronic pulmonary obstruction and increased chances of infection. Chronic airway inflammation in patients with CF has been largely attributed to this increased susceptibility to infections, but studies have demonstrated that even in the absence of any bacterial, fungal, or viral insult, an ER stress-related inflammatory response occurs, suggesting a plausible role of the ER stress response in initiating and propagating inflammation in CF lung disease.15 Furthermore, Kerbiriou et al16 have illustrated that the UPR markers glucose-regulated protein 78 (GRP78) and ATF6 are induced in human cells transfected with DF508-CFTR. The converse has been shown to occur as well, where UPR induction reduced the expression of wild-type CFTR at the transcriptional, translational, and maturational levels in various cell lines,17 suggesting that CF-like symptoms in pathologies with nonmutant CFTR (eg, chronic lung inflammation) may be regulated through the UPR. The research focus of CF treatment strategies has shifted from addressing symptoms to targeting the molecular components involved in maintaining proteostasis. For instance, the chemical chaperone sodium 4-phenylbutyrate (4-PBA) improves trafficking of both DF508- and wild-type CFTR in bronchial epithelial cells.18 Furthermore, selective inhibition of ERAD through interference with valosin-containing protein (VCP), a component of the ER export and proteasomal degradation machinery, was shown to rescue DF508-CFTR from ERAD and increase its cell surface expression in human bronchial epithelial cells.19 Further research is necessary to develop treatment strategies centered on resolving misfolding events and/or regulating the UPR to correct for CF lung disease. AAT Deficiency AAT, produced mainly in the liver, is the most prevalent antiprotease found in serum. When transported
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Figure 3. ER stress and the unfolded protein response. A, In addition to the internal cellular contributors shown in Figure 2, external pathogens and toxins, such as bacterial and viral infections, cigarette smoke, or other environmental components originating from the air or through the circulation, may induce ER stress in the lungs. B, This might result in an accumulation of misfolded proteins in the ER, leading to a distended ER and downstream activation of the unfolded protein response (UPR) through the PERK, IRE1, and ATF6 pathways. The UPR may be activated by dissociation of GRP78 from the three ER transmembrane-bound molecules, PERK, IRE1, and ATF6, or through binding of misfolded proteins directly to PERK or IRE1. The binding of GRP78 to these molecules is thought to block its activation and for this reason GRP78 is often called the master regulator of the UPR. C, The activated UPR aims to restore cell homeostasis through halting protein translation, increasing protein degradation, and elevating the expression of chaperones and other ER stress response genes. When homeostasis is not reached, the UPR might induce cell death. Taken together with its observed role in cell differentiation processes, the activated UPR is probably involved in both disease onset and progression. ATF6 5 activating transcription factor 6; ERAD 5 endoplasmic reticulum-associated degradation; GRP78 5 glucosergulated protein 78; IRE1 5 inositol-requiring kinase 1; PERK 5 protein kinase RNA-like endoplasmic reticulum kinase. See Figure 2 legend for expansion of other abbreviations.
to the lung, AAT is vital in maintaining lung integrity because its inhibition of the protease neutrophil elastase prevents degradation of the extracellular matrix in the alveolar interstitium.20 Although . 100 mutations exist in the AAT gene, the most common and severe is the Z mutation (Glu342Lys).20 Some mutants can polymerize and accumulate in the ER or be targeted for proteosomal degradation, instigating ER stress.20 AAT mutations have unique consequences in the liver and lungs. In the originating liver cells, the aggregation of mutant AAT in the ER can lead to fibrosis21 or cancer,22 and as many as 30% to 40% of patients
can expect to develop cirrhosis over their lifetime.20,21 In contrast, patients who suffer from its complete absence (null homozygotes) do not appear to develop significant liver disease,23 indicating that liver cirrhosis and cancer are most likely due to the accumulation of misfolded AAT. However, patients with the null mutation develop the most severe emphysematous changes,24 giving credence to the protease-antiprotease hypothesis that states that low levels of AAT are unable to inhibit neutrophil elastase activity, allowing for subsequent emphysematous changes. Overall, AAT deficiency showcases the deleterious effects
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of unresolved accumulation of misfolded protein and ER stress, as seen in the liver, with presentation of lung disease being a secondary effect. The definition of AAT deficiency-induced lung inflammation has expanded recently to include peripheral blood monocytes, which also express AAT. Z-AAT accumulation has been noted in these cells in Z-AAT homozygous individuals, with subsequent sustained UPR activation and the induction of proinflammatory genes,25 illustrating the involvement and contribution of protein misfolding, ER stress, and the UPR in AAT-deficiency-induced disease. The leading treatment of AAT deficiency is IV administration of AAT obtained from the plasma of healthy individuals. This line of treatment has been shown to increase plasma AAT levels to the protective threshold of 11 μmol/L in vivo. However, the effectiveness of this therapy in preventing AAT-deficiency-associated lung disease is unclear.26 Other potential treatments for correcting the lung disease aspect of Z-AAT deficiency focus on increasing secretion of Z-AAT and include 4-PBA, as well as various iminosugar compounds.27 Agents such as tauroursodeoxycholic acid and salubrinal may also serve as potential treatments of Z-AAT-associated lung disease because of their ability to modulate ER stress in monocytes and airway epithelial cells through inhibition of ER stress/UPRinduced apoptosis.28 Pulmonary Surfactant Mutation-Induced Lung Diseases Pulmonary surfactant is a complex of lipoproteins produced by alveolar type 2 cells that reduce surface tension within the lungs to optimize lung compliance and prevent collapse. Genetic mutations in one of the essential hydrophobic peptide subunits, surfactant protein C, has been shown to cause cytotoxicity known to be associated with various interstitial lung diseases.29 Specifically, mutations leading to the deletion of exon 4 or amino acid substitution L188Q result in surfactant protein C misfolding and its retention in the ER.30 This leads to ER stress, UPR-induced apoptosis, and, ultimately, epithelial cell dysfunction.31 Moreover, a mutation in the transporter molecule ABCA3 has been described in fatal respiratory distress syndrome in newborns. In vitro experiments showed that this mutant was unprocessed and trapped in the ER, whereas normal ATP-binding cassette subfamily A member 3 reached the lysosome. Treatment with 4-PBA in cultured human lung cells improved the mutants’ trafficking to lamellar body-like structures, which are involved in the secretory pathway of surfactant, indicating that protein-misfolding events may be rescued.32 Furthermore, a mutation in surfactant protein A2 exhibits the similar result of ER stress
and retention of the protein, with the addition of heterozygotes for the mutation expressing a sufficient amount of normal A2, yet disease still manifests. This suggests that it is not surfactant protein A2 insufficiency, but prolonged ER stress and UPR-activated cell death that may be the mechanistic cause behind epithelial cell dysfunction.33 COPD COPD is a progressive disease that is marked by emphysema and chronic bronchitis leading to the destruction of lung parenchyma and narrowing of the airways. As the leading cause of COPD, CS subjects the lungs to an excessive amount of reactive oxygen species, carcinogens, and free radicals, all factors known to trigger ER stress.7 In both in vitro studies in airway epithelial cells and in vivo studies in mice, CS exposure has been shown to activate key aspects of the UPR, presumably in an attempt to reduce ER stress.34 In vitro experiments have strengthened this finding by showing that CS and its aqueous extracts yield similar responses in human lung cells, suggesting a role of the ER in CS-induced COPD.35 In addition to augmenting ER stress and activating the UPR, CS also increases the levels of apoptosis.34 Studies in patients with COPD have indicated the presence of ER stress through the detection of UPR-specific markers, including GRP78, calreticulin, calnexin, and protein disulfide isomerases.36 These markers were increased in chronic smokers in comparison with nonsmokers,7 suggesting a plausible role of ER stress and prolonged UPR activation, possibly leading to cell death and disease. The UPR may mediate this activity through nuclear erythroid-2-related factor (NRF2) signaling, because NRF2 is needed to promote cell survival signals and regulate antioxidant responses37 and is a downstream target of one of the three UPR arms (PERK pathway). NRF2 expression is decreased in patients with COPD and has been suggested as a target for these patients.38 Further, the expression of VCP, a molecule that extracts proteins from the ER back to the cytosol and is also part of the ubiquitination system that tags proteins for proteosomal degradation, was witnessed to be progressively elevated in patients with more severe COPD.36 Taken together, the reduced proteasome activity but elevated ubiquitination of ER proteins seem to inhibit the ERAD function of UPR and may contribute to the chronic inflammatory phenotype seen in COPD.36 This highlights the importance of proteostasis in which expression of VCP and NRF2 are common protective responses but are characteristically altered in the pathogenesis of COPD. Experiments examining the effects of activating NRF2 and inhibiting VCP have yielded exciting results. When administered to CS-exposed human
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lung epithelial cells, sulforaphane, an activator of NRF2, reduced the cell death believed to occur by increasing degradative activity.39 Salubrinal, an ER stress inhibitor, decreased VCP expression in cell experiments, correcting the protein imbalance in acute CS-exposed mice, and is an interesting candidate for therapeutic treatment in patients with COPD.36 These observations indicate that protein misfolding and UPR activation may contribute to the pathogenesis of COPD, highlighting the need for treatment strategies centered on these pathways. Idiopathic Pulmonary Fibrosis Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by myofibroblast accumulation, extensive scarring, and continuous loss of lung function. Although the pathogenesis of IPF is not well understood, it is thought to occur from repeated epithelial injuries, epithelial-mesenchymal transition of epithelial cells to fibroblasts, and the transformation of fibroblasts into myofibroblasts that deposit excessive extracellular matrix.40 Despite the uncertainties of the disease’s origins, in fibrotic conditions of the lung and in particular in patients with IPF, an increased amount of ER stress and an increased number of UPR agents, including GRP78, ATF6, ATF4, and C/EBP homologous protein expression, as well as spliced xbox-binding protein 1 mRNA levels, have been confirmed.41 Recent publications have also indicated that ER stress and the UPR are involved in many of the molecular pathways leading to the aforementioned physiologic changes, including ER-stress-induced transforming growth factor-b expression,42 myofibroblast differentiation,43 epithelialmesenchymal transition,44 apoptosis, disruption of subcellular barriers leading to increased risks of viral infections,45 and altered surfactant production.46 TorresGonzález et al47 interestingly found that older mice, compared with younger mice, were more susceptible to g herpes virus-induced ER stress, which was associated with an increase in pulmonary fibrosis. This highlights the significance of ER stress and age in IPF, because patients are typically middle aged or older. Furthermore, experiments involving a bleomycininduced lung fibrosis model developed greater pathology when a potent chemical inducer of ER stress (tunicamycin) was added,48 reinforcing the possibility that ER stress and UPR activation may contribute to disease initiation or progression. Asthma Asthma is characterized as a chronic inflammatory disease that leads to the swelling and narrowing of the airways and symptomatically produces chest tight-
ening, wheezing, shortness of breath, and coughing. Based on genomic analysis, ORMLD3, a gene coding for an ER-localized transmembrane protein, was noted as a risk factor for asthma.49 Taken together, findings of elevated ORMLD3 expression in inflammatory response cells and knowledge that the UPR is known to activate and control various inflammatory pathways49 led investigators to look into ORMLD3, and they confirmed an association with ER stress. As mentioned previously, maintenance of a high subcellular Ca21 concentration is essential for ER function and viability. There are various pumps and channels that help mediate this process, including the sarcoendoplasmic reticulum Ca21 pump (SERCA), a membrane protein that mediates the amount of calcium entering the ER. Overexpression of ORMLD3 was found to sufficiently reduce the ER Ca21 concentration believed to occur through inhibition of SERCA, thereby activating the UPR. Meanwhile, knockdown experiments demonstrated the opposite effect and attenuated UPR activity. In addition, inadequate SERCA expression has been shown to induce airway remodeling, a hallmark of asthma pathogenesis.50 Thus, modulation of the UPR in these cells may reduce inflammatory pathways and airway remodeling, which are characteristic features of asthma. Conclusions and Future Directions Although compelling evidence suggests that prolonged ER stress and activation of the UPR are associated with disease, it remains to be elucidated how these processes contribute to disease initiation and progression. Whereas it is likely that cells already experiencing ER stress respond differently to additional stimuli, it is still unclear if the disease pathogenesis can be explained by additive exposure to various stimuli due to prolonged activation of the UPR. Increased understanding of the role of ER stress and protein misfolding in chronic lung diseases will likely yield various therapeutic prevention and intervention strategies, ranging from prevention strategies to reduce ER stress (Fig 3A), to the administration of molecules that can enhance protein folding (ie, small weight molecular chaperones) or molecules that can regulate protein degradation (ie, proteasome inhibitors). Further understanding of the implication of the ER stress/UPR axis and the interconnectedness between other molecular mechanisms involved in the disease pathogenesis are required and will ultimately enhance our ability to interfere with these complex pathways in patients suffering from various chronic respiratory diseases. Acknowledgments Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any
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companies/organizations whose products or services may be discussed in this article. Other contributions: We thank Antje Ask, MD; Mark Inman, MD, PhD; Jørn A. Holme, PhD; and Sinan Qasha, MD, for critical reading of this manuscript.
19.
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Protein Misfolding and Endoplasmic Reticulum Stress in Chronic Lung Disease James Wei, BSc; Sadaf Rahman, BSc; Ehab A. Ayaub, BSc; Jeffrey G. Dickhout, PhD; and Kjetil Ask, PhD
The pathogenesis of chronic lung disorders is poorly understood but is often thought to arise because of repeated injuries derived from exposure to exogenous or endogenous stress factors. Protein-misfolding events have been observed in a variety of genetic and nongenetic chronic lung disorders and may contribute to both the initiation and the progression of lung disease through endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). Evidence indicates that exposure to common lung irritants such as cigarette smoke, environmental pollutants, and infectious viral or bacterial agents can induce ER stress and protein misfolding. Although the UPR is thought to be a molecular mechanism involved in the repair and restoration of protein homeostasis or “proteostasis,” prolonged activation of the UPR may lead to compromised cellular functions, cellular transformation, or cell death. Here, we review literature that associates protein-misfolding events with ER stress and UPR activation and discuss how this basic molecular repair mechanism may contribute to the initiation and progression of various genetic and nongenetic chronic lung diseases. CHEST 2013; 143(4):1098–1105 Abbreviations: 4-PBA 5 4-phenylbutyrate; AAT 5 a1-antitrypsin; ATF6 5 activating transcription factor 6; CF 5 cystic fibrosis; CFTR 5 cystic fibrosis transmembrane conductance regulator; CS 5 cigarette smoke; ER 5 endoplasmic reticulum; ERAD 5 endoplasmic reticulum-associated degradation; GRP78 5 glucose-regulated protein 78; IPF 5 idiopathic pulmonary fibrosis; IRE1 5 inositol-requiring kinase 1; NRF2 5 nuclear erythroid-2-related factor; PERK 5 protein kinase RNA-like endoplasmic reticulum kinase; SERCA 5 sarcoendoplasmic reticulum Ca21 pump; UPR 5 unfolded protein response; VCP 5 valosin-containing protein
endoplasmic reticulum (ER) is a membraneThebound organelle found in eukaryotic cells. It con-
sists of smooth and rough sections: The former is responsible for the synthesis of lipids, metabolism of carbohydrates, and detoxification of xenobiotics, whereas the latter synthesizes proteins for integration into the cell membrane, use by other organelles, or secretion.1 Approximately one-third of proteins assembled in the cell travel through the rough ER (Fig 1), where they undergo folding and posttranslational modifications.1,2 Because proteins are involved Manuscript received August 28, 2012; revision accepted October 10, 2012. Affiliations: From the Department of Medicine (Messrs Wei and Ayaub, Ms Rahman, and Drs Dickhout and Ask), Department of Biochemistry and Biomedical Sciences (Dr Ask), and McMaster Immunology Research Center (Mr Ayaub and Dr Ask), McMaster University; Firestone Institute for Respiratory Health (Messrs Wei and Ayaub, Ms Rahman, and Dr Ask), St. Joseph’s Healthcare; and Hamilton Centre for Kidney Research (Drs Dickhout and Ask), Hamilton, ON, Canada.
in a variety of biologic processes, their “proteostasis” (ie, their efficient synthesis, folding, and adequate degradation) is fundamental to ensuring optimal cellular function and maintaining tissue homeostasis. For appropriate protein production to occur, it is critical that (1) the physiochemical conditions within the ER lumen are adequate and (2) its molecular transport systems are operational. The former requires the maintenance of a high Ca21 concentration, an oxidizing redox state for disulfide bond formation, and sufficient amounts of subcellular sugars and molecular chaperones, all aspects that facilitate protein folding.3 Correspondence to: Kjetil Ask, PhD, Department of Medicine, McMaster University, Firestone Institute for Respiratory Health, 50 Charlton Ave E, Room T2112, Hamilton, ON, L8N 4A6, Canada; e-mail: [email protected] © 2013 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details. DOI: 10.1378/chest.12-2133
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tional. Any external or internal perturbations of these conditions can reduce the ability of the ER to perform its native physiologic roles, causing ER stress. ER Stress in Lungs The numerous inducers of ER stress are also common causes of a wide array of lung diseases, suggesting that protein-misfolding events may serve as a central unifying mechanism. Evidence indicates that ER stress can be induced by common pathogens to which the lungs are exposed, such as viral and bacterial infections,4 environmental pollution,5 chemical compounds,6 and cigarette smoke (CS)7 (Fig 2). ER stress may also be triggered by a default in the molecules associated with the secretory pathway and in the ERAD pathway8 or by the trapping of misfolded proteins in the ER as a result of the genetic mutations discussed later. Aging may also contribute to heightened ER stress because of a loss of chaperones and folding molecules localized in the ER.9 Unfolded Protein Response
Figure 1. The secretory pathway. mRNA is transcribed from DNA and posttranscriptionally modified and exported out of the nucleus (1). Proteins that are destined to be secreted, embedded in the plasma membrane or membranes of the Golgi complex, lysosomes, and endosomes have the mRNA targeted for translation in the rough endoplasmic reticulum (RER). In the RER, newly translated proteins are folded into their three-dimensional state (2) and transported to the Golgi complex in transporting vesicles for posttranslational modifications (3). Mature protein exits the Golgi in vesicles and is either sent to its final destination in a membrane or shipped via exocytosis to the extracellular space (4). Proteins that reach the Golgi and are recognized as not properly folded can be transported back to the endoplasmic reticulum for reprocessing (5) or targeted to the endoplasmic reticulumassociated degradation pathway and sent to the proteasome for degradation.
The latter requires the secretory pathway and the ER-associated degradation (ERAD) pathway (which recycles defective molecules) to be intact and func-
The unfolded protein response (UPR) is an evolutionarily conserved biochemical pathway that restores cellular homeostasis when the ER is stressed.10 The UPR acts to resolve the accumulation of misfolded proteins that compromise ER function. It acts when unfolded proteins lead to the activation of three known ER transmembrane transducers: activating transcription factor 6 (ATF6), inositol-requiring kinase 1 (IRE1), and protein kinase RNA-like endoplasmic reticulum kinase (PERK). PERK activation can reduce global protein production by inducing the phosphorylation of eukaryotic translation initiation factor 2a. ATF6 and IRE1 activation increases the expression of chaperones to increase protein-folding capacity, and IRE1 activation increases ERAD protein expression, allowing greater misfolded protein degradation, all of which assist in restoring ER homeostasis11 (Fig 3). The UPR can also stimulate programs aimed at increasing the surface area and volume of the ER, enhancing the cellular capacity for folding and processing12 and it has been shown to have a key role in cellular differentiation processes.13 If these initiatives do not restore cellular homeostasis, the UPR can ultimately promote cell death. Thus, it is not surprising that persistent deregulation of the UPR can contribute to a wide range of diseases, including various neurodegenerative, cardiovascular, and metabolic diseases. In this article, we discuss the current knowledge of the implications of prolonged ER stress and UPR in the development and progression of chronic lung diseases,
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diseases of uncertain causes, such as COPD, idiopathic pulmonary fibrosis, and asthma. Cystic Fibrosis
Figure 2. Contributors to ER stress. Mutations in DNA (1) or defaults in the transcription machinery (2) may lead to the production of incorrectly coded mRNA sequences that may have replaced, frame-shifted, or deleted nucleotide sequences (3). Ultimately, this may lead to the translation of mutant proteins that may not fold properly in the ER. Appropriate levels of molecules must be present in the ER to maintain an optimal folding environment. This includes high Ca21 concentrations, oxidizing redox state, and sufficient amounts of sugars and molecular chaperones (4). The production of unfolded or improperly folded proteins is a hallmark of ER stress (5). Improperly folded proteins may still proceed through the secretory pathway (6). However, the cell will attempt to refold the protein when possible. If this cannot be achieved, the mutant proteins are targeted to the proteasome for degradation and recycling (7). ER 5 endoplasmic reticulum.
including the genetic disorders cystic fibrosis (CF), a1-antitrypsin (AAT) deficiency, and pulmonary surfactant mutation-induced interstitial lung disease, and
Approximately 90% of CF cases are at least partially attributable to the DF508 mutation on the ion channel protein, cystic fibrosis transmembrane conductance regulator (CFTR).14 This mutation results in a misfolded CFTR protein that is primarily targeted for degradation through the proteasome. The fraction that reaches the apical surface of epithelial cells has reduced function, and the associated compromised transmembrane ion transport and deficient mucociliary clearance have severe implications in the airway, causing chronic pulmonary obstruction and increased chances of infection. Chronic airway inflammation in patients with CF has been largely attributed to this increased susceptibility to infections, but studies have demonstrated that even in the absence of any bacterial, fungal, or viral insult, an ER stress-related inflammatory response occurs, suggesting a plausible role of the ER stress response in initiating and propagating inflammation in CF lung disease.15 Furthermore, Kerbiriou et al16 have illustrated that the UPR markers glucose-regulated protein 78 (GRP78) and ATF6 are induced in human cells transfected with DF508-CFTR. The converse has been shown to occur as well, where UPR induction reduced the expression of wild-type CFTR at the transcriptional, translational, and maturational levels in various cell lines,17 suggesting that CF-like symptoms in pathologies with nonmutant CFTR (eg, chronic lung inflammation) may be regulated through the UPR. The research focus of CF treatment strategies has shifted from addressing symptoms to targeting the molecular components involved in maintaining proteostasis. For instance, the chemical chaperone sodium 4-phenylbutyrate (4-PBA) improves trafficking of both DF508- and wild-type CFTR in bronchial epithelial cells.18 Furthermore, selective inhibition of ERAD through interference with valosin-containing protein (VCP), a component of the ER export and proteasomal degradation machinery, was shown to rescue DF508-CFTR from ERAD and increase its cell surface expression in human bronchial epithelial cells.19 Further research is necessary to develop treatment strategies centered on resolving misfolding events and/or regulating the UPR to correct for CF lung disease. AAT Deficiency AAT, produced mainly in the liver, is the most prevalent antiprotease found in serum. When transported
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Figure 3. ER stress and the unfolded protein response. A, In addition to the internal cellular contributors shown in Figure 2, external pathogens and toxins, such as bacterial and viral infections, cigarette smoke, or other environmental components originating from the air or through the circulation, may induce ER stress in the lungs. B, This might result in an accumulation of misfolded proteins in the ER, leading to a distended ER and downstream activation of the unfolded protein response (UPR) through the PERK, IRE1, and ATF6 pathways. The UPR may be activated by dissociation of GRP78 from the three ER transmembrane-bound molecules, PERK, IRE1, and ATF6, or through binding of misfolded proteins directly to PERK or IRE1. The binding of GRP78 to these molecules is thought to block its activation and for this reason GRP78 is often called the master regulator of the UPR. C, The activated UPR aims to restore cell homeostasis through halting protein translation, increasing protein degradation, and elevating the expression of chaperones and other ER stress response genes. When homeostasis is not reached, the UPR might induce cell death. Taken together with its observed role in cell differentiation processes, the activated UPR is probably involved in both disease onset and progression. ATF6 5 activating transcription factor 6; ERAD 5 endoplasmic reticulum-associated degradation; GRP78 5 glucosergulated protein 78; IRE1 5 inositol-requiring kinase 1; PERK 5 protein kinase RNA-like endoplasmic reticulum kinase. See Figure 2 legend for expansion of other abbreviations.
to the lung, AAT is vital in maintaining lung integrity because its inhibition of the protease neutrophil elastase prevents degradation of the extracellular matrix in the alveolar interstitium.20 Although . 100 mutations exist in the AAT gene, the most common and severe is the Z mutation (Glu342Lys).20 Some mutants can polymerize and accumulate in the ER or be targeted for proteosomal degradation, instigating ER stress.20 AAT mutations have unique consequences in the liver and lungs. In the originating liver cells, the aggregation of mutant AAT in the ER can lead to fibrosis21 or cancer,22 and as many as 30% to 40% of patients
can expect to develop cirrhosis over their lifetime.20,21 In contrast, patients who suffer from its complete absence (null homozygotes) do not appear to develop significant liver disease,23 indicating that liver cirrhosis and cancer are most likely due to the accumulation of misfolded AAT. However, patients with the null mutation develop the most severe emphysematous changes,24 giving credence to the protease-antiprotease hypothesis that states that low levels of AAT are unable to inhibit neutrophil elastase activity, allowing for subsequent emphysematous changes. Overall, AAT deficiency showcases the deleterious effects
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of unresolved accumulation of misfolded protein and ER stress, as seen in the liver, with presentation of lung disease being a secondary effect. The definition of AAT deficiency-induced lung inflammation has expanded recently to include peripheral blood monocytes, which also express AAT. Z-AAT accumulation has been noted in these cells in Z-AAT homozygous individuals, with subsequent sustained UPR activation and the induction of proinflammatory genes,25 illustrating the involvement and contribution of protein misfolding, ER stress, and the UPR in AAT-deficiency-induced disease. The leading treatment of AAT deficiency is IV administration of AAT obtained from the plasma of healthy individuals. This line of treatment has been shown to increase plasma AAT levels to the protective threshold of 11 μmol/L in vivo. However, the effectiveness of this therapy in preventing AAT-deficiency-associated lung disease is unclear.26 Other potential treatments for correcting the lung disease aspect of Z-AAT deficiency focus on increasing secretion of Z-AAT and include 4-PBA, as well as various iminosugar compounds.27 Agents such as tauroursodeoxycholic acid and salubrinal may also serve as potential treatments of Z-AAT-associated lung disease because of their ability to modulate ER stress in monocytes and airway epithelial cells through inhibition of ER stress/UPRinduced apoptosis.28 Pulmonary Surfactant Mutation-Induced Lung Diseases Pulmonary surfactant is a complex of lipoproteins produced by alveolar type 2 cells that reduce surface tension within the lungs to optimize lung compliance and prevent collapse. Genetic mutations in one of the essential hydrophobic peptide subunits, surfactant protein C, has been shown to cause cytotoxicity known to be associated with various interstitial lung diseases.29 Specifically, mutations leading to the deletion of exon 4 or amino acid substitution L188Q result in surfactant protein C misfolding and its retention in the ER.30 This leads to ER stress, UPR-induced apoptosis, and, ultimately, epithelial cell dysfunction.31 Moreover, a mutation in the transporter molecule ABCA3 has been described in fatal respiratory distress syndrome in newborns. In vitro experiments showed that this mutant was unprocessed and trapped in the ER, whereas normal ATP-binding cassette subfamily A member 3 reached the lysosome. Treatment with 4-PBA in cultured human lung cells improved the mutants’ trafficking to lamellar body-like structures, which are involved in the secretory pathway of surfactant, indicating that protein-misfolding events may be rescued.32 Furthermore, a mutation in surfactant protein A2 exhibits the similar result of ER stress
and retention of the protein, with the addition of heterozygotes for the mutation expressing a sufficient amount of normal A2, yet disease still manifests. This suggests that it is not surfactant protein A2 insufficiency, but prolonged ER stress and UPR-activated cell death that may be the mechanistic cause behind epithelial cell dysfunction.33 COPD COPD is a progressive disease that is marked by emphysema and chronic bronchitis leading to the destruction of lung parenchyma and narrowing of the airways. As the leading cause of COPD, CS subjects the lungs to an excessive amount of reactive oxygen species, carcinogens, and free radicals, all factors known to trigger ER stress.7 In both in vitro studies in airway epithelial cells and in vivo studies in mice, CS exposure has been shown to activate key aspects of the UPR, presumably in an attempt to reduce ER stress.34 In vitro experiments have strengthened this finding by showing that CS and its aqueous extracts yield similar responses in human lung cells, suggesting a role of the ER in CS-induced COPD.35 In addition to augmenting ER stress and activating the UPR, CS also increases the levels of apoptosis.34 Studies in patients with COPD have indicated the presence of ER stress through the detection of UPR-specific markers, including GRP78, calreticulin, calnexin, and protein disulfide isomerases.36 These markers were increased in chronic smokers in comparison with nonsmokers,7 suggesting a plausible role of ER stress and prolonged UPR activation, possibly leading to cell death and disease. The UPR may mediate this activity through nuclear erythroid-2-related factor (NRF2) signaling, because NRF2 is needed to promote cell survival signals and regulate antioxidant responses37 and is a downstream target of one of the three UPR arms (PERK pathway). NRF2 expression is decreased in patients with COPD and has been suggested as a target for these patients.38 Further, the expression of VCP, a molecule that extracts proteins from the ER back to the cytosol and is also part of the ubiquitination system that tags proteins for proteosomal degradation, was witnessed to be progressively elevated in patients with more severe COPD.36 Taken together, the reduced proteasome activity but elevated ubiquitination of ER proteins seem to inhibit the ERAD function of UPR and may contribute to the chronic inflammatory phenotype seen in COPD.36 This highlights the importance of proteostasis in which expression of VCP and NRF2 are common protective responses but are characteristically altered in the pathogenesis of COPD. Experiments examining the effects of activating NRF2 and inhibiting VCP have yielded exciting results. When administered to CS-exposed human
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lung epithelial cells, sulforaphane, an activator of NRF2, reduced the cell death believed to occur by increasing degradative activity.39 Salubrinal, an ER stress inhibitor, decreased VCP expression in cell experiments, correcting the protein imbalance in acute CS-exposed mice, and is an interesting candidate for therapeutic treatment in patients with COPD.36 These observations indicate that protein misfolding and UPR activation may contribute to the pathogenesis of COPD, highlighting the need for treatment strategies centered on these pathways. Idiopathic Pulmonary Fibrosis Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by myofibroblast accumulation, extensive scarring, and continuous loss of lung function. Although the pathogenesis of IPF is not well understood, it is thought to occur from repeated epithelial injuries, epithelial-mesenchymal transition of epithelial cells to fibroblasts, and the transformation of fibroblasts into myofibroblasts that deposit excessive extracellular matrix.40 Despite the uncertainties of the disease’s origins, in fibrotic conditions of the lung and in particular in patients with IPF, an increased amount of ER stress and an increased number of UPR agents, including GRP78, ATF6, ATF4, and C/EBP homologous protein expression, as well as spliced xbox-binding protein 1 mRNA levels, have been confirmed.41 Recent publications have also indicated that ER stress and the UPR are involved in many of the molecular pathways leading to the aforementioned physiologic changes, including ER-stress-induced transforming growth factor-b expression,42 myofibroblast differentiation,43 epithelialmesenchymal transition,44 apoptosis, disruption of subcellular barriers leading to increased risks of viral infections,45 and altered surfactant production.46 TorresGonzález et al47 interestingly found that older mice, compared with younger mice, were more susceptible to g herpes virus-induced ER stress, which was associated with an increase in pulmonary fibrosis. This highlights the significance of ER stress and age in IPF, because patients are typically middle aged or older. Furthermore, experiments involving a bleomycininduced lung fibrosis model developed greater pathology when a potent chemical inducer of ER stress (tunicamycin) was added,48 reinforcing the possibility that ER stress and UPR activation may contribute to disease initiation or progression. Asthma Asthma is characterized as a chronic inflammatory disease that leads to the swelling and narrowing of the airways and symptomatically produces chest tight-
ening, wheezing, shortness of breath, and coughing. Based on genomic analysis, ORMLD3, a gene coding for an ER-localized transmembrane protein, was noted as a risk factor for asthma.49 Taken together, findings of elevated ORMLD3 expression in inflammatory response cells and knowledge that the UPR is known to activate and control various inflammatory pathways49 led investigators to look into ORMLD3, and they confirmed an association with ER stress. As mentioned previously, maintenance of a high subcellular Ca21 concentration is essential for ER function and viability. There are various pumps and channels that help mediate this process, including the sarcoendoplasmic reticulum Ca21 pump (SERCA), a membrane protein that mediates the amount of calcium entering the ER. Overexpression of ORMLD3 was found to sufficiently reduce the ER Ca21 concentration believed to occur through inhibition of SERCA, thereby activating the UPR. Meanwhile, knockdown experiments demonstrated the opposite effect and attenuated UPR activity. In addition, inadequate SERCA expression has been shown to induce airway remodeling, a hallmark of asthma pathogenesis.50 Thus, modulation of the UPR in these cells may reduce inflammatory pathways and airway remodeling, which are characteristic features of asthma. Conclusions and Future Directions Although compelling evidence suggests that prolonged ER stress and activation of the UPR are associated with disease, it remains to be elucidated how these processes contribute to disease initiation and progression. Whereas it is likely that cells already experiencing ER stress respond differently to additional stimuli, it is still unclear if the disease pathogenesis can be explained by additive exposure to various stimuli due to prolonged activation of the UPR. Increased understanding of the role of ER stress and protein misfolding in chronic lung diseases will likely yield various therapeutic prevention and intervention strategies, ranging from prevention strategies to reduce ER stress (Fig 3A), to the administration of molecules that can enhance protein folding (ie, small weight molecular chaperones) or molecules that can regulate protein degradation (ie, proteasome inhibitors). Further understanding of the implication of the ER stress/UPR axis and the interconnectedness between other molecular mechanisms involved in the disease pathogenesis are required and will ultimately enhance our ability to interfere with these complex pathways in patients suffering from various chronic respiratory diseases. Acknowledgments Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any
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companies/organizations whose products or services may be discussed in this article. Other contributions: We thank Antje Ask, MD; Mark Inman, MD, PhD; Jørn A. Holme, PhD; and Sinan Qasha, MD, for critical reading of this manuscript.
19.
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