p16-3MR: A Novel Model to Study Cellular Senescence in Cigarette Smoke-Induced Lung Injuries.

Cellular senescence and lung aging are associated with the pathogenesis of chronic obstructive pulmonary disease (COPD). COPD progresses with aging, and chronic smoking is the key susceptibility factor in lung pathological changes concurrent with mitochondrial dysfunction and biological aging. However, these processes involving cigarette smoke (CS)-mediated lung cellular senescence are difficult to distinguish. One of the impediments to studying cellular senescence in relation to age-related lung pathologies is the lack of a suitable in vivo model. In view of this, we provide evidence that supports the suitability of p16-3MR mice to studying cellular senescence in CS-mediated and age-related lung pathologies. p16-3MR mice have a trimodal reporter fused to the promoter of the p16INK4a gene that enables detection, isolation, and selective elimination of senescent cells, thus making them a suitable model to study cellular senescence. To determine their suitability in CS-mediated lung pathologies, we exposed young (12-14 months) and old (17-20 months) p16-3MR mice to 30 day CS exposure and studied the expression of senescent genes (p16, p21, and p53) and SASP-associated markers (MMP9, MMP12, PAI-1, and FN-1) in air- and CS-exposed mouse lungs. Our results showed that this model could detect cellular senescence using luminescence and isolate cells undergoing senescence with the help of tissue fluorescence in CS-exposed young and old mice. Our results from the expression of senescence markers and SASP-associated genes in CS-exposed young and old p16-3MR mice were comparable with increased lung cellular senescence and SASP in COPD. We further showed alteration in the; (i) tissue luminescence and fluorescence, (ii) mRNA and protein expressions of senescent markers and SASP genes, and (iii) SA-ß-gal activity in CS-exposed young and old p16-3MR mice as compared to their air controls. Overall, we showed that p16-3MR is a competent model for studying the cellular senescence in CS-induced pathologies. Hence, the p16-3MR reporter mouse model may be used as a novel tool for understanding the pathobiology of cellular senescence and other underlying mechanisms involved in COPD and fibrosis.

Prenatal Exposure to Electronic-Cigarette Aerosols Leads to Sex-Dependent Pulmonary Extracellular-Matrix Remodeling and Myogenesis in Offspring Mice.

Electronic-cigarette (e-cig) vaping is a serious concern, as many pregnant women who vape consider it safe. However, little is known about the harmful effects of prenatal e-cig exposure on adult offspring, especially on extracellular-matrix (ECM) deposition and myogenesis in the lungs of offspring. We evaluated the biochemical and molecular implications of maternal exposure during pregnancy to e-cig aerosols on the adult offspring of both sexes, with a particular focus on pulmonary ECM remodeling and myogenesis. Pregnant CD-1 mice were exposed to e-cig aerosols with or without nicotine, throughout gestation, and lungs were collected from adult male and female offspring. Compared with the air-exposed control group, female mice exposed to e-cig aerosols, with or without nicotine, demonstrated increased lung protein abundance of LEF-1 (lymphoid enhancer-binding factor 1), fibronectin, and E-cadherin, whereas altered E-cadherin and PPARγ (peroxisome proliferator-activated receptor γ) levels were observed only in males exposed to e-cig aerosols with nicotine. Moreover, lipogenic and myogenic mRNAs were dysregulated in adult offspring in a sex-dependent manner. PAI-1 (plasminogen activator inhibitor-1), one of the ECM regulators, was significantly increased in females exposed prenatally to e-cig aerosols with nicotine and in males exposed to e-cig aerosols compared with control animals exposed to air. MMP9 (matrix metalloproteinase 9), a downstream target of PAI-1, was downregulated in both sexes exposed to e-cig aerosols with nicotine. No differences in lung histology were observed among any of the treatment groups. Overall, adult mice exposed prenatally to e-cig aerosols could be predisposed to developing pulmonary disease later in life. Thus, these findings suggest that vaping during pregnancy is unsafe and increases the propensity for later-life interstitial lung diseases.

E-cigarette-induced pulmonary inflammation and dysregulated repair are mediated by nAChR α7 receptor: role of nAChR α7 in SARS-CoV-2 Covid-19 ACE2 receptor regulation.

Electronic cigarette (e-cig) vaping is increasing rapidly in the United States, as e-cigs are considered less harmful than combustible cigarettes. However, limited research has been conducted to understand the possible mechanisms that mediate toxicity and pulmonary health effects of e-cigs. We hypothesized that sub-chronic e-cig exposure induces inflammatory response and dysregulated repair/extracellular matrix (ECM) remodeling, which occur through the α7 nicotinic acetylcholine receptor (nAChRα7). Adult wild-type (WT), nAChRα7 knockout (KO), and lung epithelial cell-specific KO (nAChRα7 CreCC10) mice were exposed to e-cig aerosol containing propylene glycol (PG) with or without nicotine. Bronchoalveolar lavage fluids (BALF) and lung tissues were collected to determine e-cig induced inflammatory response and ECM remodeling, respectively. Sub-chronic e-cig exposure with nicotine increased inflammatory cellular influx of macrophages and T-lymphocytes including increased pro-inflammatory cytokines in BALF and increased SARS-Cov-2 Covid-19 ACE2 receptor, whereas nAChRα7 KO mice show reduced inflammatory responses associated with decreased ACE2 receptor. Interestingly, matrix metalloproteinases (MMPs), such as MMP2, MMP8 and MMP9, were altered both at the protein and mRNA transcript levels in female and male KO mice, but WT mice exposed to PG alone showed a sex-dependent phenotype. Moreover, MMP12 was increased significantly in male mice exposed to PG with or without nicotine in a nAChRα7-dependent manner. Additionally, sub-chronic e-cig exposure with or without nicotine altered the abundance of ECM proteins, such as collagen and fibronectin, significantly in a sex-dependent manner, but without the direct role of nAChRα7 gene. Overall, sub-chronic e-cig exposure with or without nicotine affected lung inflammation and repair responses/ECM remodeling, which were mediated by nAChRα7 in a sex-dependent manner.

Association of smoking and electronic cigarette use with wheezing and related respiratory symptoms in adults: cross-sectional results from the Population Assessment of Tobacco and Health (PATH) study, wave 2.

BACKGROUND: Wheezing is a symptom of potential respiratory disease and known to be associated with smoking. Electronic cigarette use ('vaping') has increased exponentially in recent years. This study examined the cross-sectional association of vaping with wheezing and related respiratory symptoms and compare this association with smokers and dual users. METHODS: The Population Assessment of Tobacco and Health study wave 2 data collected from October 2014 to October 2015 with 28 171 adults were used. The cross-sectional association of vaping with self-reported wheezing and related respiratory symptoms relative to smokers and dual users of tobacco and electronic cigarettes were studied using multivariable logistic and cumulative logistic regression models with consideration of complex sampling design. RESULTS: Among the 28 171 adult participants, 641 (1.2%) were current vapers who used e-cigarettes exclusively, 8525 (16.6%) were current exclusive smokers, 1106 (2.0%) were dual users and 17 899 (80.2%) were non-users. Compared with non-users, risks of wheezing and related respiratory symptoms were significantly increased in current vapers (adjusted OR (aOR)=1.67, 95% CI: 1.23 to 2.15). Current vapers had significantly lower risk in wheezing and related respiratory symptoms compared with current smokers (aOR=0.68, 95% CI: 0.53 to 0.87). No significant differences were found between dual users and current smokers in risk of wheezing and related respiratory symptoms (aOR=1.06, 95% CI: 0.91 to 1.24). CONCLUSIONS: Vaping was associated with increased risk of wheezing and related respiratory symptoms. Current vapers had lower risk in wheezing and related respiratory symptoms than current smokers or dual users but higher than non-users. Both dual use and smoking significantly increased the risk of wheezing and related respiratory symptoms.

Genetic ablation of histone deacetylase 2 leads to lung cellular senescence and lymphoid follicle formation in COPD/emphysema.

Histone deacetylase 2 (HDAC2), a critical determinant of chromatin remodeling, is reduced as a consequence of oxidative stress-mediated DNA damage and impaired repair. Cigarette smoke (CS) exposure causes DNA damage and cellular senescence. However, no information is available on the role of HDAC2 in CS-induced DNA damage, stress-induced premature senescence (SIPS), and senescence-associated secretory phenotype (SASP) during the pathogenesis of chronic obstructive pulmonary disease (COPD)/emphysema. We hypothesized that CS causes persistent DNA damage and cellular senescence via HDAC2-dependent mechanisms. We used HDAC2 global knockout (KO) and HDAC2 lung epithelial cell-specific KO [Clara cell-specific HDAC2 deletion (HDAC2 CreCC10)] mice to determine whether HDAC2 is a major player in CS-induced oxidative stress, SIPS, and SASP. HDAC2 KO mice exposed to CS show exaggerated DNA damage, inflammatory response, and decline in lung function leading to airspace enlargement. Chronic CS exposure augments lung senescence-associated ß-galactosidase activity in HDAC2 KO, but not in HDAC2 CreCC10 mice. HDAC2 lung epithelial cell-specific KO did not further augment CS-induced inflammatory response and airspace enlargement but instead caused an increase in lymphoid aggregate formation. Our study reveals that HDAC2 is a key player regulating CS-induced DNA damage, inflammatory response, and cellular senescence leading to COPD/emphysema.-Sundar, I. K., Rashid, K., Gerloff, J., Rangel-Moreno, J., Li, D., Rahman, I. Genetic ablation of histone deacetylase 2 leads to lung cellular senescence and lymphoid follicle formation in COPD/emphysema.

Genetic Ablation of p16INK4a Does Not Protect against Cellular Senescence in Mouse Models of Chronic Obstructive Pulmonary Disease/Emphysema.

Cigarette smoke (CS) affects DNA damage and cellular senescence signaling pathways in the pathogenesis of chronic obstructive pulmonary disease (COPD). p16INK4a (p16: a cyclin-dependent kinase inhibitor) is a key marker of cellular senescence, which is induced by CS in lung cells. It is thought that removal of p16 attenuates premature aging by removing senesced cells. However, the role of p16 in CS-induced stress-induced premature senescence (SIPS) and senescence-associated secretory phenotype (SASP) during the development of COPD/emphysema is not known. We hypothesize that p16 regulates cellular senescence and DNA damage/repair molecular signaling targets during chronic CS-induced inflammation and airspace enlargement in mouse models of COPD. We used p16 global knockout (KO) and p16 lung epithelial cell-specific KO (p16CreCC10) mice to determine whether p16 removal in lung epithelium augments or protects against cellular senescence (SIPS and SASP) in chronic CS- and elastase-induced development of COPD/emphysema in mice. p16 KO mice exposed to chronic CS and p16 lung epithelial cell-specific KO mice exposed to elastase did not show attenuation of lung inflammation, altered lung function, or airspace enlargement. p16 KO and p16CreCC10 exposed to CS and elastase showed increases in lung senescence-associated ß-galactosidase activity. Thus, removal of p16-positive cells did not protect against airspace enlargement and decline in lung function induced in COPD mouse models. Our findings suggest that p16 is not the only key player associated with CS-induced cellular senescence phenotypes (SIPS and SASP), decline in lung function, and airspace enlargement in COPD/emphysema.

Blockade of RAGE ameliorates elastase-induced emphysema development and progression via RAGE-DAMP signaling.

The receptor for advanced glycan end products (RAGE) has been identified as a susceptibility gene for chronic obstructive pulmonary disease (COPD) in genome-wide association studies (GWASs). However, less is known about how RAGE is involved in the pathogenesis of COPD. To determine the molecular mechanism by which RAGE influences COPD in experimental COPD models, we investigated the efficacy of the RAGE-specific antagonist FPS-ZM1 administration in in vivo and in vitro COPD models. We injected elastase intratracheally and the RAGE antagonist FPS-ZM1 in mice, and the infiltrated inflammatory cells and cytokines were assessed by ELISA. Cellular expression of RAGE was determined in protein, serum, and bronchoalveolar lavage fluid of mice and lungs and serum of human donors and patients with COPD. Downstream damage-associated molecular pattern (DAMP) pathway activation in vivo and in vitro and in patients with COPD was assessed by immunofluorescence staining, Western blot analysis, and ELISA. The expression of membrane RAGE in initiating the inflammatory response and of soluble RAGE acting as a decoy were associated with up-regulation of the DAMP-related signaling pathway via Nrf2. FPS-ZM1 administration significantly reversed emphysema in the lung of mice. Moreover, FPS-ZM1 treatment significantly reduced lung inflammation in Nrf2+/+ , but not in Nrf2-/- mice. Thus, our data indicate for the first time that RAGE inhibition has an essential protective role in COPD. Our observation of RAGE inhibition provided novel insight into its potential as a therapeutic target in emphysema/COPD.-Lee, H., Park, J.-R., Kim, W. J., Sundar, I. K., Rahman, I., Park, S.-M., Yang. S.-R. Blockade of RAGE ameliorates elastase-induced emphysema development and progression via RAGE-DAMP signaling.

Shelterin Telomere Protection Protein 1 Reduction Causes Telomere Attrition and Cellular Senescence via Sirtuin 1 Deacetylase in Chronic Obstructive Pulmonary Disease.

Lung cellular senescence and inflammatory response are the key events in the pathogenesis of chronic obstructive pulmonary disease (COPD) when cigarette smoke (CS) is the main etiological factor. Telomere dysfunction is induced by either critical shortening or disruption of the shelterin complex, leading to cellular senescence. However, it remains unknown whether disruption of the shelterin complex is responsible for CS-induced lung cellular senescence. Here we show that telomere protection protein 1 (TPP1) levels are reduced on telomeres in lungs from mice with emphysema, as well as in lungs from smokers and from patients with COPD. This is associated with persistent telomeric DNA damage, leading to cellular senescence. CS disrupts the interaction of TPP1 with the Sirtuin 1 (Sirt1) complex, leading to increased TPP1 acetylation and degradation. Lung fibroblasts deficient in Sirt1 or treated with a selective Sirt1 inhibitor exhibit increased cellular senescence and decreased TPP1 levels, whereas Sirt1 overexpression and pharmacological activation protect against CS-induced TPP1 reduction and telomeric DNA damage. Our findings support an essential role of TPP1 in protecting CS-induced telomeric DNA damage and cellular senescence, and therefore provide a rationale for a potential therapy for COPD, on the basis of the shelterin complex, in attenuating cellular senescence.

The nuclear receptor and clock gene REV-ERBα regulates cigarette smoke-induced lung inflammation.

REV-ERBα is a nuclear heme receptor, transcriptional repressor and critical component of the molecular clock that drives daily rhythms of metabolism. Evidence reveals that REV-ERBα also plays an important regulatory role in clock-dependent lung physiology and inflammatory responses. We hypothesize that cigarette smoke (CS) exposure influences REV-ERBα abundance in the lungs, facilitating a pro-inflammatory phenotype. To determine the impact of REV-ERBα activation in the CS-induced inflammatory response we treated primary human small airway epithelial cells (SAECs) with CS extract (CSE) or lipopolysaccharide (LPS) in the absence or presence of pre-treatment with the REV-ERBα agonist GSK 4112. We also exposed adult C57BL/6J (WT) and Rev-erbα global KO mice to CS (10 and 30 days) and measured pro-inflammatory cytokine release. Our data reveal that pre-treatment with GSK 4112 reduced CSE/LPS induced pro-inflammatory cytokines release from both SAECs and mouse lung fibroblasts (MLFs). Furthermore, REV-ERBα KO mice show a greater inflammatory response to 10 and 30 days of CS, including increased neutrophil lung influx, pro-inflammatory cytokine (IL-6, MCP-1 and KC) release, and pro-senescence marker (p16) when compared to WT mice. These data demonstrate that REV-ERBα is a critical regulator of CS-induced lung inflammatory responses.

Gene expression profiling of epigenetic chromatin modification enzymes and histone marks by cigarette smoke: implications for COPD and lung cancer.

Chromatin-modifying enzymes mediate DNA methylation and histone modifications on recruitment to specific target gene loci in response to various stimuli. The key enzymes that regulate chromatin accessibility for maintenance of modifications in DNA and histones, and for modulation of gene expression patterns in response to cigarette smoke (CS), are not known. We hypothesize that CS exposure alters the gene expression patterns of chromatin-modifying enzymes, which then affects multiple downstream pathways involved in the response to CS. We have, therefore, analyzed chromatin-modifying enzyme profiles and validated by quantitative real-time PCR (qPCR). We also performed immunoblot analysis of targeted histone marks in C57BL/6J mice exposed to acute and subchronic CS, and of lungs from nonsmokers, smokers, and patients with chronic obstructive pulmonary disease (COPD). We found a significant increase in expression of several chromatin modification enzymes, including DNA methyltransferases, histone acetyltransferases, histone methyltransferases, and SET domain proteins, histone kinases, and ubiquitinases. Our qPCR validation data revealed a significant downregulation of Dnmt1, Dnmt3a, Dnmt3b, Hdac2, Hdac4, Hat1, Prmt1, and Aurkb We identified targeted chromatin histone marks (H3K56ac and H4K12ac), which are induced by CS. Thus CS-induced genotoxic stress differentially affects the expression of epigenetic modulators that regulate transcription of target genes via DNA methylation and site-specific histone modifications. This may have implications in devising epigenetic-based therapies for COPD and lung cancer.

Electronic cigarette aerosols and copper nanoparticles induce mitochondrial stress and promote DNA fragmentation in lung fibroblasts.

Oxidants or nanoparticles have recently been identified as constituents of aerosols released from various styles of electronic cigarettes (E-cigs). Cells in the lung may be directly exposed to these constituents and harbor reactive properties capable of incurring acute cell injury. Our results show mitochondria are sensitive to both E-cig aerosols and aerosol containing copper nanoparticles when exposed to human lung fibroblasts (HFL-1) using an Air-Liquid Interface culture system, evident by elevated levels of mitochondrial ROS (mtROS). Increased mtROS after aerosol exposure is associated with reduced stability of OxPhos electron transport chain (ETC) complex IV subunit and nuclear DNA fragmentation. Increased levels of IL-8 and IL-6 in HFL-1 conditioned media were also observed. These findings reveal both mitochondrial, genotoxic, and inflammatory stresses are features of direct cell exposure to E-cig aerosols which are ensued by inflammatory duress, raising a concern on deleterious effect of vaping.

Impaired mitophagy leads to cigarette smoke stress-induced cellular senescence: implications for chronic obstructive pulmonary disease.

Cigarette smoke (CS)-induced cellular senescence is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The molecular mechanism by which CS induces cellular senescence is unknown. Here, we show that CS stress (exposure of primary lung cells to CS extract 0.2-0.75% with a half-maximal inhibitory concentration of ∼0.5%) led to impaired mitophagy and perinuclear accumulation of damaged mitochondria associated with cellular senescence in both human lung fibroblasts and small airway epithelial cells (SAECs). Impaired mitophagy was attributed to reduced Parkin translocation to damaged mitochondria, which was due to CS-induced cytoplasmic p53 accumulation and its interaction with Parkin. Impaired Parkin translocation to damaged mitochondria was also observed in mouse lungs with emphysema (6 months CS exposure, 100 mg TPM/m(3)) as well as in lungs of chronic smokers and patients with COPD. Primary SAECs from patients with COPD also exhibited impaired mitophagy and increased cellular senescence via suborganellar signaling. Mitochondria-targeted antioxidant (Mito-Tempo) restored impaired mitophagy, decreased mitochondrial mass accumulation, and delayed cellular senescence in Parkin-overexpressing cells. In conclusion, defective mitophagy leads to CS stress-induced lung cellular senescence, and restoring mitophagy delays cellular senescence, which provides a promising therapeutic intervention in chronic airway diseases.

Circadian clock-coupled lung cellular and molecular functions in chronic airway diseases.

Airway diseases are associated with abnormal circadian rhythms of lung function, reflected in daily changes of airway caliber, airway resistance, respiratory symptoms, and abnormal immune-inflammatory responses. Circadian rhythms are generated at the cellular level by an autoregulatory feedback loop of interlocked transcription factors collectively referred to as clock genes. The molecular clock is altered by cigarette smoke, LPS, and bacterial and viral infections in mouse and human lungs and in patients with chronic airway diseases. Stress-mediated post-translational modification of molecular clock proteins, brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (BMAL1) and PERIOD 2, is associated with a reduction in the activity/level of the deacetylase sirtuin 1 (SIRT1). Similarly, the levels of the nuclear receptor REV-ERBα and retinoic acid receptor-related orphan receptor α (ROR α), critical regulators of Bmal1 expression, are altered by environmental stresses. Molecular clock dysfunction is implicated in immune and inflammatory responses, DNA damage response, and cellular senescence. The molecular clock in the lung also regulates the timing of glucocorticoid sensitivity and phasic responsiveness to inflammation. Herein, we review our current understanding of clock-controlled cellular and molecular functions in the lungs, the impact of clock dysfunction in chronic airway disease, and the response of the pulmonary clock to different environmental perturbations. Furthermore, we discuss the evidence for candidate signaling pathways, such as the SIRT1-BMAL1-REV-ERBα axis, as novel targets for chronopharmacological management of chronic airway diseases.

Disruption of Sirtuin 1-Mediated Control of Circadian Molecular Clock and Inflammation in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is the fourth most common cause of death, and it is characterized by abnormal inflammation and lung function decline. Although the circadian molecular clock regulates inflammatory responses, there is no information available regarding the impact of COPD on lung molecular clock function and its regulation by sirtuin 1 (SIRT1). We hypothesize that the molecular clock in the lungs is disrupted, leading to increased inflammatory responses in smokers and patients with COPD and its regulation by SIRT1. Lung tissues, peripheral blood mononuclear cells (PBMCs), and sputum cells were obtained from nonsmokers, smokers, and patients with COPD for measurement of core molecular clock proteins (BMAL1, CLOCK, PER1, PER2, and CRY1), clock-associated nuclear receptors (REV-ERBα, REV-ERBß, and RORα), and SIRT1 by immunohistochemistry, immunofluorescence, and immunoblot. PBMCs were treated with the SIRT1 activator SRT1720 followed by LPS treatment, and supernatant was collected at 6-hour intervals. Levels of IL-8, IL-6, and TNF-α released from PBMCs were determined by ELISA. Expression of BMAL1, PER2, CRY1, and REV-ERBα was reduced in PBMCs, sputum cells, and lung tissues from smokers and patients with COPD when compared with nonsmokers. SRT1720 treatment attenuated LPS-mediated reduction of BMAL1 and REV-ERBα in PBMCs from nonsmokers. Additionally, LPS differentially affected the timing and amplitude of cytokine (IL-8, IL-6, and TNF-α) release from PBMCs in nonsmokers, smokers, and patients with COPD. Moreover, SRT1720 was able to inhibit LPS-induced cytokine release from cultured PBMCs. In conclusion, disruption of the molecular clock due to SIRT1 reduction contributes to abnormal inflammatory response in smokers and patients with COPD.

Circadian molecular clock in lung pathophysiology.

Disrupted daily or circadian rhythms of lung function and inflammatory responses are common features of chronic airway diseases. At the molecular level these circadian rhythms depend on the activity of an autoregulatory feedback loop oscillator of clock gene transcription factors, including the BMAL1:CLOCK activator complex and the repressors PERIOD and CRYPTOCHROME. The key nuclear receptors and transcription factors REV-ERBα and RORα regulate Bmal1 expression and provide stability to the oscillator. Circadian clock dysfunction is implicated in both immune and inflammatory responses to environmental, inflammatory, and infectious agents. Molecular clock function is altered by exposomes, tobacco smoke, lipopolysaccharide, hyperoxia, allergens, bleomycin, as well as bacterial and viral infections. The deacetylase Sirtuin 1 (SIRT1) regulates the timing of the clock through acetylation of BMAL1 and PER2 and controls the clock-dependent functions, which can also be affected by environmental stressors. Environmental agents and redox modulation may alter the levels of REV-ERBα and RORα in lung tissue in association with a heightened DNA damage response, cellular senescence, and inflammation. A reciprocal relationship exists between the molecular clock and immune/inflammatory responses in the lungs. Molecular clock function in lung cells may be used as a biomarker of disease severity and exacerbations or for assessing the efficacy of chronotherapy for disease management. Here, we provide a comprehensive overview of clock-controlled cellular and molecular functions in the lungs and highlight the repercussions of clock disruption on the pathophysiology of chronic airway diseases and their exacerbations. Furthermore, we highlight the potential for the molecular clock as a novel chronopharmacological target for the management of lung pathophysiology.

Circadian clock function is disrupted by environmental tobacco/cigarette smoke, leading to lung inflammation and injury via a SIRT1-BMAL1 pathway.

Patients with obstructive lung diseases display abnormal circadian rhythms in lung function. We determined the mechanism whereby environmental tobacco/cigarette smoke (CS) modulates expression of the core clock gene BMAL1, through Sirtuin1 (SIRT1) deacetylase during lung inflammatory and injurious responses. Adult C57BL6/J and various mice mutant for SIRT1 and BMAL1 were exposed to both chronic (6 mo) and acute (3 and 10 d) CS, and we measured the rhythmic expression of clock genes, circadian rhythms of locomotor activity, lung function, and inflammatory and emphysematous responses in the lungs. CS exposure (100-300 mg/m(3) particulates) altered clock gene expression and reduced locomotor activity by disrupting the central and peripheral clocks and increased lung inflammation, causing emphysema in mice. BMAL1 was acetylated and degraded in the lungs of mice exposed to CS and in patients with chronic obstructive pulmonary disease (COPD), compared with lungs of the nonsmoking controls, linking it mechanistically to CS-induced reduction of SIRT1. Targeted deletion of Bmal1 in lung epithelium augmented inflammation in response to CS, which was not attenuated by the selective SIRT1 activator SRT1720 (EC50=0.16 µM) in these mice. Thus, the circadian clock, specifically the enhancer BMAL1 in epithelium, plays a pivotal role, mediated by SIRT1-dependent BMAL1, in the regulation of CS-induced lung inflammatory and injurious responses.

Cigarette smoke induces distinct histone modifications in lung cells: implications for the pathogenesis of COPD and lung cancer.

Cigarette smoke (CS)-mediated oxidative stress induces several signaling cascades, including kinases, which results in chromatin modifications (histone acetylation/deacetylation and histone methylation/demethylation). We have previously reported that CS induces chromatin remodeling in pro-inflammatory gene promoters; however, the underlying site-specific histone marks formed in histones H3 and H4 during CS exposure in lungs in vivo and in lung cells in vitro, which can either drive gene expression or repression, are not known. We hypothesize that CS exposure in mouse and human bronchial epithelial cells (H292) can cause site-specific posttranslational histone modifications (PTMs) that may play an important role in the pathogenesis of CS-induced chronic lung diseases. We used a bottom-up mass spectrometry approach to identify some potentially novel histone marks, including acetylation, monomethylation, and dimethylation, in specific lysine and arginine residues of histones H3 and H4 in mouse lungs and H292 cells. We found that CS-induced distinct posttranslational histone modification patterns in histone H3 and histone H4 in lung cells, which may be considered as usable biomarkers for CS-induced chronic lung diseases. These identified histone marks (histone H3 and histone H4) may play an important role in the epigenetic state during the pathogenesis of smoking-induced chronic lung diseases, such as chronic obstructive pulmonary disease and lung cancer.

SIRT1 protects against cigarette smoke-induced lung oxidative stress via a FOXO3-dependent mechanism.

Oxidative and carbonyl stress is increased in lungs of smokers and patients with chronic obstructive pulmonary disease (COPD), as well as in cigarette smoke (CS)-exposed rodent lungs. We previously showed that sirtuin1 (SIRT1), an antiaging protein, is reduced in lungs of CS-exposed mice and patients with COPD and that SIRT1 attenuates CS-induced lung inflammation and injury. It is not clear whether SIRT1 protects against CS-induced lung oxidative stress. Therefore, we determined the effect of SIRT1 on lung oxidative stress and antioxidants in response to CS exposure using loss- and gain-of-function approaches, as well as a pharmacological SIRT1 activation by SRT1720. We found that CS exposure increased protein oxidation and lipid peroxidation in lungs of wild-type (WT) mice, which was further augmented in SIRT1-deficient mice. Furthermore, both SIRT1 genetic overexpression and SRT1720 treatment significantly decreased oxidative stress induced by CS exposure. FOXO3 deletion augmented lipid peroxidation products but reduced antioxidants in response to CS exposure, which was not affected by SRT1720. Interestingly, SRT1720 treatment exhibited a similar effect on lipid peroxidation and antioxidants (i.e., manganese superoxide dismutase, heme oxygenase-1, and NADPH quinone oxidoreductase-1) in WT and nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-deficient mice in response to CS exposure. This indicates that SIRT1 protects against CS-induced oxidative stress, which is mediated by FOXO3, but is independent of Nrf2. Overall, these findings reveal a novel function of SIRT1, which is to reduce CS-induced oxidative stress, and this may contribute to its protective effects against lung inflammation and subsequent development of COPD.

SIRT1 redresses the imbalance of tissue inhibitor of matrix metalloproteinase-1 and matrix metalloproteinase-9 in the development of mouse emphysema and human COPD.

Sirtuin1 (SIRT1), a protein/histone deacetylase, protects against the development of pulmonary emphysema. However, the molecular mechanisms underlying this observation remain elusive. The imbalance of tissue inhibitor of matrix metalloproteinases (TIMPs)/matrix metalloproteinases (MMPs) plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD)/emphysema. We hypothesized that SIRT1 protects against emphysema by redressing the imbalance between MMPs and TIMPs. To test this hypothesis, SIRT1-deficient and overexpressing/transgenic mice were exposed to cigarette smoke (CS). The protein level and activity of MMP-9 were increased in lungs of SIRT1-deficient mice exposed to CS compared with wild-type (WT) littermates, and these effects were attenuated by SIRT1 overexpression. SIRT1 deficiency decreased the level of TIMP-1, which was augmented in SIRT1 transgenic mice compared with WT littermates by CS. However, the level of MMP-2, MMP-12, TIMP-2, TIMP-3, or TIMP-4 was not altered by SIRT1 in response to CS exposure. SIRT1 reduction was associated with imbalance of TIMP-1 and MMP-9 in lungs of smokers and COPD patients. Mass spectrometry and immunoprecipitation analyses revealed that TIMP-1 acetylation on specific lysine residues was increased, whereas its interaction with SIRT1 and MMP-9 was reduced in mouse lungs with emphysema, as well as in lungs of smokers and COPD patients. SIRT1 deficiency increased CS-induced TIMP-1 acetylation, and these effects were attenuated by SIRT1 overexpression. These results suggest that SIRT1 protects against COPD/emphysema, in part, via redressing the TIMP-1/MMP-9 imbalance involving TIMP-1 deacetylation. Thus redressing the TIMP-1/MMP-9 imbalance by pharmacological activation of SIRT1 is an attractive approach in the intervention of COPD.

FOXO3 deficiency leads to increased susceptibility to cigarette smoke-induced inflammation, airspace enlargement, and chronic obstructive pulmonary disease.

Forkhead box class O 3a (FOXO3) is a member of the FoxO transcription factor subfamily, which regulates the expression of target genes not only through DNA binding as a transcription factor, but also through protein-protein interaction. Although FoxO3 is a well-known transcription factor involved in diverse biological processes, the role of FoxO3 in cigarette smoke (CS)-induced lung inflammation and injury has not been studied. It is, therefore, hypothesized that deficiency of FoxO3 leads to increased susceptibility to CS-induced lung inflammatory response and airspace enlargement. In this article, we show that the levels of FOXO3 are significantly decreased in lungs of smokers and patients with chronic obstructive pulmonary disease, as well as in lungs of mice exposed to CS. Genetic ablation of FoxO3 led to pulmonary emphysema and exaggerated inflammatory response in lungs of mice exposed to CS. We further showed that CS induced the translocation of FoxO3 into the nucleus where FoxO3 interacted with NF-κB and disrupted NF-κB DNA-binding ability, leading to inhibition of its activity. Targeted disruption of FoxO3 also resulted in downregulation of antioxidant genes in mouse lungs in response to CS exposure. These results suggest that FoxO3 plays a pivotal role in regulation of lung inflammatory response and antioxidant genes, and deficiency of FoxO3 results in development of chronic obstructive pulmonary disease/emphysema.