Characterization of a novel long noncoding RNA, SCAL1, induced by cigarette smoke and elevated in lung cancer cell lines.
ABSTRACT: The incidence of lung diseases and cancer caused by cigarette smoke is increasing. The molecular mechanisms of gene regulation induced by cigarette smoke that ultimately lead to cancer remain unclear. This report describes a novel long noncoding RNA (lncRNA) that is induced by cigarette smoke extract (CSE) both in vitro and in vivo and is elevated in numerous lung cancer cell lines. We have termed this lncRNA the smoke and cancer-associated lncRNA-1 (SCAL1). This lncRNA is located in chromosome 5, and initial sequencing analysis reveals a transcript with four exons and three introns. The expression of SCAL1 is regulated transcriptionally by nuclear factor erythroid 2-related factor (NRF2), as determined by the small, interfering RNA (siRNA) knockdown of NRF2 and kelch-like ECH-associated protein 1 (KEAP1). A nuclear factor erythroid-derived 2 (NF-E2) motif was identified in the promoter region that shows binding to NRF2 after its activation. Functionally, the siRNA knockdown of SCAL1 in human bronchial epithelial cells shows a significant potentiation of cytotoxicity induced by CSE in vitro. Altogether, these results identify a novel and intriguing new noncoding RNA that may act downstream of NRF2 to regulate gene expression and mediate oxidative stress protection in airway epithelial cells.
Project description:The oxidative stress and cellular apoptosis by environmental factor including cigarette smoke induces alveolar airway remodeling leading to chronic obstructive pulmonary disease (COPD). Recently, the receptor for advanced glycan end products (RAGE) which is highly expressed in alveolar epithelium is emerging as a biomarker for COPD susceptibility or progression. However, it still remains unknown how RAGE plays a role in cigarette smoke extract (CSE)-exposed human alveolar type II epithelial cell line. Therefore, we determined the efficacy of RAGE-specific antagonist FPS-ZM1 in response to CSE-induced lung epithelial cells. CSE induced the elevated generation of RONS and release of pro-inflammatory cytokines, and impaired the cellular antioxidant defense system. Further, CSE induced the alteration of RAGE distribution via the activation of redox-sensitive DAMP (Damage-associated molecular patterns) signaling through Nrf2 in cells. Although pre-treatment with SB202190 (p38 inhibitor) or SP600125 (JNK inhibitor) failed to recover the alteration of RAGE distribution, treatment of FPS-ZM1 significantly exhibited anti-inflammatory and anti-oxidative/nitrosative effects, also inhibited the activation of redox-sensitive DAMP signaling through Nrf2 (nuclear factor erythroid 2-related factor 2) migration in the presence of CSE. Taken together, our data demonstrate that RAGE and Nrf2 play a pivotal role in maintenance of alveolar epithelial integrity.
Project description:Naringenin is found mainly in citrus fruits, and is thought to be beneficial in the prevention and control of lung diseases. This study aims to investigate the mechanisms of naringenin against the damage in the lung caused by cigarette smoke. A system bioinformatic approach was proposed to predict the mechanisms of naringenin for protecting lung health. Then, we validated this prediction in BEAS-2B cells treated with cigarette smoke extract (CSE). System bioinformatic analysis indicated that naringenin exhibits protective effects on lung through the inhibition of inflammation and suppression of oxidative stress based on a multi-pathways network, mainly including oxidative stress pathway, Nrf2 pathway, Lung fibrosis pathway, IL-3 signaling pathway, and Aryl hydrocarbon receptor pathway. The in vitro results showed that naringenin significantly attenuated CSE-induced up-regulation of IL-8 and TNF-?. CSE stimulation increased the mRNA expressions of Nrf2, HO-1, and NQO1; the levels of total protein and nuclear protein of Nrf2; and the activity of SOD on days 2 and 4; but decreased these indexes on day 6. Naringenin can balance the antioxidant system by regulating Nrf2 and its downstream genes, preliminarily validating that Nrf2 pathway is involved in the protection offered by naringenin against cigarette smoke-induced damage to the lung. It suggests that dietary naringenin shows possible potential use in the management of lung health.
Project description:Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and abnormal inflammatory response. Wnt/?-catenin and AMP-activated protein kinase (AMPK) have been shown to modulate lung inflammatory responses and injury. However, it remains elusive whether Wnt/?-catenin and AMPK modulate nuclear factor erythroid-2 related factor-2 (Nrf2)-mediated protective responses during the development of emphysema. Here we showed that treatment with a Wnt pathway activator (LiCl) reduced elastase-induced airspace enlargement and cigarette smoke extract (CSE)-induced lung inflammatory responses in WT mice, which was associated with increased activation of Nrf2 pathway. Interestingly, these effects of LiCl were not observed in Nrf2-/- mice exposed to elastase. In normal human bronchial epithelial (NHBE) cells, Wnt3a overexpression up-regulated, whereas Wnt3a knockdown further down-regulated the levels of Nrf2 and its target proteins heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) by CSE treatment. In contrast, Nrf2 deficiency did not have any effects on Wnt/?-catenin pathway in mouse lungs and NHBE cells. Both elastase and CSE exposures reduced AMPK phosphorylation. A specific AMPK activator metformin increased Wnt3a, ?-catenin, Nrf2 phosphorylation and activation but reduced the levels of IL-6 and IL-8 in NHBE cells and mouse lungs exposed to CSE. Furthermore, Nrf2 deficiency abolished the protection of metformin against CSE-induced increase in IL-6 and IL-8 in NHBE cells. In conclusion, Nrf2 mediates the protective effects of both Wnt3a/?-catenin and AMPK on lung inflammatory responses during the development of COPD/emphysema. These findings provide potential therapeutic targets for the intervention of COPD/emphysema.
Project description:Cigarette smoke (CS) is a highly complex mixture and many of its components are known carcinogens, mutagens, and other toxic substances. CS induces oxidative stress and cell death, and this cell toxicity plays a key role in the pathogenesis of several pulmonary diseases.We studied the effect of cigarette smoke extract (CSE) in human alveolar epithelial type I-like (ATI-like) cells. These are isolated type II cells that are differentiating toward the type I cell phenotype in vitro and have lost many type II cell markers and express type I cell markers. ATI-like cells were more sensitive to CSE than alveolar type II cells, which maintained their differentiated phenotype in vitro. We observed disruption of mitochondrial membrane potential, apoptosis and necrosis that were detected by double staining with acridine orange and ethidium bromide or Hoechst 33342 and propidium iodide and TUNEL assay after treatment with CSE. We also detected caspase 3 and caspase 7 activities and lipid peroxidation. CSE induced nuclear translocation of Nrf2 and increased expression of Nrf2, HO-1, Hsp70 and Fra1. Moreover, we found that Nrf2 knockdown sensitized ATI-like cells to CSE and Nrf2 overexpression provided protection against CSE-induced cell death. We also observed that two antioxidant compounds N-acetylcysteine and trolox protected ATI-like cells against injury by CSE.Our study indicates that Nrf2 activation is a major factor in cellular defense of the human alveolar epithelium against CSE-induced toxicity and oxidative stress. Therefore, antioxidant agents that modulate Nrf2 would be expected to restore antioxidant and detoxifying enzymes and to prevent CS-related lung injury and perhaps lessen the development of emphysema.
Project description:It is generally acknowledged that e-cigarettes are unlikely to be as harmful as conventional cigarettes, but there is little data that quantifies their relative harms. We investigated the biological response to e-cigarette aerosol exposure (versus conventional cigarette smoke exposure) at the cellular level, by exposing human coronary artery endothelial cells (HCAEC) to aqueous filtered extracts of e-cigarette aerosol or cigarette smoke and looking at gene expression changes consistent with a stress response. This included genes controlled by the oxidant-stress sensing transcription factor NFR2 (NFE2L2), and cytochrome P450 family members.Cigarette smoke extract (CSE) was created using mainstream smoke from a single cigarette drawn through 10ml of endothelial cell growth media MV2. Electronic cigarette aerosol extract (eCAE) was created using the same apparatus, using a constant power output of 10.8w (4.2V) and 18mg/ml nicotine solution. eCAE was generated using 5 cycles of 5s heat with at least 10s in between each puff to allow the coil to cool, air being drawn through the device at 70ml/minute.HCAEC responded to the noxious components in CSE, resulting in activation of NRF2 and upregulation of cytochrome p450. However, eCAE did not induce NRF2 nuclear localisation, upregulation of NRF2-activated genes, or the upregulation of cytochrome p450.The use of e-cigarettes as a substitute for conventional cigarettes is likely to reduce immediate tobacco-related harm, at least with respect to cardiovascular harms.
Project description:Whereas cigarette smoking (CS) and dysregulated complement are thought to play central roles in age-related macular degeneration (AMD), their exact roles are unknown. The aim of this study was to determine if CS activates complement and if the antioxidant transcription factor Nrf2 modulates this response. In AMD specimens, Nrf2 immunolabeling was strong in the cytoplasm, with scattered nuclear labeling of macular retinal pigmented epithelial (RPE) cells that appeared normal, but was decreased and without nuclear labeling in dysmorphic cells overlying drusen, a hallmark AMD lesion. Cigarette smoke extract (CSE) induced Nrf2 nuclear translocation in RPE cells with increased antioxidant and complement gene expression. Whereas CFH protein was not altered by CSE, the cell membrane regulator proteins CD46, CD55, and CD59 were decreased, and C3a and C3b, but not iC3b, were increased compared to controls. C5b-9 was increased by CSE, but at sublytic levels, only after addition of normal human serum. Nrf2 knockdown enhanced the increase in C3a and C3b from CSE, but not iC3b, C5a, or C5b-9. CSE also increased IL-1b expression and secretion after C3a generation and was reduced by a C3aR antagonist. In contrast, the Nrf2 activator CDDO-Im restored complement gene expression in RPE cells exposed to CSE. We provide evidence of altered Nrf2 in human AMD and that CSE induces a proinflammatory environment specifically by generating C3a and C3b, and Nrf2 deficiency magnifies this specific complement response.
Project description:BACKGROUND & AIMS:Cigarette smoking is a major risk factor for pancreatic cancer. Aggressive pancreatic tumors contain cancer cells with stem cell features. We investigated whether cigarette smoke induces stem cell features in pancreatic cancer cells. METHODS:KrasG12D; Pdx1-Cre mice were exposed to cigarette smoke or clean air (controls) for up to 20 weeks; pancreata were collected and analyzed by histology, quantitative reverse transcription polymerase chain reaction, and confocal immunofluorescence microscopy. HPNE and Capan1 cells were exposed to cigarette smoke extract (CSE), nicotine and nicotine-derived carcinogens (NNN or NNK), or clean air (controls) for 80 days and evaluated for stem cell markers and features using flow cytometry-based autofluorescence, sphere formation, and immunoblot assays. Proteins were knocked down in cells with small interfering RNAs. We performed RNA sequencing analyses of CSE-exposed cells. We used chromatin immunoprecipitation assays to confirm the binding of FOS-like 1, AP-1 transcription factor subunit (FOSL1) to RNA polymerase II-associated factor (PAF1) promoter. We obtained pancreatic ductal adenocarcinoma (PDAC) and matched nontumor tissues (n = 15) and performed immunohistochemical analyses. RESULTS:Chronic exposure of HPNE and Capan1 cells to CSE caused them to increase markers of stem cells, including autofluorescence and sphere formation, compared with control cells. These cells increased expression of ABCG2, SOX9, and PAF1, via cholinergic receptor nicotinic alpha 7 subunit (CHRNA7) signaling to mitogen-activated protein kinase 1 and FOSL1. CSE-exposed pancreatic cells with knockdown of PAF1 did not show stem cell features. Exposure of cells to NNN and NNK led to increased expression of CHRNA7, FOSL1, and PAF1 along with stem cell features. Pancreata from KrasG12D; Pdx1-Cre mice exposed to cigarette smoke had increased levels of PAF1 mRNA and protein, compared with control mice, as well as increased expression of SOX9. Levels of PAF1 and FOSL1 were increased in PDAC tissues, especially those from smokers, compared with nontumor pancreatic tissue. CSE exposure increased expression of PHD-finger protein 5A, a pluripotent transcription factor and its interaction with PAF1. CONCLUSIONS:Exposure to cigarette smoke activates stem cell features of pancreatic cells, via CHRNA7 signaling and FOSL1 activation of PAF1 expression. Levels of PAF1 are increased in pancreatic tumors of humans and mice with chronic cigarette smoke exposure.
Project description:Expression of the pro-oncogenic mucin MUC1 is elevated by inflammation in airway epithelial cells, but the contributions of MUC1 to the development of lung cancer are uncertain. In this study, we developed our finding that cigarette smoke increases Muc1 expression in mouse lung macrophages, where we hypothesized MUC1 may contribute to cigarette smoke-induced transformation of bronchial epithelial cells. In human macrophages, cigarette smoke extract (CSE) strongly induced MUC1 expression through a mechanism involving the nuclear receptor PPAR-?. CSE-induced extracellular signal-regulated kinase (ERK) activation was also required for MUC1 expression, but it had little effect on MUC1 transcription. RNA interference-mediated attenuation of MUC1 suppressed CSE-induced secretion of TNF-? from macrophages, by suppressing the activity of the TNF-?-converting enzyme (TACE), arguing that MUC1 is required for CSE-induced and TACE-mediated TNF-? secretion. Similarly, MUC1 blockade after CSE induction through suppression of PPAR-? or ERK inhibited TACE activity and TNF-? secretion. Conditioned media from CSE-treated macrophages induced MUC1 expression and potentiated CSE-induced transformation of human bronchial epithelial cells in a TNF-?-dependent manner. Together, our results identify a signaling pathway involving PPAR-?, ERK, and MUC1 for TNF-? secretion induced by CSE from macrophages. Furthermore, our results show how MUC1 contributes to smoking-induced lung cancers that are driven by inflammatory signals from macrophages.
Project description:Chronic Obstructive Pulmonary Disease (COPD) is a highly prevalent condition characterized by inflammation and progressive obstruction of the airways. At present, there is no treatment that suppresses the chronic inflammation of the disease, and COPD patients often succumb to the condition. Excessive oxidative stress caused by smoke inhalation is a major driving force of the disease. The transcription factor NRF2 is a critical player in the battle against oxidative stress and its function is impaired in COPD. Increasing NRF2 activity may therefore be a viable therapeutic option for COPD treatment. We show that down regulation of KEAP1, a NRF2 inhibitor, protects primary human lung epithelial cells from cigarette-smoke-extract (CSE) induced cell death in an established in vitro model of radical stress. To identify new potential drug targets with a similar effect, we performed a siRNA screen of the 'druggable' genome using a NRF2 transcriptional reporter cell line. This screen identified multiple genes that when down regulated increased NRF2 transcriptional activity and provided a survival benefit in the in vitro model. Our results suggest that inhibiting components of the ubiquitin-proteasome system will have the strongest effects on NRF2 transcriptional activity by increasing NRF2 levels. We also find that down regulation of the small GTPase Rab28 or the Estrogen Receptor ESRRA provide a survival benefit. Rab28 knockdown increased NRF2 protein levels, indicating that Rab28 may regulate NRF2 proteolysis. Conversely ESRRA down regulation increased NRF2 transcriptional activity without affecting NRF2 levels, suggesting a proteasome-independent mechanism.
Project description:BACKGROUND:Aging is affected by genetic and environmental factors, and cigarette smoking is strongly associated with accumulation of senescent cells. In this study, we wanted to identify genes that may potentially be beneficial for cell survival in response to cigarette smoke and thereby may contribute to development of cellular senescence. RESULTS:Primary human bronchial epithelial cells from five healthy donors were cultured, treated with or without 1.5% cigarette smoke extract (CSE) for 24?h or were passaged into replicative senescence. Transcriptome changes were monitored using RNA-seq in CSE and non-CSE exposed cells and those passaged into replicative senescence. We found that, among 1534 genes differentially regulated during senescence and 599 after CSE exposure, 243 were altered in both conditions, representing strong enrichment. Pathways and gene sets overrepresented in both conditions belonged to cellular processes that regulate reactive oxygen species, proteasome degradation, and NF-?B signaling. CONCLUSIONS:Our results offer insights into gene expression responses during cellular aging and cigarette smoke exposure, and identify potential molecular pathways that are altered by cigarette smoke and may also promote airway epithelial cell senescence.