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1.
Am J Physiol Lung Cell Mol Physiol ; 326(5): L651-L659, 2024 May 01.
Article in English | MEDLINE | ID: mdl-38529552

ABSTRACT

Airway smooth muscle cell (ASM) is renowned for its involvement in airway hyperresponsiveness through impaired ASM relaxation and bronchoconstriction in asthma, which poses a significant challenge in the field. Recent studies have explored different targets in ASM to alleviate airway hyperresponsiveness, however, a sizeable portion of patients with asthma still experience poor control. In our study, we explored protein phosphatase 2 A (PP2A) in ASM as it has been reported to regulate cellular contractility by controlling intracellular calcium ([Ca2+]i), ion channels, and respective regulatory proteins. We obtained human ASM cells and lung tissues from healthy and patients with asthma and evaluated PP2A expression using RNA-Seq data, immunofluorescence, and immunoblotting. We further investigated the functional importance of PP2A by determining its role in bronchoconstriction using mouse bronchus and human ASM cell [Ca2+]i regulation. We found robust expression of PP2A isoforms in human ASM cells with PP2Aα being highly expressed. Interestingly, PP2Aα was significantly downregulated in asthmatic tissue and human ASM cells exposed to proinflammatory cytokines. Functionally, FTY720 (PP2A agonist) inhibited acetylcholine- or methacholine-induced bronchial contraction in mouse bronchus and further potentiated isoproterenol-induced bronchial relaxation. Mechanistically, FTY720 inhibited histamine-evoked [Ca2+]i response and myosin light chain (MLC) phosphorylation in the presence of interleukin-13 (IL-13) in human ASM cells. To conclude, we for the first time established PP2A signaling in ASM, which can be further explored to develop novel therapeutics to alleviate airway hyperresponsiveness in asthma.NEW & NOTEWORTHY This novel study deciphered the expression and function of protein phosphatase 2Aα (PP2Aα) in airway smooth muscle (ASM) during asthma and/or inflammation. We showed robust expression of PP2Aα in human ASM while its downregulation in asthmatic ASM. Similarly, we demonstrated reduced PP2Aα expression in ASM exposed to proinflammatory cytokines. PP2Aα activation inhibited bronchoconstriction of isolated mouse bronchi. In addition, we unveiled that PP2Aα activation inhibits the intracellular calcium release and myosin light chain phosphorylation in human ASM.


Subject(s)
Asthma , Bronchoconstriction , Down-Regulation , Myocytes, Smooth Muscle , Protein Phosphatase 2 , Asthma/metabolism , Asthma/pathology , Humans , Protein Phosphatase 2/metabolism , Protein Phosphatase 2/genetics , Animals , Mice , Down-Regulation/drug effects , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Myocytes, Smooth Muscle/drug effects , Bronchoconstriction/drug effects , Muscle, Smooth/metabolism , Muscle, Smooth/pathology , Muscle, Smooth/drug effects , Male , Bronchi/pathology , Bronchi/metabolism , Bronchi/drug effects , Calcium/metabolism , Female , Mice, Inbred C57BL
2.
Am J Physiol Regul Integr Comp Physiol ; 327(1): R79-R87, 2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38766774

ABSTRACT

Sulfur dioxide (SO2), a common environmental and industrial air pollutant, possesses a potent effect in eliciting cough reflex, but the primary type of airway sensory receptors involved in its tussive action has not been clearly identified. This study was carried out to determine the relative roles of three major types of vagal bronchopulmonary afferents [slowly adapting receptors (SARs), rapidly adapting receptors (RARs), and C-fibers] in regulating the cough response to inhaled SO2. Our results showed that inhalation of SO2 (300 or 600 ppm for 8 min) evoked an abrupt and intense stimulatory effect on bronchopulmonary C-fibers, which continued for the entire duration of inhalation challenge and returned toward the baseline in 1-2 min after resuming room air-breathing in anesthetized and mechanically ventilated mice. In stark contrast, the same SO2 inhalation challenge generated a distinct and consistent inhibitory effect on both SARs and phasic RARs; their phasic discharges synchronized with respiratory cycles during the baseline (breathing room air) began to decline progressively within 1-3 min after the onset of SO2 inhalation, ceased completely before termination of the 8-min inhalation challenge, and then slowly returned toward the baseline after >40 min. In a parallel study in awake mice, inhalation of SO2 at the same concentration and duration as that in the nerve recording experiments evoked cough responses in a pattern and time course similar to that observed in the C-fiber responses. Based on these results, we concluded that stimulation of vagal bronchopulmonary C-fibers is primarily responsible for triggering the cough response to inhaled SO2.NEW & NOTEWORTHY This study demonstrated that inhalation of a high concentration of sulfur dioxide, an irritant gas and common air pollutant, completely and reversibly inhibited the neural activities of both slowly adapting receptor and rapidly adapting receptor, two major types of mechanoreceptors in the lungs with their activities conducted by myelinated fibers. Furthermore, the results of this study suggested that stimulation of vagal bronchopulmonary C-fibers is primarily responsible for triggering the cough reflex responses to inhaled sulfur dioxide.


Subject(s)
Cough , Nerve Fibers, Unmyelinated , Sulfur Dioxide , Vagus Nerve , Animals , Sulfur Dioxide/administration & dosage , Cough/physiopathology , Cough/chemically induced , Vagus Nerve/drug effects , Vagus Nerve/physiology , Mice , Male , Nerve Fibers, Unmyelinated/drug effects , Mice, Inbred C57BL , Reflex/drug effects , Administration, Inhalation , Bronchi/innervation , Bronchi/drug effects , Lung/innervation , Lung/drug effects , Neurons, Afferent/drug effects
3.
Drug Metab Dispos ; 52(8): 836-846, 2024 Jul 16.
Article in English | MEDLINE | ID: mdl-38772712

ABSTRACT

This study investigated an association between the cytochrome P450 (CYP) 2C8*3 polymorphism with asthma symptom control in children and changes in lipid metabolism and pro-inflammatory signaling by human bronchial epithelial cells (HBECs) treated with cigarette smoke condensate (CSC). CYP genes are inherently variable in sequence, and while such variations are known to produce clinically relevant effects on drug pharmacokinetics and pharmacodynamics, the effects on endogenous substrate metabolism and associated physiologic processes are less understood. In this study, CYP2C8*3 was associated with improved asthma symptom control among children: Mean asthma control scores were 3.68 (n = 207) for patients with one or more copies of the CYP2C8*3 allele versus 4.42 (n = 965) for CYP2C8*1/*1 (P = 0.0133). In vitro, CYP2C8*3 was associated with an increase in montelukast 36-hydroxylation and a decrease in linoleic acid metabolism despite lower mRNA and protein expression. Additionally, CYP2C8*3 was associated with reduced mRNA expression of interleukin-6 (IL-6) and C-X-C motif chemokine ligand 8 (CXCL-8) by HBECs in response to CSC, which was replicated using the soluble epoxide hydrolase inhibitor, 12-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]-dodecanoic acid. Interestingly, 9(10)- and 12(13)- dihydroxyoctadecenoic acid, the hydrolyzed metabolites of 9(10)- and 12(13)- epoxyoctadecenoic acid, increased the expression of IL-6 and CXCL-8 mRNA by HBECs. This study reveals previously undocumented effects of the CYP2C8*3 variant on the response of HBECs to exogenous stimuli. SIGNIFICANCE STATEMENT: These findings suggest a role for CYP2C8 in regulating the epoxyoctadecenoic acid:dihydroxyoctadecenoic acid ratio leading to a change in cellular inflammatory responses elicited by environmental stimuli that exacerbate asthma.


Subject(s)
Asthma , Bronchi , Cytochrome P-450 CYP2C8 , Epithelial Cells , Humans , Asthma/drug therapy , Asthma/genetics , Asthma/metabolism , Cytochrome P-450 CYP2C8/genetics , Cytochrome P-450 CYP2C8/metabolism , Child , Male , Female , Bronchi/drug effects , Bronchi/metabolism , Bronchi/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Adolescent , Lipid Metabolism/drug effects , Lipid Metabolism/genetics , Inflammation/genetics , Inflammation/metabolism , Cells, Cultured , Quinolines/pharmacology , Polymorphism, Single Nucleotide , Acetates , Cyclopropanes , Sulfides
4.
Respir Res ; 25(1): 227, 2024 May 29.
Article in English | MEDLINE | ID: mdl-38812021

ABSTRACT

BACKGROUND: Steroid insensitivity in Chronic Obstructive Pulmonary Disease (COPD) presents a problem for controlling the chronic inflammation of the airways. The glucocorticoid receptor (GR) mediates the intracellular signaling of inhaled corticosteroids (ICS) by interacting with transcription factors and histone deacetylases (HDACs). The aim of this study was to assess if COPD patients' response to ICS in vivo, may be associated with the expression of GR, the complex of GR with transcription factors, and the expression of various HDACs in vitro. METHODS: Primary airway smooth muscle cells (ASMC) were established from endobronchial biopsies obtained from patients with asthma (n = 10), patients with COPD (n = 10) and subjects that underwent diagnostic bronchoscopy without pathological findings and served as controls (n = 6). ASMC were also established from 18 COPD patients, 10 responders and 8 non-responders to ICS, who participated in the HISTORIC study, an investigator-initiated and driven clinical trial that proved the hypothesis that COPD patients with high ASMC in their endobronchial biopsies respond better to ICS than patients with low ASMC. Expression of GR and its isoforms GRα and GRß and HDACs was investigated in primary ASMC in the absence or in the presence of dexamethasone (10- 8M) by western blotting. The complex formation of GR with transcription factors was assessed by co-immunoprecipitation. RESULTS: Expression of GR and its isoform GRα but not GRß was significantly reduced in ASMC from COPD patients as compared to controls. There were no significant differences in the expression of GR, GRα and GRß between responders and non-responders to ICS. However, treatment with dexamethasone upregulated the expression of total GR (p = 0.004) and GRα (p = 0.005) after 30 min in responders but not in non-responders. Τhe formation of the complex GR-c-Jun was increased 60 min after treatment with dexamethasone only in responders who exhibited significantly lower expression of HDAC3 (p = 0.005) and HDAC5 (p < 0.0001) as compared to non-responders. CONCLUSIONS: These data suggest that ASMC from COPD patients who do not respond to treatment with ICS, are characterized by reduced GR-c-Jun complex formation and increased expression of HDAC3 and HDAC5. TRIAL REGISTRATION: ISRCTN11017699 (Registration date: 15/11/2016).


Subject(s)
Histone Deacetylases , Myocytes, Smooth Muscle , Pulmonary Disease, Chronic Obstructive , Receptors, Glucocorticoid , Humans , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/drug therapy , Pulmonary Disease, Chronic Obstructive/pathology , Receptors, Glucocorticoid/metabolism , Receptors, Glucocorticoid/biosynthesis , Histone Deacetylases/metabolism , Histone Deacetylases/biosynthesis , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Male , Middle Aged , Female , Aged , Cells, Cultured , Adrenal Cortex Hormones/therapeutic use , Glucocorticoids/pharmacology , Dexamethasone/pharmacology , Treatment Outcome , Administration, Inhalation , Bronchi/drug effects , Bronchi/metabolism , Bronchi/pathology , Bronchi/enzymology
5.
Respir Res ; 25(1): 269, 2024 Jul 10.
Article in English | MEDLINE | ID: mdl-38982492

ABSTRACT

BACKGROUND: Cystic Fibrosis causing mutations in the gene CFTR, reduce the activity of the CFTR channel protein, and leads to mucus aggregation, airway obstruction and poor lung function. A role for CFTR in the pathogenesis of other muco-obstructive airway diseases such as Chronic Obstructive Pulmonary Disease (COPD) has been well established. The CFTR modulatory compound, Ivacaftor (VX-770), potentiates channel activity of CFTR and certain CF-causing mutations and has been shown to ameliorate mucus obstruction and improve lung function in people harbouring these CF-causing mutations. A pilot trial of Ivacaftor supported its potential efficacy for the treatment of mucus obstruction in COPD. These findings prompted the search for CFTR potentiators that are more effective in ameliorating cigarette-smoke (CS) induced mucostasis. METHODS: Small molecule potentiators, previously identified in CFTR binding studies, were tested for activity in augmenting CFTR channel activity using patch clamp electrophysiology in HEK-293 cells, a fluorescence-based assay of membrane potential in Calu-3 cells and in Ussing chamber studies of primary bronchial epithelial cultures. Addition of cigarette smoke extract (CSE) to the solutions bathing the apical surface of Calu-3 cells and primary bronchial airway cultures was used to model COPD. Confocal studies of the velocity of fluorescent microsphere movement on the apical surface of CSE exposed airway epithelial cultures, were used to assess the effect of potentiators on CFTR-mediated mucociliary movement. RESULTS: We showed that SK-POT1, like VX-770, was effective in augmenting the cyclic AMP-dependent channel activity of CFTR. SK-POT-1 enhanced CFTR channel activity in airway epithelial cells previously exposed to CSE and ameliorated mucostasis on the surface of primary airway cultures. CONCLUSION: Together, this evidence supports the further development of SK-POT1 as an intervention in the treatment of COPD.


Subject(s)
Aminophenols , Bronchi , Cystic Fibrosis Transmembrane Conductance Regulator , Epithelial Cells , Quinolones , Humans , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Quinolones/pharmacology , Aminophenols/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Bronchi/drug effects , Bronchi/metabolism , Smoke/adverse effects , Cells, Cultured , HEK293 Cells , Chloride Channel Agonists/pharmacology , Chloride Channel Agonists/therapeutic use , Respiratory Mucosa/drug effects , Respiratory Mucosa/metabolism
6.
Chem Res Toxicol ; 37(6): 957-967, 2024 Jun 17.
Article in English | MEDLINE | ID: mdl-38771128

ABSTRACT

Lung cancer is the main cause of cancer deaths around the world. Nitrosamine 4-(methyl nitrosamine)-1-(3-pyridyl)-1-butanone (NNK) is a tobacco-specific carcinogen of lung cancer. Abundant evidence implicates long noncoding RNAs (lncRNAs) in tumorigenesis. Yet, the effects and mechanisms of lncRNAs in NNK-induced carcinogenesis are still unclear. In this study, we discovered that NNK-induced transformed Beas-2B cells (Beas-2B-NNK) showed increased cell migration and proliferation while decreasing rates of apoptosis. RNA sequencing and differentially expressed lncRNAs analyses showed that lncRNA PSMB8-AS1 was obviously upregulated. Interestingly, silencing the lncRNA PSMB8-AS1 in Beas-2B-NNK cells reduced cell proliferation and migration and produced cell cycle arrest in the G2/M phase along with a decrease in CDK1 expression. Conclusively, our results demonstrate that lncRNA PSMB8-AS1 could promote the malignant characteristics of Beas-2B-NNK cells by regulating CDK1 and affecting the cell cycle, suggesting that it may supply a new prospective epigenetic mechanism for lung cancer.


Subject(s)
Bronchi , Carcinogens , Cell Cycle , Cell Proliferation , Epithelial Cells , Nicotiana , RNA, Long Noncoding , Humans , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Bronchi/cytology , Bronchi/pathology , Bronchi/drug effects , Cell Proliferation/drug effects , Nicotiana/adverse effects , Cell Cycle/drug effects , Carcinogens/toxicity , Nitrosamines/toxicity , Cell Line , Cell Movement/drug effects
7.
Prostaglandins Other Lipid Mediat ; 172: 106833, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38460760

ABSTRACT

Smoking causes several diseases such as chronic obstructive pulmonary disease (COPD). Aspirin-triggered-resolvin D1 (AT-RvD1) is a lipid mediator produced during the resolution of inflammation and demonstrates anti-inflammatory and pro-resolution effects in several inflammatory experimental models including in the airways. Here we evaluated the role of AT-RvD1 (100 nM) in bronchial epithelial cells (BEAS-2B) stimulated by cigarette smoke extract (CSE; 1%; 1 cigarette) for 24 h. CSE induced the productions of IL-1ß, TNF-α, IL-10, IL-4 and IFN-γ as well as the activations of NF-κB and STAT3 and the expression of ALX/FPR2 receptor. AT-RvD1 reduced the IL-1ß and TNF-α production and increased the production of IFN-γ. These effects were reversed BOC2, an antagonist of ALX/FPR2 receptor for AT-RvD1. The production of IL-4 and IL-10 were not altered by AT-RvD1. In addition, AT-RvD1 reduced the phosphorylation of NF-κB and STAT3 when compared to CSE-stimulated BEAS-2B cells. No alteration of ALX/FPR2 expression was observed by AT-RvD1 when compared to CSE group. In the human monocytic leukemia cell line, the relative number of copies of IL-1ß and IL-4 was significantly higher in CSE + AT-RvD1 group compared CSE group, however, the expression of M1 cytokine was more pronounced than M2 profile. AT-RvD1 could be an important target for the reduction of inflammation in the airways associated with smoking.


Subject(s)
Anti-Inflammatory Agents , Aspirin , Bronchi , Docosahexaenoic Acids , Epithelial Cells , Humans , Docosahexaenoic Acids/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Bronchi/drug effects , Bronchi/cytology , Bronchi/metabolism , Aspirin/pharmacology , Anti-Inflammatory Agents/pharmacology , NF-kappa B/metabolism , STAT3 Transcription Factor/metabolism , Cell Line , Smoke/adverse effects , Cytokines/metabolism , Nicotiana , Receptors, Lipoxin/metabolism
8.
Mol Cell ; 64(4): 746-759, 2016 11 17.
Article in English | MEDLINE | ID: mdl-27863227

ABSTRACT

Excitation-transcription coupling, linking stimulation at the cell surface to changes in nuclear gene expression, is conserved throughout eukaryotes. How closely related coexpressed transcription factors are differentially activated remains unclear. Here, we show that two Ca2+-dependent transcription factor isoforms, NFAT1 and NFAT4, require distinct sub-cellular InsP3 and Ca2+ signals for physiologically sustained activation. NFAT1 is stimulated by sub-plasmalemmal Ca2+ microdomains, whereas NFAT4 additionally requires Ca2+ mobilization from the inner nuclear envelope by nuclear InsP3 receptors. NFAT1 is rephosphorylated (deactivated) more slowly than NFAT4 in both cytoplasm and nucleus, enabling a more prolonged activation phase. Oscillations in cytoplasmic Ca2+, long considered the physiological form of Ca2+ signaling, play no role in activating either NFAT protein. Instead, effective sustained physiological activation of NFAT4 is tightly linked to oscillations in nuclear Ca2+. Our results show how gene expression can be controlled by coincident yet geographically distinct Ca2+ signals, generated by a freely diffusible InsP3 message.


Subject(s)
Calcium Signaling , Calcium/metabolism , Inositol Phosphates/metabolism , NFATC Transcription Factors/genetics , Recombinant Fusion Proteins/genetics , Animals , Basophils/cytology , Basophils/drug effects , Basophils/metabolism , Bronchi/cytology , Bronchi/drug effects , Bronchi/metabolism , Cell Line , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Gene Expression Regulation , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Inositol 1,4,5-Trisphosphate Receptors/genetics , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Leukotriene C4/pharmacology , NFATC Transcription Factors/metabolism , Protein Transport , Rats , Recombinant Fusion Proteins/metabolism , Thapsigargin/pharmacology , Transcription, Genetic
9.
Int J Mol Sci ; 25(13)2024 Jul 05.
Article in English | MEDLINE | ID: mdl-39000481

ABSTRACT

Pollen, in addition to allergens, comprise low molecular weight components (LMC) smaller than 3 kDa. Emerging evidence indicates the relevance of LMC in allergic immune responses. However, the interaction of birch pollen (BP)-derived LMC and epithelial cells has not been extensively studied. We investigated epithelial barrier modifications induced by exposure to BP LMC, using the human bronchial epithelial cell line 16HBE14o-. Epithelial cell monolayers were apically exposed to the major BP allergen Bet v 1, aqueous BP extract or BP-derived LMC. Barrier integrity after the treatments was monitored by measuring transepithelial electrical resistance at regular intervals and by using the xCELLigence Real-Time Cell Analysis system. The polarized release of cytokines 24 h following treatment was measured using a multiplex immunoassay. Epithelial barrier integrity was significantly enhanced upon exposure to BP LMC. Moreover, BP LMC induced the repair of papain-mediated epithelial barrier damage. The apical release of CCL5 and TNF-α was significantly reduced after exposure to BP LMC, while the basolateral release of IL-6 significantly increased. In conclusion, the results of our study demonstrate that BP-derived LMC modify the physical and immunological properties of bronchial epithelial cells and thus regulate airway epithelial barrier responses.


Subject(s)
Betula , Bronchi , Epithelial Cells , Molecular Weight , Pollen , Humans , Bronchi/metabolism , Bronchi/cytology , Bronchi/drug effects , Epithelial Cells/metabolism , Epithelial Cells/drug effects , Cell Line , Allergens , Cytokines/metabolism , Respiratory Mucosa/metabolism , Respiratory Mucosa/drug effects
10.
Int J Mol Sci ; 25(8)2024 Apr 13.
Article in English | MEDLINE | ID: mdl-38673911

ABSTRACT

One of the most significant challenges in human health risk assessment is to evaluate hazards from exposure to environmental chemical mixtures. Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous contaminants typically found as mixtures in gaseous and particulate phases in ambient air pollution associated with petrochemicals from Superfund sites and the burning of fossil fuels. However, little is understood about how PAHs in mixtures contribute to toxicity in lung cells. To investigate mixture interactions and component additivity from environmentally relevant PAHs, two synthetic mixtures were created from PAHs identified in passive air samplers at a legacy creosote site impacted by wildfires. The primary human bronchial epithelial cells differentiated at the air-liquid interface were treated with PAH mixtures at environmentally relevant proportions and evaluated for the differential expression of transcriptional biomarkers related to xenobiotic metabolism, oxidative stress response, barrier integrity, and DNA damage response. Component additivity was evaluated across all endpoints using two independent action (IA) models with and without the scaling of components by toxic equivalence factors. Both IA models exhibited trends that were unlike the observed mixture response and generally underestimated the toxicity across dose suggesting the potential for non-additive interactions of components. Overall, this study provides an example of the usefulness of mixture toxicity assessment with the currently available methods while demonstrating the need for more complex yet interpretable mixture response evaluation methods for environmental samples.


Subject(s)
Epithelial Cells , Polycyclic Aromatic Hydrocarbons , Humans , Polycyclic Aromatic Hydrocarbons/toxicity , Polycyclic Aromatic Hydrocarbons/metabolism , Epithelial Cells/metabolism , Epithelial Cells/drug effects , Oxidative Stress/drug effects , DNA Damage/drug effects , Models, Biological , Air Pollutants/toxicity , Cells, Cultured , Bronchi/metabolism , Bronchi/cytology , Bronchi/drug effects , Biomarkers
11.
Toxicol Appl Pharmacol ; 436: 115849, 2022 02 01.
Article in English | MEDLINE | ID: mdl-34974052

ABSTRACT

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a Group 1 human carcinogen, as classified by the International Agency for Research of Cancer (IARC), and plays a significant role in lung carcinogenesis. However, its carcinogenic mechanism has not yet been fully elucidated. In this study, we performed colony formation assays, soft-agar assays, and tumor growth in nude mice to show that 100 mg/L NNK facilitates the malignant transformation of human bronchial epithelial Beas-2B cells. Transcriptome sequencing showed that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), a post-transcriptional regulator, was differentially expressed in NNK-induced malignant transformed Beas-2B cells (2B-NNK cells). Small interfering RNA (SiRNA) was used to downregulate the expression of the IGF2BP1 gene. The reduction in protein expression, cell proliferation rate, and colony-forming ability and the increase in the apoptosis rate of Beas-2B cells transfected with the SiRNA indicated a role for IGF2BP1 in NNK-induced malignant transformation. IGF2BP1 is an N6-methyladenosine (m6A) regulatory factor, but it is not known whether its association with m6A mediates the malignant transformation of cells. Therefore, we measured the overall levels of m6A in Beas-2B cells. We found that the overall m6A level was lower in 2B-NNK cells, and knocking down IGF2BP1, the overall level of m6A was restored. Hence, we concluded that IGF2BP1 is involved in the NNK-induced malignant transformation of Beas-2B cells, possibly via m6A modification. This study therefore contributes novel insights into the environmental pathogenesis of lung cancer and the gene regulatory mechanisms of chemical carcinogenesis.


Subject(s)
Bronchi/drug effects , Butanones/pharmacology , Cell Transformation, Neoplastic/genetics , Epithelial Cells/drug effects , Methyltransferases/metabolism , Nicotiana/adverse effects , Nitrosamines/pharmacology , RNA-Binding Proteins/genetics , Adult , Aged , Animals , Apoptosis/drug effects , Apoptosis/genetics , Carcinogens/pharmacology , Cell Line , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Transformation, Neoplastic/chemically induced , Down-Regulation/physiology , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Humans , Lung/drug effects , Lung Neoplasms/chemically induced , Lung Neoplasms/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Middle Aged , Transfection/methods
12.
Toxicol Appl Pharmacol ; 436: 115884, 2022 02 01.
Article in English | MEDLINE | ID: mdl-35031324

ABSTRACT

Arsenic (As3+), a metalloid abundant in environment, is classified as a group I carcinogen associated with several common human cancers, including cancers in lung, skin, bladder, liver, and prostate (Wei et al., 2019). The mechanisms of As3+-induced carcinogenesis had been extensively studied, and different mechanisms might be involved in different types of cancer (Wei et al., 2019). Recent studies showed that exposure to a high dose of arsenic is able to induce lung cancer. Meanwhile, prolonged exposure to a low concentration of arsenic can increase the risk of lung cancer also (Liao et al., 2009; Fernández et al., 2012). Emerging evidence indicated that prolonged exposure to arsenic promotes malignant transformation and some of the transformed cells have cancer-stem-like properties (Ngalame et al., 2014). In the present report, we revealed that exposure to As3+ for short time period inhibited tyrosine-705 phosphorylation of signal transducer and activator of transcription 3 (pSTAT3Y705) and induced Src homology region 2 domain-containing phosphatase-1 (SHP-1) in bronchial epithelial cell line, BEAS-2B. In addition, we found that long term exposure of the cells to As3+ activates phosphorylation of STAT3 at serine 727 (pSTAT3S727) as well as pSTAT3Y705. Moreover, As3+ is able to induce the expression of miRNA-21 (miR-21) and decrease the expression of PDCD4. Taken together, our data suggest that activation of STAT3 and induction of miR-21 are important contributing factors to the reduced expression of PDCD4, which may play significant role in As3+-induced transformation of BEAS-2B cells.


Subject(s)
Arsenic/adverse effects , Bronchi/drug effects , Cell Transformation, Neoplastic/drug effects , Epithelial Cells/drug effects , STAT3 Transcription Factor/genetics , Signal Transduction/drug effects , Apoptosis Regulatory Proteins/genetics , Bronchi/metabolism , Carcinogenesis/chemically induced , Carcinogenesis/genetics , Cell Transformation, Neoplastic/genetics , Cells, Cultured , Epithelial Cells/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Humans , Lung Neoplasms/chemically induced , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Phosphorylation/drug effects , Phosphorylation/genetics , RNA-Binding Proteins/genetics , Reactive Oxygen Species/metabolism , Signal Transduction/genetics
13.
Ecotoxicol Environ Saf ; 236: 113486, 2022 May 01.
Article in English | MEDLINE | ID: mdl-35397445

ABSTRACT

Methanethiol is a widely existing malodorous pollutant with health effects on the human population. However, the cytotoxicity mechanism of methanethiol in vitro and its metabolic transformation (bioactivation or detoxification) have not been fully elucidated. Herein, the metabolites of methanethiol during cell culture and the cytotoxicity of methanethiol in human bronchial epithelial (16HBE) cells were investigated. Results indicate that methanethiol (10-50 µM) was partially converted into dimethyl sulfide, mainly catalyzed by thiol S-methyltransferase in the 16HBE cells, and then it induced potent cytotoxicity and cell membrane permeability. Moreover, methanethiol induced intracellular reactive oxygen species (ROS) up to 50 µM and further activated the tumor necrosis factor (TNF) signaling pathway, which eventually led to the decline in the mitochondrial membrane potential (MMP) and cell necrosis. However, all these effects were significantly alleviated with gene silencing of the methyltransferase-like protein 7B (METTL7B). These results indicate that methanethiol may induce cell necrosis in human respiratory tract cells mainly mediated by S-methyltransferase with interfering TNF and ROS induction. Non-target metabolomics results suggest that methanethiol potently affects expression of endogenous small molecule metabolites in 16HBE cells. To some extent, this work shows the possible conversion path and potential injury mechanism of human respiratory tract cells exposed to methanethiol.


Subject(s)
Bronchi , Carrier Proteins , Methyltransferases , Sulfhydryl Compounds , Bronchi/drug effects , Bronchi/enzymology , Bronchi/pathology , Carrier Proteins/metabolism , Enzyme Activation , Epithelial Cells/drug effects , Epithelial Cells/enzymology , Epithelial Cells/pathology , Humans , Methyltransferases/genetics , Methyltransferases/metabolism , Necrosis , Reactive Oxygen Species/metabolism , Sulfhydryl Compounds/pharmacology
14.
Int J Mol Sci ; 23(3)2022 Feb 04.
Article in English | MEDLINE | ID: mdl-35163691

ABSTRACT

Exposure of the airways epithelium to environmental insults, including cigarette smoke, results in increased oxidative stress due to unbalance between oxidants and antioxidants in favor of oxidants. Oxidative stress is a feature of inflammation and promotes the progression of chronic lung diseases, including Chronic Obstructive Pulmonary Disease (COPD). Increased oxidative stress leads to exhaustion of antioxidant defenses, alterations in autophagy/mitophagy and cell survival regulatory mechanisms, thus promoting cell senescence. All these events are amplified by the increase of inflammation driven by oxidative stress. Several models of bronchial epithelial cells are used to study the molecular mechanisms and the cellular functions altered by cigarette smoke extract (CSE) exposure, and to test the efficacy of molecules with antioxidant properties. This review offers a comprehensive synthesis of human in-vitro and ex-vivo studies published from 2011 to 2021 describing the molecular and cellular mechanisms evoked by CSE exposure in bronchial epithelial cells, the most used experimental models and the mechanisms of action of cellular antioxidants systems as well as natural and synthetic antioxidant compounds.


Subject(s)
Cigarette Smoking/adverse effects , Epithelial Cells/drug effects , Oxidative Stress , Animals , Bronchi/drug effects , Bronchi/metabolism , Bronchi/physiopathology , Epithelial Cells/metabolism , Epithelial Cells/physiology , Humans , Inflammation
15.
Am J Respir Cell Mol Biol ; 64(4): 426-440, 2021 04.
Article in English | MEDLINE | ID: mdl-33444514

ABSTRACT

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the United States and is primarily caused by cigarette smoking. Increased numbers of mucus-producing secretory ("goblet") cells, defined as goblet cell metaplasia or hyperplasia (GCMH), contributes significantly to COPD pathophysiology. The objective of this study was to determine whether NOTCH signaling regulates goblet cell differentiation in response to cigarette smoke. Primary human bronchial epithelial cells (HBECs) from nonsmokers and smokers with COPD were differentiated in vitro on air-liquid interface and exposed to cigarette smoke extract (CSE) for 7 days. NOTCH signaling activity was modulated using 1) the NOTCH/γ-secretase inhibitor dibenzazepine (DBZ), 2) lentiviral overexpression of the NICD3 (NOTCH3-intracellular domain), or 3) NOTCH3-specific siRNA. Cell differentiation and response to CSE were evaluated by quantitative PCR, Western blotting, immunostaining, and RNA sequencing. We found that CSE exposure of nonsmoker airway epithelium induced goblet cell differentiation characteristic of GCMH. Treatment with DBZ suppressed CSE-dependent induction of goblet cell differentiation. Furthermore, CSE induced NOTCH3 activation, as revealed by increased NOTCH3 nuclear localization and elevated NICD3 protein levels. Overexpression of NICD3 increased the expression of goblet cell-associated genes SPDEF and MUC5AC, whereas NOTCH3 knockdown suppressed CSE-mediated induction of SPDEF and MUC5AC. Finally, CSE exposure of COPD airway epithelium induced goblet cell differentiation in a NOTCH3-dependent manner. These results identify NOTCH3 activation as one of the important mechanisms by which cigarette smoke induces goblet cell differentiation, thus providing a novel potential strategy to control GCMH-related pathologies in smokers and patients with COPD.


Subject(s)
Bronchi/drug effects , Cell Differentiation/drug effects , Cigarette Smoking/adverse effects , Goblet Cells/drug effects , Pulmonary Disease, Chronic Obstructive/etiology , Receptor, Notch3/agonists , Smoke/adverse effects , Tobacco Products/adverse effects , Bronchi/metabolism , Bronchi/pathology , Case-Control Studies , Cells, Cultured , Goblet Cells/metabolism , Goblet Cells/pathology , Humans , Non-Smokers , Primary Cell Culture , Pulmonary Disease, Chronic Obstructive/genetics , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/pathology , Receptor, Notch3/genetics , Receptor, Notch3/metabolism , Signal Transduction , Smokers , Time Factors , Transcriptome
16.
Mol Pharmacol ; 99(3): 197-216, 2021 03.
Article in English | MEDLINE | ID: mdl-33376135

ABSTRACT

In 2019, the Global Initiative for Asthma treatment guidelines were updated to recommend that inhaled corticosteroid (ICS)/long-acting ß 2-adrenoceptor agonist (LABA) combination therapy should be a first-in-line treatment option for asthma. Although clinically superior to ICS, mechanisms underlying the efficacy of this combination therapy remain unclear. We hypothesized the existence of transcriptomic interactions, an effect that was tested in BEAS-2B and primary human bronchial epithelial cells (pHBECs) using formoterol and budesonide as representative LABA and ICS, respectively. In BEAS-2B cells, formoterol produced 267 (212 induced; 55 repressed) gene expression changes (≥2/≤0.5-fold) that were dominated by rapidly (1 to 2 hours) upregulated transcripts. Conversely, budesonide induced 370 and repressed 413 mRNAs, which occurred predominantly at 6-18 hours and was preceded by transcripts enriched in transcriptional regulators. Significantly, genes regulated by both formoterol and budesonide were over-represented in the genome; moreover, budesonide plus formoterol induced and repressed 609 and 577 mRNAs, respectively, of which ∼one-third failed the cutoff criterion for either treatment alone. Although induction of many mRNAs by budesonide plus formoterol was supra-additive, the dominant (and potentially beneficial) effect of budesonide on formoterol-induced transcripts, including those encoding many proinflammatory proteins, was repression. Gene ontology analysis of the budesonide-modulated transcriptome returned enriched terms for transcription, apoptosis, proliferation, differentiation, development, and migration. This "functional" ICS signature was augmented in the presence of formoterol. Thus, LABAs modulate glucocorticoid action, and comparable transcriptome-wide interactions in pHBECs imply that such effects may be extrapolated to individuals with asthma taking combination therapy. Although repression of formoterol-induced proinflammatory mRNAs should be beneficial, the pathophysiological consequences of other interactions require investigation. SIGNIFICANCE STATEMENT: In human bronchial epithelial cells, formoterol, a long-acting ß 2-adrenoceptor agonist (LABA), enhanced the expression of inflammatory genes, and many of these changes were reduced by the glucocorticoid budesonide. Conversely, the ability of formoterol to enhance both gene induction and repression by budesonide provides mechanistic insight as to how adding a LABA to an inhaled corticosteroid may improve clinical outcomes in asthma.


Subject(s)
Adrenergic beta-2 Receptor Agonists/pharmacology , Bronchi/cytology , Budesonide/pharmacology , Formoterol Fumarate/pharmacology , Gene Expression Profiling/methods , Gene Regulatory Networks/drug effects , Glucocorticoids/pharmacology , Administration, Inhalation , Bronchi/drug effects , Bronchi/metabolism , Cell Line , Drug Synergism , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Gene Expression Regulation/drug effects , Gene Ontology , Humans , Oligonucleotide Array Sequence Analysis
17.
Carcinogenesis ; 42(1): 136-147, 2021 02 11.
Article in English | MEDLINE | ID: mdl-32710611

ABSTRACT

Hexavalent chromium [Cr(VI)] is a potent human lung carcinogen. Multiple mechanisms have been proposed that contribute to Cr(VI)-induced lung carcinogenesis including oxidative stress, DNA damage, genomic instability and epigenetic modulation. However, the molecular mechanisms and pathways mediating Cr(VI) carcinogenicity have not been fully elucidated. Hedgehog (Hh) signaling is a key pathway that plays important roles in the formation of multiple tissues during embryogenesis and in the maintenance of stem cell populations in adults. Dysregulation of Hh signaling pathway has been reported in many human cancers. Here, we report a drastic reduction in both mRNA and protein levels of hedgehog-interacting protein (HHIP), a downstream target and a negative regulator of Hh signaling, in Cr(VI)-transformed cells. These findings point to a potential role of Hh signaling in Cr(VI)-induced malignant transformation and lung carcinogenesis. Cr(VI)-transformed cells exhibited DNA hypermethylation and silencing histone marks in the promoter region of HHIP, indicating that an epigenetic mechanism mediates Cr(VI)-induced silencing of HHIP. In addition, the major targets of Hh signaling (GLI1-3 and PTCH1) were significantly increased in Cr(VI)-transformed cells, suggesting an aberrant activation of Hh signaling in these cells. Moreover, ectopically expressing HHIP not only suppressed Hh signaling but also inhibited cell proliferation and anchorage-independent growth in Cr(VI)-transformed cells. In conclusion, these findings establish a novel regulatory mechanism underlying Cr(VI)-induced lung carcinogenesis and provide new insights for developing a better diagnostic and prognostic strategy for Cr(VI)-related human lung cancer.


Subject(s)
Carrier Proteins/genetics , Cell Transformation, Neoplastic/genetics , Chromium/toxicity , Gene Expression Regulation, Neoplastic/drug effects , Lung Neoplasms/chemically induced , Membrane Glycoproteins/genetics , Bronchi/cytology , Bronchi/drug effects , Bronchi/pathology , Cell Line , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Transformation, Neoplastic/chemically induced , DNA Methylation/drug effects , Down-Regulation/drug effects , Epithelial Cells/drug effects , Epithelial Cells/pathology , Gene Silencing/drug effects , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Respiratory Mucosa/cytology , Respiratory Mucosa/drug effects , Respiratory Mucosa/pathology , Signal Transduction/drug effects
18.
Pflugers Arch ; 473(2): 287-311, 2021 02.
Article in English | MEDLINE | ID: mdl-33386991

ABSTRACT

TGF-ß1 is a major mediator of airway tissue remodelling during atopic asthma and affects tight junctions (TJs) of airway epithelia. However, its impact on TJs of ciliated epithelia is sparsely investigated. Herein we elaborated effects of TGF-ß1 on TJs of primary human bronchial epithelial cells. We demonstrate that TGF-ß1 activates TGF-ß1 receptors TGFBR1 and TGFBR2 resulting in ALK5-mediated phosphorylation of SMAD2. We observed that TGFBR1 and -R2 localize specifically on motile cilia. TGF-ß1 activated accumulation of phosphorylated SMAD2 (pSMAD2-C) at centrioles of motile cilia and at cell nuclei. This triggered an increase in paracellular permeability via cellular redistribution of claudin 3 (CLDN3) from TJs into cell nuclei followed by disruption of epithelial integrity and formation of epithelial lesions. Only ciliated cells express TGF-ß1 receptors; however, nuclear accumulations of pSMAD2-C and CLDN3 redistribution were observed with similar time course in ciliated and non-ciliated cells. In summary, we demonstrate a role of motile cilia in TGF-ß1 sensing and showed that TGF-ß1 disturbs TJ permeability of conductive airway epithelia by redistributing CLDN3 from TJs into cell nuclei. We conclude that the observed effects contribute to loss of epithelial integrity during atopic asthma.


Subject(s)
Bronchi/drug effects , Cilia/drug effects , Claudin-3/metabolism , Epithelial Cells/drug effects , Tight Junctions/drug effects , Transforming Growth Factor beta1/pharmacology , Bronchi/metabolism , Cells, Cultured , Cilia/metabolism , Claudin-3/genetics , Electric Impedance , Epithelial Cells/metabolism , Humans , Permeability , Phosphorylation , Protein Transport , Receptor, Transforming Growth Factor-beta Type I/agonists , Receptor, Transforming Growth Factor-beta Type I/metabolism , Receptor, Transforming Growth Factor-beta Type II/agonists , Receptor, Transforming Growth Factor-beta Type II/metabolism , Signal Transduction , Smad2 Protein/metabolism , Tight Junctions/genetics , Tight Junctions/metabolism
19.
Am J Physiol Lung Cell Mol Physiol ; 320(4): L600-L614, 2021 04 01.
Article in English | MEDLINE | ID: mdl-33295836

ABSTRACT

The growing interest in regulating flavored E-liquids must incorporate understanding of the "flavoring profile" of each E-liquid-which flavorings (flavoring chemicals) are present and at what concentrations not just focusing on the flavor on the label. We investigated the flavoring profile of 10 different flavored E-liquids. We assessed bronchial epithelial cell viability and apoptosis, phagocytosis of bacteria and apoptotic cells by macrophages after exposure to E-cigarette vapor extract (EVE). We validated our data in normal human bronchial epithelial cells (NHBE) and alveolar macrophages (AM) from healthy donors. We also assessed cytokine release and validated in the saliva from E-cigarette users. Increased necrosis/apoptosis (16.1-64.5% apoptosis) in 16HBE cells was flavor dependent, and NHBEs showed an increased susceptibility to flavors. In THP-1 differentiated macrophages phagocytosis was also flavor dependent, with AM also showing increased susceptibility to flavors. Further, Banana and Chocolate were shown to reduce surface expression of phagocytic target recognition receptors on alveolar macrophages. Banana and Chocolate increased IL-8 secretion by NHBE, whereas all 4 flavors reduced AM IL-1ß secretion, which was also reduced in the saliva of E-cigarette users compared with healthy controls. Flavorant profiles of E-liquids varied from simple 2 compound mixtures to complex mixtures containing over a dozen flavorants. E-liquids with high benzene content, complex flavoring profiles, high chemical concentration had the greatest impacts. The Flavorant profile of E-liquids is key to disruption of the airway status quo by increasing bronchial epithelial cell apoptosis, causing alveolar macrophage phagocytic dysfunction, and altering airway cytokines.


Subject(s)
Apoptosis , Bronchi/pathology , Cytokines/metabolism , Electronic Nicotine Delivery Systems/statistics & numerical data , Flavoring Agents/adverse effects , Macrophages/pathology , Phagocytosis , Bronchi/drug effects , Bronchi/metabolism , Humans , Macrophages/drug effects , Risk Factors
20.
Am J Physiol Lung Cell Mol Physiol ; 321(6): L1147-L1160, 2021 12 01.
Article in English | MEDLINE | ID: mdl-34668421

ABSTRACT

Aberrant anion secretion across the bronchial epithelium is associated with airway disease, most notably in cystic fibrosis. Although the cystic fibrosis transmembrane conductance regulator (CFTR) is recognized as the primary source of airway anion secretion, alternative anion transport mechanisms play a contributing role. An alternative anion transporter of growing interest is SLC26A9, a constitutively active chloride channel that has been shown to interact with CFTR and may also contribute to bicarbonate secretion. Interest in SLC26A9 has been fueled by genome-wide association studies that suggest it is a significant modifier of CF disease severity. Despite this growing evidence that SLC26A9 plays an important role in the airway, its presence and function in bronchial epithelia remain poorly understood, in part, because its activity is difficult to separate from the activity of CFTR. Here, we present results using primary human bronchial epithelia (HBE) from multiple patient sources to confirm that SLC26A9 mRNA is present in HBE and that its constitutive channel activity is unaffected by knockdown of CFTR. Furthermore, SLC26A9 and CFTR show differential responses to common inhibitors of anion secretion. Finally, we assess the impact of bicarbonate on the activity of SLC26A9 and CFTR. These results confirm that SLC26A9 is the primary source of constitutive anion secretion across HBE, and should inform future studies focused on activation of SLC26A9 as an alternative anion channel in CF. These results should provide a strong foundation to investigate how single-nucleotide polymorphisms in SLC26A9 modulate airway disease.


Subject(s)
Antiporters/metabolism , Bicarbonates/metabolism , Bronchi/metabolism , Chlorides/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/metabolism , Epithelial Cells/metabolism , Sulfate Transporters/metabolism , Antiporters/genetics , Antiporters/pharmacology , Biological Transport , Bronchi/drug effects , Cells, Cultured , Cystic Fibrosis/drug therapy , Cystic Fibrosis/pathology , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Epithelial Cells/drug effects , Humans , Sulfate Transporters/genetics
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