Multiplexed quantitative high content screening reveals that cigarette smoke condensate induces changes in cell structure and function through alterations in cell signaling pathways in human bronchial cells

Carter, Charleata A.; Hamm, Jon

doi:10.1016/j.tox.2009.04.039

Cited by 27 publications

(25 citation statements)

References 80 publications

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“…indirectly supporting the endosome/lysosome compartment as the temporary DCB-230 destination after uptake. Relocation of these compartments to the perinuclear region has been observed after PM uptake (40) and usually accompanies loss of focal adhesions (41). DCB-230 mimicked these responses.…”

Section: Discussionmentioning

confidence: 88%

Radical-Containing Ultrafine Particulate Matter Initiates Epithelial-to-Mesenchymal Transitions in Airway Epithelial Cells

Thevenot

Saravia

Jin

et al. 2013

Am J Respir Cell Mol Biol

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Environmentally persistent free radicals (EPFRs) in combustiongenerated particulate matter (PM) are capable of inducing pulmonary pathologies and contributing to the development of environmental asthma. In vivo exposure of infant rats to EPFRs demonstrates their ability to induce airway hyperresponsiveness to methacholine, a hallmark of asthma. However, the mechanisms by which combustionderived EPFRs elicit in vivo responses remain elusive. In this study, we used a chemically defined EPFR consisting of approximately 0.2 mm amorphrous silica containing 3% cupric oxide with the organic pollutant 1,2-dichlorobenzene (DCB-230). DCB-230 possesses similar radical content to urban-collected EPFRs but offers several advantages, including lack of contaminants and chemical uniformity. DCB-230 was readily taken up by BEAS-2B and at high doses (200 mg/cm 2 ) caused substantial necrosis. At low doses (20 mg/cm 2 ), DCB-230 particles caused lysosomal membrane permeabilization, oxidative stress, and lipid peroxidation within 24 hours of exposure. During this period, BEAS-2B underwent epithelial-to-mesenchymal transition (EMT), including loss of epithelial cell morphology, decreased E-cadherin expression, and increased a-smooth muscle actin (a-SMA) and collagen I production. Similar results were observed in neonatal air-liquid interface culture (i.e., disruption of epithelial integrity and EMT). Acute exposure of infant mice to DCB-230 resulted in EMT, as confirmed by lineage tracing studies and evidenced by coexpression of epithelial E-cadherin and mesenchymal a-SMA proteins in airway cells and increased SNAI1 expression in the lungs. EMT in neonatal mouse lungs after EPFR exposure may provide an explanation for epidemiological evidence supporting PM exposure and increased risk of asthma.Keywords: particulate matter; epithelial-mesenchymal transition; environmental asthma; pediatric Combustion-generated particulate matter (PM) from industrial processes and burning of biomass and fossil fuels has been linked with adverse pulmonary health effects (1). Environmental PM, both fine and ultrafine, is capable of airway deposition, alveolar penetration, respiratory distress, and exacerbation of preexisting pulmonary conditions. Previous studies highlight the potential roles of PM exposure in predisposing to asthma and pulmonary fibrosis (2-4). Additionally, PM has adjuvant effects when combined with innocuous antigen (5-7) and induces cellular damage, stimulating fibrotic remodeling in adult rodent exposure models (2). The developing pulmonary and immune systems are particularly vulnerable (8). We have developed a model for studying particulate exposures in neonatal rodents (, 7 d of age) (9), which we apply here to understand the effects of combustiongenerated environmentally persistent free radicals (EPFRs) on pulmonary airway remodeling.Delineation of the influences of particulate burden from the reactive chemical species complexed with the particulate has proven difficult. The nature of the chemical species drastically influences ...

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Section: Discussionmentioning

confidence: 88%

Radical-Containing Ultrafine Particulate Matter Initiates Epithelial-to-Mesenchymal Transitions in Airway Epithelial Cells

Thevenot

Saravia

Jin

et al. 2013

Am J Respir Cell Mol Biol

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“…Our data showed that cigarette smoke-exposed rat lung tissues displayed an increase in activated PKC-a staining and an increase in the number of activated PKC-a-stained alveolar macrophages compared with controls. Cigarette smoke exposure activates PKC-a in human bronchial BEAS-2B cells (Carter and Hamm 2009) and in tracheal epithelial cells from tracheal ring explants from Sprague Dawley rats (Vander Top, Wyatt, and Gentry-Nielsen 2005). Alterations in PKC occur in lung cancers (Bae et al 2007) and PKC-a is highly expressed in patients with lung adenocarcinoma (Lahn et al 2004).…”

Section: Discussionmentioning

confidence: 99%

Effects of Short-Term Cigarette Smoke Exposure on Fischer 344 Rats and on Selected Lung Proteins

Carter¹,

Misra²

2010

Toxicol Pathol

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A short-term 5-day cigarette smoke exposure study was conducted in Fischer 344 rats to identify smoke-induced lung protein changes. Groups of 10 male and 10 female rats at 5 weeks of age were randomly assigned to one of four exposure groups. Animals received filtered air (control) or 75, 200, or 400 mg total particulate matter (TPM)/m 3 of diluted Kentucky reference 3R4F cigarette smoke. Nose-only exposures were conducted for 3 hours/day for 5 consecutive days. Mean body weights were significantly reduced only in male rats exposed to 400 mg TPM/m 3 . Body weight gains were significantly reduced in 200-and 400-mg TPM/m 3 -exposed males and in all smoke-exposed females compared with controls. Alveolar histiocytosis increased slightly in all smoke exposed-females and 200-and 400-mg TPM/m 3 -exposed males. Cyclooxygenase-2 staining increased at 400 mg TPM/m 3 . Matrix metalloproteinase-12 staining of alveolar macrophages and bronchiolar epithelia increased in smoke-exposed animals, especially 400-mg TPM/m 3 -exposed females. Protein kinase C-a staining increased in macrophages at 200-and 400-mg TPM/m 3 doses. c-Jun NH 2 -terminal kinases staining decreased in smoke-exposed tissues. The identified changed proteins play roles in inflammation, transformation, proliferation, stress activation, and apoptosis.

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“…For F‐actin analysis, images were collected in 2 channels with the appropriate filter selected for analysis of each fluorochrome. Channel 1 was used for nuclear labeling (DAPI staining), to establish the plane of focus for the cells (21, 22), and channel 2 was used for labeling the area of F‐actin stress fibers with rhodamine‐phalloidin. The Morphology Explorer Bio‐application (Thermo Fisher—Cellomics) was used to quantify fluorescent images of the cytoskeleton, and 2 thresholds were set: fluorescence intensity larger than 60 and F‐actin stress fiber length larger than 100 pixels.…”

Section: Methodsmentioning

confidence: 99%

NHERF1 regulates actin cytoskeleton organization through modulation of α‐actinin‐4 stability

et al. 2015

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The actin cytoskeleton is composed of a highly dynamic network of filamentous proteins, yet the molecular mechanism that regulates its organization and remodeling remains elusive. In this study, Na + /H + exchanger regulatory factor (NHERF)-1 loss-of-function and gain-of-function experiments reveal that polymerized actin cytoskeleton (F-actin) in HeLa cells is disorganized by NHERF1, whereas actin protein expression levels exhibit no detectable change. To elucidate the molecular mechanism underlying actin cytoskeleton disorganization by NHERF1, a combined 2-dimensional electrophoresismatrix-assisted laser desorption/ionization-time of flight mass spectrometry approach was used to screen for proteins regulated by NHERF1 in HeLa cells. a-Actinin-4, an actin cross-linking protein, was identified. Glutathione S-transferase pull-down and coimmunoprecipitation studies showed the a-actinin-4 carboxyl-terminal region specifically interacted with the NHERF1 postsynaptic density 95/disc-large/zona occludens-1 domain. The NHERF1/ a-actinin-4 interaction increased a-actinin-4 ubiquitination and decreased its expression levels, resulting in actin cytoskeleton disassembly. Our study identified a-actinin-4 as a novel NHERF1 interaction partner and provided new insights into the regulatory mechanism of the actin cytoskeleton by NHERF1

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Multiplexed quantitative high content screening reveals that cigarette smoke condensate induces changes in cell structure and function through alterations in cell signaling pathways in human bronchial cells

Cited by 27 publications

References 80 publications

Radical-Containing Ultrafine Particulate Matter Initiates Epithelial-to-Mesenchymal Transitions in Airway Epithelial Cells

Radical-Containing Ultrafine Particulate Matter Initiates Epithelial-to-Mesenchymal Transitions in Airway Epithelial Cells

Effects of Short-Term Cigarette Smoke Exposure on Fischer 344 Rats and on Selected Lung Proteins

NHERF1 regulates actin cytoskeleton organization through modulation of α‐actinin‐4 stability

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