scholarly journals BACH1, the master regulator of oxidative stress, has a dual effect on CFTR expression.

2021 ◽  
Author(s):  
Monali NandyMazumdar ◽  
Alekh Paranjapye ◽  
James Browne ◽  
Shiyi Yin ◽  
Shih-Hsing Leir ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cellular Response ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Airway Epithelial Cells ◽  
Cftr Gene ◽  
Airway Epithelial ◽  
Dual Effect ◽  
Master Regulator ◽  
Specific Expression

The cystic fibrosis transmembrane conductance regulator (CFTR) gene lies within a TAD in which multiple cis-regulatory elements (CREs) and transcription factors (TFs) regulate its cell-specific expression. The CREs are recruited to the gene promoter by a looping mechanism that depends upon both architectural proteins and specific TFs. An siRNA screen to identify TFs coordinating CFTR expression in airway epithelial cells suggested an activating role for BTB Domain and CNC Homolog 1 (BACH1). BACH1 is a ubiquitous master regulator of the cellular response to oxidative stress. Here we show that BACH1 may have a dual effect on CFTR expression by direct occupancy of CREs at physiological oxygen (~8%), while indirectly modulating expression under conditions of oxidative stress. Hence BACH1, can activate or repress the same gene, to fine tune expression in response to environmental cues such as cell stress. Furthermore, our 4C-seq data suggest that BACH1 can also directly regulate CFTR gene expression by modulating locus architecture through occupancy at known enhancers and structural elements, and depletion of BACH1 alters the higher order chromatin structure.

scholarly journals Looping of upstream cis-regulatory elements is required for CFTR expression in human airway epithelial cells

2020 ◽  
Vol 48 (7) ◽  
pp. 3513-3524 ◽  
Author(s):  
Monali NandyMazumdar ◽  
Shiyi Yin ◽  
Alekh Paranjapye ◽  
Jenny L Kerschner ◽  
Hannah Swahn ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Airway Epithelial Cells ◽  
Control Mechanisms ◽  
Airway Epithelial ◽  
Cell Type ◽  
Long Range Interactions ◽  
Cell Type Specific

Abstract The CFTR gene lies within an invariant topologically associated domain (TAD) demarcated by CTCF and cohesin, but shows cell-type specific control mechanisms utilizing different cis-regulatory elements (CRE) within the TAD. Within the respiratory epithelium, more than one cell type expresses CFTR and the molecular mechanisms controlling its transcription are likely divergent between them. Here, we determine how two extragenic CREs that are prominent in epithelial cells in the lung, regulate expression of the gene. We showed earlier that these CREs, located at −44 and −35 kb upstream of the promoter, have strong cell-type-selective enhancer function. They are also responsive to inflammatory mediators and to oxidative stress, consistent with a key role in CF lung disease. Here, we use CRISPR/Cas9 technology to remove these CREs from the endogenous locus in human bronchial epithelial cells. Loss of either site extinguished CFTR expression and abolished long-range interactions between these sites and the gene promoter, suggesting non-redundant enhancers. The deletions also greatly reduced promoter interactions with the 5′ TAD boundary. We show substantial recruitment of RNAPII to the −35 kb element and identify CEBPβ as a key activator of airway expression of CFTR, likely through occupancy at this CRE and the gene promoter.

scholarly journals Ozone effect on inflammatory and proteomic profile of human macrophages and airway epithelial cells

2020 ◽  
Vol 30 (Supplement_5) ◽  
Author(s):  
L Falcone ◽  
E Aruffo ◽  
P Di Carlo ◽  
P Del Boccio ◽  
M C Cufaro ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Air Pollution ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Urban Areas ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Proteomics Analysis ◽  
Human Macrophages ◽  
Tnf Α

Abstract Background Reactive oxygen species (ROS) and oxidative stress in the respiratory system are involved in lung inflammation and tumorigenesis. Ozone (O3) is one of the main components of air pollution in urban areas able to act as strong pro-oxidant agent, however its effects on human health is still poorly investigated. In this study the effect of O3 has been evaluated in THP-1 monocytes differentiated into macrophages with PMA and in HBEpC (primary human bronchial epithelial) cells, two model systems for in vitro studies and translational research. Methods Cell viability, ROS and pro-inflammatory cytokines like interleukin-8(IL-8) and tumor necrosis factor(TNF-α) have been tested in the above-mentioned cell lines not exposed to any kind of pollution (basal condition-b.c.) or exposed to O3 at a concentration of 120 ppb. In HBEpC a labelfree shotgun proteomics analysis has been also performed in the same conditions. Results Ozone significantly increased the production of IL-8 and TNF-α in THP-1 whereas no changes were shown in HBEpC. In both cell lines lipopolysaccharide(LPS) caused an increase of IL-8 and TNF-α production in b.c. and O3 treatment potentiated this effect. Ozone exposure increased ROS formation in a time dependent manner in both cell lines and in THP-1 cells a decrease in catalase activity was also shown. Finally, according to these data, functional proteomics analysis revealed that in HBEpC exposure to O3 many differential proteins are related to oxidative stress and inflammation. Conclusions Our results indicate that O3, at levels that can be reached in urban areas, causes an increase of pro-inflammatory agents either per se or potentiating the effect of immune response stimulators in cell models of human macrophages and human airway epithelial cells. Interestingly, the proteomic analysis showed that besides the dysregulated proteins, O3 induced the expression of AKR1D1 and AKR1B10, proteins recognized to play a significant role in cancer development. Key messages This study adds new pieces of information on the association between O3 exposure and detrimental effects on respiratory system. This study suggests the need for further research on the mechanisms involved and for a continued monitoring/re-evaluation of air pollution standards aimed at safeguarding human health.

Glutathione permeability of CFTR

1998 ◽  
Vol 275 (1) ◽  
pp. C323-C326 ◽  
Author(s):  
Paul Linsdell ◽  
John W. Hanrahan
Keyword(s):  
Oxidative Stress ◽  
Cystic Fibrosis ◽  
Ion Channel ◽  
Airway Epithelial Cells ◽  
Transmembrane Conductance Regulator ◽  
Airway Epithelial ◽  
Transmembrane Conductance ◽  
High Concentrations ◽  
Cystic Fibrosis Transmembrane ◽  
Glutathione Transport

The cystic fibrosis transmembrane conductance regulator (CFTR) forms an ion channel that is permeable both to Cl− and to larger organic anions. Here we show, using macroscopic current recording from excised membrane patches, that the anionic antioxidant tripeptide glutathione is permeant in the CFTR channel. This permeability may account for the high concentrations of glutathione that have been measured in the surface fluid that coats airway epithelial cells. Furthermore, loss of this pathway for glutathione transport may contribute to the reduced levels of glutathione observed in airway surface fluid of cystic fibrosis patients, which has been suggested to contribute to the oxidative stress observed in the lung in cystic fibrosis. We suggest that release of glutathione into airway surface fluid may be a novel function of CFTR.

scholarly journals Cell-Selective Regulation of CFTR Gene Expression: Relevance to Gene Editing Therapeutics

Genes ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 235 ◽  
Author(s):  
Hannah Swahn ◽  
Ann Harris
Keyword(s):  
Gene Expression ◽  
Cystic Fibrosis ◽  
Gene Editing ◽  
Genetic Diseases ◽  
3D Structure ◽  
Three Dimensional ◽  
Cell Types ◽  
Regulatory Elements ◽  
Cftr Gene ◽  
Specific Expression

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is an attractive target for gene editing approaches, which may yield novel therapeutic approaches for genetic diseases such as cystic fibrosis (CF). However, for gene editing to be effective, aspects of the three-dimensional (3D) structure and cis-regulatory elements governing the dynamic expression of CFTR need to be considered. In this review, we focus on the higher order chromatin organization required for normal CFTR locus function, together with the complex mechanisms controlling expression of the gene in different cell types impaired by CF pathology. Across all cells, the CFTR locus is organized into an invariant topologically associated domain (TAD) established by the architectural proteins CCCTC-binding factor (CTCF) and cohesin complex. Additional insulator elements within the TAD also recruit these factors. Although the CFTR promoter is required for basal levels of expression, cis-regulatory elements (CREs) in intergenic and intronic regions are crucial for cell-specific and temporal coordination of CFTR transcription. These CREs are recruited to the promoter through chromatin looping mechanisms and enhance cell-type-specific expression. These features of the CFTR locus should be considered when designing gene-editing approaches, since failure to recognize their importance may disrupt gene expression and reduce the efficacy of therapies.

Induction of Oxidative DNA Damage and Epithelial Mesenchymal Transitions in Small Airway Epithelial Cells Exposed to Cosmetic Aerosols

2020 ◽  
Vol 177 (1) ◽  
pp. 248-262
Author(s):  
Kaitlin M Pearce ◽  
Imoh Okon ◽  
Christa Watson-Wright
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Epithelial Cells ◽  
Oxidative Dna Damage ◽  
Airway Epithelial Cells ◽  
Consumer Products ◽  
Small Airway ◽  
Airway Epithelial ◽  
Western Blots ◽  
Small Airway Epithelial Cells

Abstract Engineered metal nanoparticles (ENPs) are frequently incorporated into aerosolized consumer products, known as nano-enabled products (NEPs). Concern for consumer pulmonary exposures grows as NEPs produce high concentrations of chemically modified ENPs. A significant knowledge gap still exists surrounding NEP aerosol respiratory effects as previous research focuses on pristine/unmodified ENPs. Our research evaluated metal-containing aerosols emitted from nano-enabled cosmetics and their induction of oxidative stress and DNA damage, which may contribute to epithelial mesenchymal transitions (EMT) within primary human small airway epithelial cells. We utilized an automated NEP generation system to monitor and gravimetrically collect aerosols from two aerosolized cosmetic lines. Aerosol monitoring data were inputted into modeling software to determine potential inhaled dose and in vitro concentrations. Toxicological profiles of aerosols and comparable pristine ENPs (TiO2 and Fe2O3) were used to assess reactive oxygen species and oxidative stress by fluorescent-based assays. Single-stranded DNA (ssDNA) damage and 8-oxoguanine were detected using the CometChip assay after 24-h exposure. Western blots were conducted after 21-day exposure to evaluate modulation of EMT markers. Results indicated aerosols possessed primarily ultrafine particles largely depositing in tracheobronchial lung regions. Significant increases in oxidative stress, ssDNA damage, and 8-oxoguanine were detected post-exposure to aerosols versus pristine ENPs. Western blots revealed statistically significant decreases in E-cadherin and increases in vimentin, fascin, and CD44 for two aerosols, indicating EMT. This work suggests certain prolonged NEP inhalation exposures cause oxidative DNA damage, which may play a role in cellular changes associated with reduced respiratory function and should be of concern.

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