Loss of Gadd45a does not modify the pulmonary response to oxidative stress

2005 ◽  
Vol 288 (4) ◽  
pp. L663-L671 ◽  
Author(s):  
Jason M. Roper ◽  
Sean C. Gehen ◽  
Rhonda J. Staversky ◽  
M. Christine Hollander ◽  
Albert J. Fornace ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Epithelial Cells ◽  
Oxidative Dna Damage ◽  
Genotoxic Stress ◽  
Clara Cell ◽  
Heme Oxygenase 1 ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii

It is well established that exposure to high levels of oxygen (hyperoxia) injures and kills microvascular endothelial and alveolar type I epithelial cells. In contrast, significant death of airway and type II epithelial cells is not observed at mortality, suggesting that these cell types may express genes that protect against oxidative stress and damage. During a search for genes induced by hyperoxia, we previously reported that airway and alveolar type II epithelial cells uniquely express the growth arrest and DNA damage ( Gadd) 45a gene. Because Gadd45a has been implicated in protection against genotoxic stress, adult Gadd45a (+/+) and Gadd45a (−/−) mice were exposed to hyperoxia to investigate whether it protected epithelial cells against oxidative stress. During hyperoxia, Gadd45a deficiency did not affect loss of airway epithelial expression of Clara cell secretory protein or type II epithelial cell expression of pro-surfactant protein C. Likewise, Gadd45a deficiency did not alter recruitment of inflammatory cells, edema, or overall mortality. Consistent with Gadd45a not affecting the oxidative stress response, p21Cip1/WAF1 and heme oxygenase-1 were comparably induced in Gadd45a (+/+) and Gadd45a (−/−) mice. Additionally, Gadd45a deficiency did not affect oxidative DNA damage or apoptosis as assessed by oxidized guanine and terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling staining. Overexpression of Gadd45a in human lung adenocarcinoma cells did not affect viability or survival during exposure, whereas it was protective against UV-radiation. We conclude that increased tolerance of airway and type II epithelial cells to hyperoxia is not attributed solely to expression of Gadd45a.

scholarly journals Dynamic Compartmentalization of Base Excision Repair Proteins in Response to Nuclear and Mitochondrial Oxidative Stress

2008 ◽  
Vol 29 (3) ◽  
pp. 794-807 ◽  
Author(s):  
Lyra M. Griffiths ◽  
Dan Swartzlander ◽  
Kellen L. Meadows ◽  
Keith D. Wilkinson ◽  
Anita H. Corbett ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Dna Repair ◽  
Base Excision Repair ◽  
Oxidative Dna Damage ◽  
Excision Repair ◽  
Intracellular Localization ◽  
Genotoxic Stress ◽  
Mitochondrial Oxidative Stress ◽  
Base Excision

ABSTRACT DNAs harbored in both nuclei and mitochondria of eukaryotic cells are subject to continuous oxidative damage resulting from normal metabolic activities or environmental insults. Oxidative DNA damage is primarily reversed by the base excision repair (BER) pathway, initiated by N-glycosylase apurinic/apyrimidinic (AP) lyase proteins. To execute an appropriate repair response, BER components must be distributed to accommodate levels of genotoxic stress that may vary considerably between nuclei and mitochondria, depending on the growth state and stress environment of the cell. Numerous examples exist where cells respond to signals, resulting in relocalization of proteins involved in key biological transactions. To address whether such dynamic localization contributes to efficient organelle-specific DNA repair, we determined the intracellular localization of the Saccharomyces cerevisiae N-glycosylase/AP lyases, Ntg1 and Ntg2, in response to nuclear and mitochondrial oxidative stress. Fluorescence microscopy revealed that Ntg1 is differentially localized to nuclei and mitochondria, likely in response to the oxidative DNA damage status of the organelle. Sumoylation is associated with targeting of Ntg1 to nuclei containing oxidative DNA damage. These studies demonstrate that trafficking of DNA repair proteins to organelles containing high levels of oxidative DNA damage may be a central point for regulating BER in response to oxidative stress.

scholarly journals Transitional human alveolar type II epithelial cells suppress extracellular matrix and growth factor gene expression in lung fibroblasts

2019 ◽  
Vol 317 (2) ◽  
pp. L283-L294 ◽  
Author(s):  
Kelly A. Correll ◽  
Karen E. Edeen ◽  
Rachel L. Zemans ◽  
Elizabeth F. Redente ◽  
Karina A. Serban ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Surfactant Protein ◽  
Lung Fibroblasts ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
I Cells

Epithelial-fibroblast interactions are thought to be very important in the adult lung in response to injury, but the specifics of these interactions are not well defined. We developed coculture systems to define the interactions of adult human alveolar epithelial cells with lung fibroblasts. Alveolar type II cells cultured on floating collagen gels reduced the expression of type 1 collagen (COL1A1) and α-smooth muscle actin (ACTA2) in fibroblasts. They also reduced fibroblast expression of hepatocyte growth factor (HGF), fibroblast growth factor 7 (FGF7, KGF), and FGF10. When type II cells were cultured at an air-liquid interface to maintain high levels of surfactant protein expression, this inhibitory activity was lost. When type II cells were cultured on collagen-coated tissue culture wells to reduce surfactant protein expression further and increase the expression of some type I cell markers, the epithelial cells suppressed transforming growth factor-β (TGF-β)-stimulated ACTA2 and connective tissue growth factor (CTGF) expression in lung fibroblasts. Our results suggest that transitional alveolar type II cells and likely type I cells but not fully differentiated type II cells inhibit matrix and growth factor expression in fibroblasts. These cells express markers of both type II cells and type I cells. This is probably a normal homeostatic mechanism to inhibit the fibrotic response in the resolution phase of wound healing. Defining how transitional type II cells convert activated fibroblasts into a quiescent state and inhibit the effects of TGF-β may provide another approach to limiting the development of fibrosis after alveolar injury.

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.

Identification and isolation of mouse type II cells on the basis of intrinsic expression of enhanced green fluorescent protein

2003 ◽  
Vol 285 (3) ◽  
pp. L691-L700 ◽  
Author(s):  
Jason M. Roper ◽  
Rhonda J. Staversky ◽  
Jacob N. Finkelstein ◽  
Peter C. Keng ◽  
Michael A. O'Reilly
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Airway Epithelial Cells ◽  
Clara Cell ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Green Fluorescence ◽  
Experimental Conditions ◽  
Green Fluorescent

The unique morphology and cell-specific expression of surfactant genes have been used to identify and isolate alveolar type II epithelial cells. Because these attributes can change during lung injury, a novel method was developed for detecting and isolating mouse type II cells on the basis of transgenic expression of enhanced green fluorescence protein (EGFP). A line of transgenic mice was created in which EGFP was targeted to type II cells under control of the human surfactant protein (SP)-C promoter. Green fluorescent cells that colocalized by immunostaining with endogenous pro-SP-C were scattered throughout the parenchyma. EGFP was not detected in Clara cell secretory protein-expressing airway epithelial cells or other nonlung tissues. Pro-SP-C immunostaining diminished in lungs exposed to hyperoxia, consistent with decreased expression and secretion of intracellular precursor protein. In contrast, type II cells could still be identified by their intrinsic green fluorescence, because EGFP is not secreted. Type II cells could also be purified from single-cell suspensions of lung homogenates using fluorescence-activated cell sorting. Less than 1% of presorted cells exhibited green fluorescence compared with >95% of the sorted population. As expected for type II cells, ultrastructural analysis revealed that the sorted cells contained numerous lamellar bodies. SP-A, SP-B, and SP-C mRNAs were detected in the sorted population, but T1α and CD31 (platelet endothelial cell adhesion molecule) were not, indicating enrichment of type II epithelial cells. This method will be invaluable for detecting and isolating mouse type II cells under a variety of experimental conditions.

In vivo exposure to hyperoxia induces DNA damage in a population of alveolar type II epithelial cells

2004 ◽  
Vol 286 (5) ◽  
pp. L1045-L1054 ◽  
Author(s):  
Jason M. Roper ◽  
Dawn J. Mazzatti ◽  
Richard H. Watkins ◽  
William M. Maniscalco ◽  
Peter C. Keng ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Transmembrane Protein ◽  
Dna Integrity ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Cells ◽  
Endogenous Expression

It is well established that hyperoxia injures and kills alveolar endothelial and type I epithelial cells of the lung. Although type II epithelial cells remain morphologically intact, it remains unclear whether they are also damaged. DNA integrity was investigated in adult mice whose type II cells were identified by their endogenous expression of pro-surfactant protein C or transgenic expression of enhanced green fluorescent protein. In mice exposed to room air, punctate perinuclear 8-oxoguanine staining was detected in ∼4% of all alveolar cells and in 30% of type II cells. After 48 or 72 h of hyperoxia, 8-oxoguanine was detected in 11% of all alveolar cells and in >60% of type II cells. 8-Oxoguanine colocalized by confocal microscopy with the mitochondrial transmembrane protein cytochrome oxidase subunit 1. Type II cells isolated from hyperoxic lungs exhibited nuclear DNA strand breaks by comet assay even though they were viable and morphologically indistinguishable from cells isolated from lungs exposed to room air. These data reveal that type II cells exposed to in vivo hyperoxia have oxidized and fragmented DNA. Because type II cells are essential for lung remodeling, our findings raise the possibility that they are proficient in DNA repair.

Hydrogen peroxide induces oxidative DNA damage in rat type II pulmonary epithelial cells

1999 ◽  
Vol 33 (4) ◽  
pp. 273-278 ◽  
Author(s):  
William J. Meehan ◽  
Jeremy P.E. Spencer ◽  
D. Eugene Rannels ◽  
Danny R. Welch ◽  
Edward T. Knobbe ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Epithelial Cells ◽  
Oxidative Dna Damage ◽  
Type Ii ◽  
Pulmonary Epithelial Cells

Temporal/spatial expression of nuclear receptor coactivators in the mouse lung

2000 ◽  
Vol 279 (6) ◽  
pp. L1066-L1074 ◽  
Author(s):  
Angela Naltner ◽  
Susan Wert ◽  
Jeffrey A. Whitsett ◽  
Cong Yan
Keyword(s):  
Retinoic Acid ◽  
Epithelial Cells ◽  
Nuclear Receptor ◽  
Binding Protein ◽  
Mouse Lung ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii ◽  
Creb Binding Protein ◽  
Nuclear Receptor Coactivators

Our laboratory has previously demonstrated that retinoic acid nuclear receptor, thyroid transcription factor-1 (TTF-1), and nuclear receptor coactivators such as cAMP response element binding protein (CREB) binding protein (CBP)/p300 and steroid receptor coactivator-1 (SRC-1) form an enhanceosome on the 5′-enhancer region of the human surfactant protein B gene. Immunohistochemistry was used to identify cells that coexpressed CBP/p300, SRC-1, retinoid X receptor, and TTF-1 in the developing and mature lung. CBP/p300 and SRC-1 were expressed in the adult mouse lung, CBP and p300 being present in both alveolar type I and type II epithelial cells and SRC-1 and TTF-1 being restricted to type II epithelial cells. CBP/p300, SRC-1, and TTF-1 were readily detected in the nuclei of developing respiratory epithelial tubules in fetal mice from embryonic days 10 to 18.CBP/p300 and SRC-1 were also detected in developing mesenchymal cells. These coactivators were coexpressed with TTF-1 and SP-B in human pulmonary adenocarcinoma cells (H441 cells) in vitro. Interaction assays with a two-hybrid reporter analysis demonstrated direct interactions among TTF-1, SRC-1, and CBP/p300 in H441 cells. These findings support a role for retinoic acid receptor and nuclear receptor coactivators in the regulation of SP-B gene expression in the respiratory epithelium.

Ozone induces oxidative stress in rat alveolar type II and type I-like cells

2006 ◽  
Vol 40 (11) ◽  
pp. 1914-1928 ◽  
Author(s):  
Jieru Wang ◽  
Shuanglin Wang ◽  
Rizwan Manzer ◽  
Glen McConville ◽  
Robert J. Mason
Keyword(s):  
Oxidative Stress ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii

scholarly journals Heme oxygenase 1 protects human colonocytes against ROS formation, oxidative DNA damage and cytotoxicity induced by heme iron, but not inorganic iron

2020 ◽  
Vol 11 (9) ◽  
Author(s):  
Nina Seiwert ◽  
Sabine Wecklein ◽  
Philipp Demuth ◽  
Solveig Hasselwander ◽  
Talke A. Kemper ◽  
...  
Keyword(s):  
Dna Damage ◽  
Epithelial Cells ◽  
Heme Oxygenase ◽  
Oxidative Dna Damage ◽  
Red Meat ◽  
Heme Iron ◽  
Heme Oxygenase 1 ◽  
Cytotoxic Effects ◽  
Intestinal Organoids ◽  
Increased Risk

Abstract The consumption of red meat is probably carcinogenic to humans and is associated with an increased risk to develop colorectal cancer (CRC). Red meat contains high amounts of heme iron, which is thought to play a causal role in tumor formation. In this study, we investigated the genotoxic and cytotoxic effects of heme iron (i.e., hemin) versus inorganic iron in human colonic epithelial cells (HCEC), human CRC cell lines and murine intestinal organoids. Hemin catalyzed the formation of reactive oxygen species (ROS) and induced oxidative DNA damage as well as DNA strand breaks in both HCEC and CRC cells. In contrast, inorganic iron hardly affected ROS levels and only slightly increased DNA damage. Hemin, but not inorganic iron, caused cell death and reduced cell viability. This occurred preferentially in non-malignant HCEC, which was corroborated in intestinal organoids. Both hemin and inorganic iron were taken up into HCEC and CRC cells, however with differential kinetics and efficiency. Hemin caused stabilization and nuclear translocation of Nrf2, which induced heme oxygenase-1 (HO-1) and ferritin heavy chain (FtH). This was not observed after inorganic iron treatment. Chemical inhibition or genetic knockdown of HO-1 potentiated hemin-triggered ROS generation and oxidative DNA damage preferentially in HCEC. Furthermore, HO-1 abrogation strongly augmented the cytotoxic effects of hemin in HCEC, revealing its pivotal function in colonocytes and highlighting the toxicity of free intracellular heme iron. Taken together, this study demonstrated that hemin, but not inorganic iron, induces ROS and DNA damage, resulting in a preferential cytotoxicity in non-malignant intestinal epithelial cells. Importantly, HO-1 conferred protection against the detrimental effects of hemin.

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