Regulation of ACE-2 enzyme by hyperoxia in lung epithelial cells by post-translational modification

Background: Bronchopulmonary Dysplasia (BPD) occurs in premature neonates with respiratory distress who require supplemental oxygen in the first days after birth. BPD involves uniform arrest of alveolar development and variable interstitial cellularity and/or fibroproliferation. Previous studies by our lab showed that the enzyme, angiotensin converting enzyme-2 (ACE-2) and its product Ang1-7 exerting action on the receptor Mas oncogene in what is known as ACE-2/Mas axis is protective to lung cells. We also showed that ACE-2 is expressed in fetal human lung fibroblasts but is significantly decreased by hyperoxic gas lung injury, an effect caused by ACE-2 enzyme shedding mediated by TNF-alpha-converting enzyme (TACE/ADAM17). However, no reports yet exist about the regulation of ACE-2 in the alveolar epithelia in hyperoxic lung injury. Objective: In this study we aim to define the effects of hyperoxic lung injury on the protective ACE-2 enzyme in the human lung alveolar epithelial cell line A549. Design/Methods: Cultured A549 cells were exposed to hyperoxia (95% O2) or normoxia (21% O2) for 3 or 7 days in serum-free nutrient media. Cells were lysed and culture media were collected to test for cellular ACE-2 enzymatic activity and for ACE-2, Mas receptor, TACE/ADAM17, and ubiquitin proteins abundance by immunoblotting. Cells were harvested in Trizol for RNA extraction and ACE-2 qRT-PCR. Whole cell extracts of A549 cell line was used for ACE-2 immunoprecipitation and subsequent ubiquitin immunoblotting. Whole cell extracts of A549 cell line was used for ACE-2 immunoprecipitation and subsequent ubiquitin immunoblotting. Results: Total ubiquitinated proteins were increased by hyperoxia treatment, while ACE-2 and Mas receptor proteins abundance and ACE-2 enzymatic activity were decreased significantly in A549 cells exposed to hyperoxia relative to the normoxia controls. The percent decrease in ACE-2 activity corresponded with increased time of hyperoxic gas exposure. However, in contrast to our data from lung fibroblasts, no significant change was noted in ACE-2 protein released into the media or in ACE-2 mRNA levels by the hyperoxic treatment. Ubiquitin immunoreactive bands were detectable in the ACE-2 immunoprecipitate. Conclusion(s): These data suggest that hyperoxic exposure of the lung epithelial cells decreases the protective enzyme ACE-2 by cell type specific mechanisms independent of shedding by TACE/ADAM17. The data also suggest a regulatory level of ACE-2 downstream of transcription may involve ACE-2 ubiquitination and targeting for degradation.

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Ionizing radiation enhances matrix metalloproteinase-2 production in human lung epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.280.1.l30 ◽

2001 ◽

Vol 280 (1) ◽

pp. L30-L38 ◽

Cited By ~ 41

Author(s):

Jun Araya ◽

Muneharu Maruyama ◽

Kazuhiko Sassa ◽

Tadashi Fujita ◽

Ryuji Hayashi ◽

...

Keyword(s):

Epithelial Cells ◽

Ionizing Radiation ◽

Basement Membrane ◽

Lung Injury ◽

Human Lung ◽

A549 Cells ◽

Lung Epithelial Cells ◽

Lung Epithelial ◽

Radiation Induced ◽

Human Lung Epithelial Cells

Radiation pneumonitis is a major complication of radiation therapy. However, the detailed cellular mechanisms have not been clearly defined. Based on the recognition that basement membrane disruption occurs in acute lung injury and that matrix metalloproteinase (MMP)-2 can degrade type IV collagen, one of the major components of the basement membrane, we hypothesized that ionizing radiation would modulate MMP-2 production in human lung epithelial cells. To evaluate this, the modulation of MMP-2 with irradiation was investigated in normal human bronchial epithelial cells as well as in A549 cells. We measured the activity of MMP-2 in the conditioned medium with zymography and the MMP-2 mRNA level with RT-PCR. Both of these cells constitutively expressed 72-kDa gelatinolytic activity, corresponding to MMP-2, and exposure to radiation increased this activity. Consistent with the data of zymography, ionizing radiation increased the level of MMP-2 mRNA. This radiation-induced increase in MMP-2 expression was mediated via p53 because the p53 antisense oligonucleotide abolished the increase in MMP-2 activity as well as the accumulation of p53 after irradiation in A549 cells. These results indicate that MMP-2 expression by human lung epithelial cells is involved in radiation-induced lung injury.

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Puerarin Inhibits Ferroptosis and Inflammation of Lung Injury Caused by Sepsis in LPS Induced Lung Epithelial Cells

10.21203/rs.3.rs-504436/v1 ◽

2021 ◽

Author(s):

Baiye Xu ◽

Haidao Wang ◽

Zhen Chen

Keyword(s):

Epithelial Cells ◽

Lung Injury ◽

Cell Viability ◽

A549 Cell ◽

A549 Cells ◽

Total Iron ◽

Lung Epithelial Cells ◽

Expression Level ◽

Lung Epithelial ◽

Related Proteins

Abstract Background: Ferroptosis is a new type of programmed cell death, which plays an important role in lung injury caused by sepsis. Studies have reported that Puerarin (Pue) can treat lung injury caused by sepsis in children, but whether it plays a role by regulating iron death has not been reported.Methods: LPS induced human alveolar epithelial cell A549 to form a model of lung injury caused by sepsis. MTT detected the effect of Pue on A549 cell viability and the effect of Pue on LPS-induced A549 cell viability. The effects of Pue on LPS-induced inflammatory cytokines TNF-α, IL-8, IL-1β in A549 cells were determined by ELISA assay. The expression level of MDA was detected by TBARS colorimetric quantitative detection kit. GSH kit was used to detect the expression of GSH in cells. The iron kit detected the total iron level and the expression level of ferric divalent ions in the cells. DCFH-DA fluorescent probe was used to detect ROS levels. Western blot was used to detect the expression of ferroptosis-related proteins in cells. Results: Pue alleviated LPS-induced injury and inflammatory response in A549 cells, and Pue reduced the expression of ROS, MDA and GSH in LPS-induced A549 cells. In addition, Pue reduced total iron levels and ferrous ion levels in LPS-induced A549 cells, and decreased the expression of iron ferroptosis-related proteins. Conclusion: Puerarin inhibited ferroptosis and inflammation of lung injury caused by sepsis in children in LPS induced lung epithelial cells.

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Puerarin Inhibits Ferroptosis and Inflammation of Lung Injury Caused by Sepsis in LPS Induced Lung Epithelial Cells

Frontiers in Pediatrics ◽

10.3389/fped.2021.706327 ◽

2021 ◽

Vol 9 ◽

Author(s):

Baiye Xu ◽

Haidao Wang ◽

Zhen Chen

Keyword(s):

Epithelial Cells ◽

Lung Injury ◽

Cell Viability ◽

A549 Cell ◽

A549 Cells ◽

Total Iron ◽

Lung Epithelial Cells ◽

Expression Level ◽

Lung Epithelial ◽

Related Proteins

Background: Ferroptosis is a new type of programmed cell death, which plays an important role in lung injury caused by sepsis. Studies have reported that Puerarin (Pue) can treat lung injury caused by sepsis in children, but whether it plays a role by regulating iron death has not been reported.Methods: LPS induced human alveolar epithelial cell A549 to form a model of lung injury caused by sepsis. MTT detected the effect of Pue on A549 cell viability and the effect of Pue on LPS-induced A549 cell viability. The effects of Pue on LPS-induced inflammatory cytokines TNF-α, IL-8, IL-1β in A549 cells were determined by ELISA assay. The expression level of MDA was detected by TBARS colorimetric quantitative detection kit. GSH kit was used to detect the expression of GSH in cells. The iron kit detected the total iron level and the expression level of ferric divalent ions in the cells. DCFH-DA fluorescent probe was used to detect ROS levels. Western blot was used to detect the expression of ferroptosis-related proteins in cells.Results: Pue alleviated LPS-induced injury and inflammatory response in A549 cells, and Pue reduced the expression of ROS, MDA and GSH in LPS-induced A549 cells. In addition, Pue reduced total iron levels and ferrous ion levels in LPS-induced A549 cells, and decreased the expression of iron ferroptosis-related proteins.Conclusion: Puerarin inhibited ferroptosis and inflammation of lung injury caused by sepsis in children in LPS induced lung epithelial cells.

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Protein C and thrombomodulin in human acute lung injury

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00442.2002 ◽

2003 ◽

Vol 285 (3) ◽

pp. L514-L521 ◽

Cited By ~ 150

Author(s):

Lorraine B. Ware ◽

Xiaohui Fang ◽

Michael A. Matthay

Keyword(s):

Acute Lung Injury ◽

Lung Injury ◽

Clinical Outcomes ◽

Protein C ◽

A549 Cells ◽

Lung Epithelial Cells ◽

Alveolar Type ◽

Soluble Thrombomodulin ◽

Edema Fluid ◽

Lung Epithelial

Decreased circulating protein C and increased circulating thrombomodulin are markers of the prothrombotic, antifibrinolytic state associated with poor outcomes in sepsis but have not been measured in patients with ALI (acute lung injury)/ARDS (acute respiratory distress syndrome). We measured circulating and intra-alveolar protein C and thrombomodulin in 45 patients with ALI/ARDS from septic and nonseptic causes and correlated the levels with clinical outcomes. Plasma protein C levels were lower in ALI/ARDS compared with normal. Lower levels of protein C were associated with worse clinical outcomes, including death, fewer ventilator-free days, and more nonpulmonary organ failures, even when only patients without sepsis were analyzed. Levels of thrombomodulin in pulmonary edema fluid from ALI/ARDS patients were >10-fold higher than normal plasma and 2-fold higher than ALI/ARDS plasma. Higher edema fluid thrombomodulin levels were associated with worse clinical outcomes. The higher levels in edema fluid compared with plasma suggest local release of soluble thrombomodulin in the lung, possibly from a lung epithelial source. To determine whether lung epithelial cells can release thrombomodulin, A549 cells and primary isolates of human alveolar type II cells were exposed to H2O2or inflammatory cytokines. Both epithelial cell types released thrombomodulin into the media. In summary, the protein C system is markedly disrupted in patients with ALI/ARDS from both septic and nonseptic causes. The protein C system may be a potential therapeutic target in patients with ALI/ARDS.

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Matrine Attenuates COX-2 and ICAM-1 Expressions in Human Lung Epithelial Cells and Prevents Acute Lung Injury in LPS-Induced Mice

Mediators of Inflammation ◽

10.1155/2016/3630485 ◽

2016 ◽

Vol 2016 ◽

pp. 1-12 ◽

Cited By ~ 23

Author(s):

Chian-Jiun Liou ◽

You-Rong Lai ◽

Ya-Ling Chen ◽

Yi-Hsien Chang ◽

Zih-Ying Li ◽

...

Keyword(s):

Epithelial Cells ◽

Lung Injury ◽

Human Lung ◽

A549 Cells ◽

Lung Epithelial Cells ◽

Mitogen Activated Protein Kinase ◽

Intercellular Adhesion Molecule ◽

Lung Epithelial ◽

Cox 2 ◽

Human Lung Epithelial Cells

Matrine is isolated from Sophora flavescens and shows anti-inflammatory effects in macrophages. Here we evaluated matrine’s suppressive effects on cyclooxygenase 2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) expressions in lipopolysaccharide- (LPS-) stimulated human lung epithelial A549 cells. Additionally, BALB/c mice were given various matrine doses by intraperitoneal injection, and then lung injury was induced via intratracheal instillation of LPS. In LPS-stimulated A549 cells, matrine inhibited the productions of interleukin-8 (IL-8), monocyte chemotactic protein-1, and IL-6 and decreased COX-2 expression. Matrine treatment also decreased ICAM-1 protein expression and suppressed the adhesion of neutrophil-like cells to inflammatory A549 cells. In vitro results demonstrated that matrine significantly inhibited mitogen-activated protein kinase phosphorylation and decreased nuclear transcription factor kappa-B subunit p65 protein translocation into the nucleus. In vivo data indicated that matrine significantly inhibited neutrophil infiltration and suppressed productions of tumor necrosis factor-α and IL-6 in mouse bronchoalveolar lavage fluid and serum. Analysis of lung tissue showed that matrine decreased the gene expression of proinflammatory cytokines, chemokines, COX-2, and ICAM-1. Our findings suggest that matrine improved lung injury in mice and decreased the inflammatory response in human lung epithelial cells.

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Macrophages in Lung Injury, Repair, and Fibrosis

Cells ◽

10.3390/cells10020436 ◽

2021 ◽

Vol 10 (2) ◽

pp. 436 ◽

Cited By ~ 1

Author(s):

Peiyong Cheng ◽

Shuangyan Li ◽

Huaiyong Chen

Keyword(s):

Lung Injury ◽

Macrophage Polarization ◽

Current Data ◽

Lung Epithelial Cells ◽

Lung Fibroblasts ◽

Fibrotic Response ◽

Injury Repair ◽

Lung Epithelial ◽

Activated Macrophage ◽

Potential Benefits

Fibrosis progression in the lung commonly results in impaired functional gas exchange, respiratory failure, or even death. In addition to the aberrant activation and differentiation of lung fibroblasts, persistent alveolar injury and incomplete repair are the driving factors of lung fibrotic response. Macrophages are activated and polarized in response to lipopolysaccharide- or bleomycin-induced lung injury. The classically activated macrophage (M1) and alternatively activated macrophage (M2) have been extensively investigated in lung injury, repair, and fibrosis. In the present review, we summarized the current data on monocyte-derived macrophages that are recruited to the lung, as well as alveolar resident macrophages and their polarization, pyroptosis, and phagocytosis in acute lung injury (ALI). Additionally, we described how macrophages interact with lung epithelial cells during lung repair. Finally, we emphasized the role of macrophage polarization in the pulmonary fibrotic response, and elucidated the potential benefits of targeting macrophage in alleviating pulmonary fibrosis.

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Comparative Toxic Effects of Manufactured Nanoparticles and Atmospheric Particulate Matter in Human Lung Epithelial Cells

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18010022 ◽

2020 ◽

Vol 18 (1) ◽

pp. 22

Author(s):

Yun Wu ◽

Mei Wang ◽

Shaojuan Luo ◽

Yunfeng Gu ◽

Dongyang Nie ◽

...

Keyword(s):

Particulate Matter ◽

Human Lung ◽

A549 Cells ◽

Lung Epithelial Cells ◽

Atmospheric Particulate Matter ◽

Toxic Effects ◽

Ros Generation ◽

Atmospheric Particulate ◽

Significant Difference ◽

Lung Epithelial

Although nanoparticles (NPs) have been used as simplified atmospheric particulate matter (PM) models, little experimental evidence is available to support such simulations. In this study, we comparatively assessed the toxic effects of PM and typical NPs (four carbonaceous NPs with different morphologies, metal NPs of Fe, Al, and Ti, as well as SiO2 NPs) on human lung epithelial A549 cells. The EC50 value of PM evaluated by cell viability assay was 148.7 μg/mL, closest to that of SiO2 NPs, between the values of carbonaceous NPs and metal NPs. All particles caused varying degrees of reactive oxygen species (ROS) generation and adenosine triphosphate (ATP) suppression. TiO2 NPs showed similar performance with PM in inducing ROS production (p < 0.05). Small variations between two carbonaceous NPs (graphene oxides and graphenes) and PM were also observed at 50 μg/mL. Similarly, there was no significant difference in ATP inhibition between carbonaceous NPs and PM, while markedly different effects were caused by SiO2 NP and TiO2 NP exposure. Our results indicated that carbonaceous NPs could be served as potential surrogates for urban PM. The identification of PM model may help us further explore the specific roles and mechanisms of various components in PM.

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Regulated in Development and DNA Damage Response 1 Knockdown Alleviates Lipopolysaccharide-Induced Acute Lung Injury

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2021.2691 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1333-1338

Author(s):

Han Han ◽

Zhenxi Yu ◽

Mei Feng

Keyword(s):

Acute Lung Injury ◽

Epithelial Cell ◽

Epithelial Cells ◽

Lung Injury ◽

Cell Activity ◽

Lung Epithelial Cells ◽

Lung Epithelial Cell ◽

Inflammatory Factors ◽

Lung Epithelial ◽

Damage Response

Regulated in Development and DNA Damage Response 1 (REDD1) knockdown can reduce the endoplasmic reticulum stress response in liver injury. However, its role on lipopolysaccharide (LPS)-induced acute lung injury (ALI) has not been explored. This study aimed to evaluate the effect of REDD1 on lung epithelial cells induced by LPS. Rt-qPCR and Western blot were used to detect REDD1 expression in 16HBE cells induced by LPS. The interfering REDD1 plasmid was constructed, and CCK8 was used to detect the effect of interference with REDD1 on LPS-induced lung epithelial cell activity. The expression of inflammatory factors was detected by ELISA and the apoptotic level was detected by TUNEL staining. String database was used to predict the combination of REDD1 and EP300 in lung epithelial cells, which was verified by CoIP experiment. An overexpressed plasmid of EP300 was constructed to detect the effects of EP300 on inflammatory factors and apoptosis in REDD1 lung epithelial cells. LPS-induced increased REDD1 expression in lung epithelial cells. Interference with REDD1 inhibits LPS-induced lung epithelial cell activity injury and inflammatory factor expression and inhibits LPS-induced lung epithelial cell apoptosis. After interference with REDD1, the expression of EP300 in LPS-induced lung epithelial cells was inhibited, and the overexpression of EP300 was reversed to promote the production of inflammatory factors and apoptosis. In conclusion, these results demonstrate that REDD1 knockdown alleviates LPS-induced acute lung injury.

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TRIM28 regulates SARS-CoV-2 cell entry by targeting ACE2

10.1101/2020.08.12.247825 ◽

2020 ◽

Author(s):

Yinfang Wang ◽

Yingzhe Fan ◽

Yitong Huang ◽

Tao Du ◽

Zongjun Liu ◽

...

Keyword(s):

Epithelial Cells ◽

Nk Cells ◽

Interleukin 2 ◽

A549 Cells ◽

Endogenous Retrovirus ◽

Alveolar Epithelial Cells ◽

Cell Entry ◽

Lung Epithelial Cells ◽

Lung Epithelial ◽

Ifn Γ

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), it binds to angiotensin-converting enzyme 2 (ACE2) to enter into human cells. The expression level of ACE2 potentially determine the susceptibility and severity of COVID-19, it is thus of importance to understand the regulatory mechanism of ACE2 expression. Tripartite motif containing 28 (TRIM28) is known to be involved in multiple processes including antiviral restriction, endogenous retrovirus latency and immune response, it is recently reported to be co-expressed with SARS-CoV-2 receptor in type II pneumocytes; however, the roles of TRIM28 in ACE2 expression and SARS-CoV-2 cell entry remain unclear. This study showed that knockdown of TRIM28 induces ACE2 expression and increases pseudotyped SARS-CoV-2 cell entry of A549 cells and primary pulmonary alveolar epithelial cells (PAEpiCs). In a co-culture model of NK cells and lung epithelial cells, our results demonstrated that NK cells inhibit TRIM28 and promote ACE2 expression in lung epithelial cells, which was partially reversed by depletion of interleukin-2 and blocking of granzyme B in the co-culture medium. Furthermore, TRIM28 knockdown enhanced interferon-γ (IFN-γ)-induced ACE2 expression through a mechanism involving upregulating IFN-γ receptor 2 (IFNGR2) in both A549 and PAEpiCs. Importantly, the upregulated ACE2 induced by TRIM28 knockdown and co-culture of NK cells was partially reversed by dexamethasone in A549 cells but not PAEpiCs. Our study identified TRIM28 as a novel regulator of ACE2 expression and SARS-CoV-2 cell entry.

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