scholarly journals Nuclear factor-kappaB regulates the transcription of NADPH oxidase 1 in human alveolar epithelial cells

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Weijing Wu ◽  
Li Li ◽  
Xiaoshan Su ◽  
Zhixing Zhu ◽  
Xiaoping Lin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Epithelial Cells ◽  
A549 Cells ◽  
Alveolar Epithelial Cells ◽  
Nuclear Factor ◽  
Alveolar Epithelial ◽  
Tnf Α ◽  
Potential Regulatory Role ◽  
Human Alveolar Epithelial Cells

Abstract Objective Acute lung injury (ALI) is characterized by inflammation and oxidative stress. Nuclear factor-kappaB (NF-κB) mediates the expression of various inflammation-related genes, including the NADPH oxidase family. This study aimed to identify the potential regulatory role of NF-κB on NADPH oxidases in tumor necrosis factor-α (TNF-α)-induced oxidative stress in human alveolar epithelial cells. Methods A549 cells were treated with TNF-α for 24 h to establish ALI cell models. RT-PCR, western blot, assessment of oxidative stress, Alibaba 2.1 online analysis, electrophoretic mobility shift assays and luciferase reporter analysis were employed to identify the potential regulatory role of NF-κB on NADPH oxidases in TNF-α-induced oxidative stress in human alveolar epithelial cells. Results The expression of NF-κB/p65 was notably upregulated in TNF-α-stimulated A549 cells. NF-κB knockdown by siRNA significantly inhibited the TNF-α-induced oxidative stress. Moreover, NF-κB/p65 siRNA could inhibit the activation of NOX1, NOX2 and NOX4 mRNA and protein expression in TNF-α-stimulated A549 cells. The next study demonstrated that NF-κB activated the transcription of NOX1 by binding to the -261 to -252 bp (NOX1/κB2, TAAAAATCCC) region of NOX1 promoter in TNF-α-stimulated A549 cells. Conclusion Our data demonstrated that NF-κB can aggravate TNF-α-induced ALI by regulating the oxidative stress response and the expression of NOX1, NOX2 and NOX4. Moreover, NF-κB could promote the NOX1 transcriptional activity via binding its promoter in TNF-α-stimulated A549 cells.

scholarly journals Nuclear Factor-kappaB Regulates the Transcription of NADPH Oxidase 1 in Human Alveolar Epithelial Cells

2020 ◽  
Author(s):  
Weijing Wu ◽  
Li Li ◽  
Xiaoshan Su ◽  
ZHIXING ZHU ◽  
Xiaoping Lin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Epithelial Cells ◽  
A549 Cells ◽  
Alveolar Epithelial Cells ◽  
Ros Generation ◽  
Alveolar Epithelial ◽  
Tnf Α ◽  
Potential Regulatory Role ◽  
Human Alveolar Epithelial Cells

Abstract Objective Acute lung injury (ALI) is characterized by inflammation and oxidative stress. Nuclear factor-kappaB (NF-κB) mediates the expression of various inflammation-related genes, including the NADPH oxidase family. This study aimed to identify the potential regulatory role of NF-𝜅B on NADPH oxidases in TNF-α-induced oxidative stress in human alveolar epithelial cells. Methods Type II alveolar epithelial cell-derived A549 cells were treated with TNF-α for 24 hours to establish ALI cell models. RT-PCR, western blot, DCFH-DA ROS assay, Alibaba 2.1 online analysis, electrophoretic mobility shift assays and luciferase reporter analysis were employed to identify the potential regulatory role of NF-𝜅B on NADPH oxidases in TNF-α-induced oxidative stress in human alveolar epithelial cells. Results The expression of NF-κB/p65 was notably upregulated in TNF-α-stimulated A549 cells. NF-κB knockdown by siRNA significantly inhibited the TNF-α-induced ROS generation. Moreover, NF-𝜅B/p65 siRNA could inhibite the activation of NOX1, NOX2 and NOX4 mRNA and protein expression in TNF-α-stimulated A549 cells. The next study demonstrated that NF-𝜅B activated the transcription of NOX1 by binding to the -261 to -252 bp (NOX1/κB2, TAAAAATCCC) region of NOX1 promoter in TNF-α-stimulated A549 cells. Conclusion Our data demonstrated that NF-κB can aggravate TNF-α-induced ALI by regulating the activation of ROS generation and the expression of NOX1, NOX2 and NOX4. Moreover, NF-𝜅B could promote the NOX1 transcriptional activity via binding its promoter in TNF-α-stimulated A549 cells.

scholarly journals Nrf2 Alleviates TNF-α-Induced Oxidative Stress Damage in Type II Alveolar Epithelial Cells by Down-Regulating the Expression of NOX1

2020 ◽  
Author(s):  
Weijing Wu ◽  
Jiamin Zhang ◽  
Xihua Lian ◽  
Xiaoping Lin ◽  
Xiaoshan Su ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Epithelial Cells ◽  
Binding Site ◽  
Luciferase Activity ◽  
A549 Cells ◽  
Alveolar Epithelial Cells ◽  
Type Ii ◽  
Alveolar Epithelial ◽  
Tnf Α ◽  
Stress Damage

Abstract Objective: To study the roles of Nrf2 in acute lung injury (ALI) pathogenesis by investigating the effects of Nrf2 on regulating oxidative stress damage in TNF-α-induced type II alveolar epithelial cells (T2AECs).Methods: T2AECs were transfected with Nrf2 siRNA and overexpression vectors for six hours before being induced by TNF-α for 24 hours. Subsequently, levels of interleukins (IL-6 and IL-8), reactive oxygen species (ROS), malondialdehyde (MDA), total antioxidation capability (T-AOC), Nrf2, NOX1 and NF-kB were measured. Additionally, potential Nrf2 binding site in NOX1 promoter was predicted by AliBaba2.1 and two recombinant vectors, namely “pGL3-NOX1-1500” and “pGL3-NOX1-1489, were constructed by inserting the sequence of NOX1 promoter in full-length and that in the absence of Nrf2 binding site to pGL3 basic vector. T2AECs were transfected with these vectors prior to TNF-α induction and the luciferase activity was measured.Results: Levels of IL-6, IL-8, ROS and MDA were increased (P＜0.05) while T-AOC was decreased in TNF-α-induced A549 cells after the transfection of Nrf2 siRNA vector (P＜0.05). In contrast, concentrations of IL-6, IL-8, ROS and MDA were decreased (P＜0.05) whereas T-AOC was increased after the transfection of Nrf2 overexpression vector (P＜0.05). NOX1 promoter possesses one Nrf2 binding site. Cells transfected by “pGL3-NOX1-1500” vector had the highest luciferase activity, followed by cells transfected by “pGL3-NOX1-1489” vector and the control cells (P＜0.05).Conclusion: Nrf2 modulates NOX1 expression via binding to its promoter, by which against TNF-α-induced oxidative stress damage in T2AECs. Thus, Nrf2 might be a therapeutic target for ALI.

scholarly journals LPS-induced depolymerization of cytoskeleton and its role in TNF-α production by rat pneumocytes

1999 ◽  
Vol 277 (3) ◽  
pp. L606-L615 ◽  
Author(s):  
Noritaka Isowa ◽  
Alexandre M. Xavier ◽  
Ewa Dziak ◽  
Michal Opas ◽  
Donna I. McRitchie ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Immune Cells ◽  
Protein Production ◽  
Mrna Level ◽  
Alveolar Epithelial Cells ◽  
Immunofluorescent Staining ◽  
Alveolar Epithelial ◽  
Tnf Α ◽  
Factor Α

Lipopolysaccharide (LPS) polymerizes microfilaments and microtubules in macrophages and monocytes. Disrupting microfilaments or microtubules with cytochalasin D (CytoD) or colchicine can suppress LPS-induced tumor necrosis factor-α (TNF-α) gene expression and protein production from these cells. We have recently demonstrated that primary cultured rat alveolar epithelial cells can produce TNF-α on LPS stimulation. In the present study, we found that the LPS-induced increase in TNF-α mRNA level and protein production in alveolar epithelial cells was not inhibited by CytoD or colchicine (1 nM to 10 μM). In fact, LPS-induced TNF-α production was further enhanced by CytoD (1–10 μM) and inhibited by jasplakinolide, a polymerizing agent for microfilaments. Immunofluorescent staining and confocal microscopy showed that LPS (10 μg/ml) depolymerized microfilaments and microtubules within 15 min, which was prolonged until 24 h for microfilaments. These results suggest that the effects of LPS on the cytoskeleton and the role of the cytoskeleton in mediating TNF-α production in alveolar epithelial cells are opposite to those in immune cells. This disparity may reflect the different roles between nonimmune and immune cells in host defense.

Expression of the tyrosine phosphatase LAR-PTP2 is developmentally regulated in lung epithelia

1994 ◽  
Vol 267 (3) ◽  
pp. L263-L270 ◽  
Author(s):  
D. Rotin ◽  
B. J. Goldstein ◽  
C. A. Fladd
Keyword(s):  
Epithelial Cells ◽  
Lung Development ◽  
Tyrosine Phosphatase ◽  
A549 Cells ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Type Ii ◽  
Adult Type ◽  
Alveolar Epithelial

The role of tyrosine kinases in regulating cell proliferation, differentiation, and development has been well documented. In contrast, little is known about the role of protein tyrosine phosphatases (PTPs) in mammalian development. To identify PTPs that may be involved in lung development, we have isolated (by polymerase chain reaction) from rat fetal alveolar epithelial cells a cDNA fragment which was identified as the recently cloned tyrosine phosphatase LAR-PTP2. Analysis of tissue expression of LAR-PTP2 identified a approximately 7.5-kb message in the lung, which is also expressed weakly in brain, and an alternatively spliced approximately 6.0-kb message (LAR-PTP2B) expressed in brain. In the fetal lung, LAR-PTP2 was preferentially expressed in lung epithelial (but not fibroblast) cells grown briefly in primary culture, and its expression was tightly regulated during lung development, peaking at 20 days of gestational age (term = 22 days), when mature alveolar type II epithelium first appears. Accordingly, immunoblot analysis revealed high expression of endogenous LAR-PTP2 protein in alveolar epithelial cells from 21-day gestation fetuses. LAR-PTP2 was also expressed in lungs of newborn rats, but transcripts (and protein) were barely detectable in adult lungs and in the nonproliferating adult alveolar type II cells. Interestingly, expression was restored in the transformed adult type II-like A549 cells. These results suggest that LAR-PTP2 may play a role in the proliferation and/or differentiation of epithelial cells during lung development.

scholarly journals Methemoglobin-induced signaling and chemokine responses in human alveolar epithelial cells

2014 ◽  
Vol 306 (1) ◽  
pp. L88-L100 ◽  
Author(s):  
Sharon Mumby ◽  
Latha Ramakrishnan ◽  
Timothy W. Evans ◽  
Mark J. D. Griffiths ◽  
Gregory J. Quinlan
Keyword(s):  
Epithelial Cells ◽  
Primary Cultures ◽  
A549 Cells ◽  
Alveolar Epithelial Cells ◽  
Lung Epithelial Cells ◽  
Alveolar Type ◽  
Alveolar Epithelial ◽  
Pathway Activation ◽  
Jnk Activation ◽  
Human Alveolar Epithelial Cells

Diffuse alveolar hemorrhage is characterized by the presence of red blood cells and free hemoglobin in the alveoli and complicates a number of serious medical and surgical lung conditions including the pulmonary vasculitides and acute respiratory distress syndrome. In this study we investigated the hypothesis that exposure of human alveolar epithelial cells to hemoglobin and its breakdown products regulates chemokine release via iron- and oxidant-mediated activation of the transcription factor NF-κB. Methemoglobin alone stimulated the release of IL-8 and MCP-1 from A549 cells via activation of the NF-κB pathway; additionally, IL-8 required ERK activation and MCP-1 required JNK activation. Neither antioxidants nor iron chelators and knockdown of ferritin heavy and light chains affected these responses, indicating that iron and reactive oxygen species are not involved in the response of alveolar epithelial cells to methemoglobin. Incubation of primary cultures of human alveolar type 2 cells with methemoglobin resulted in a similar pattern of chemokine release and signaling pathway activation. In summary, we have shown for the first time that methemoglobin induced chemokine release from human lung epithelial cells independent of iron- and redox-mediated signaling involving the activation of the NF-κB and MAPK pathways. Decompartmentalization of hemoglobin may be a significant proinflammatory stimulus in a variety of lung diseases.

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