TNF-α induces MUC1 gene transcription in lung epithelial cells: its signaling pathway and biological implication

2007 ◽  
Vol 293 (3) ◽  
pp. L693-L701 ◽  
Author(s):  
Takeshi Koga ◽  
Ippei Kuwahara ◽  
Erik P. Lillehoj ◽  
Wenju Lu ◽  
Takeshi Miyata ◽  
...  
Keyword(s):  
Gene Transcription ◽  
A549 Cells ◽  
Lung Epithelial Cells ◽  
Luciferase Reporter ◽  
Promoter Activation ◽  
Alveolar Cells ◽  
Intact Cells ◽  
Protein Levels ◽  
Muc1 Gene ◽  
Tnf Α

The current study was conducted to elucidate the mechanism through which TNF-α stimulates expression of MUC1, a membrane-tethered mucin. A549 human lung alveolar cells treated with TNF-α exhibited significantly higher MUC1 protein levels in detergent lysates compared with cells treated with vehicle alone. Increased MUC1 protein levels were correlated with significantly higher levels of MUC1 mRNA in TNF-α-treated cells compared with controls. However, TNF-α did not alter MUC1 transcript stability, implying increased de novo transcription induced by the cytokine. TNF-α increased MUC1 gene promoter activity in A549 cells transfected with a promoter-luciferase reporter plasmid. Both U0126, an inhibitor of MEK1/2, and dominant negative ERK1 prevented TNF-α-induced MUC1 promoter activation, and anti-TNFR1 antibody blocked TNF-α-stimulated ERK1/2 activation. MUC1 promoter activation by TNF-α also was blocked by mithramycin A, an inhibitor of Sp1, as well as either deletion or mutation of a putative Sp1 binding site in the MUC1 promoter located between nucleotides −99 and −90. TNF-α-stimulated binding of Sp1 to the MUC1 promoter in intact cells was demonstrated by chromatin immunoprecipitation assay. We conclude that TNF-α induces MUC1 gene transcription through a TNFR1 → MEK1/2 → ERK1 → Sp1 pathway.

Neutrophil elastase stimulatesMUC1gene expression through increased Sp1 binding to theMUC1promoter

2005 ◽  
Vol 289 (2) ◽  
pp. L355-L362 ◽  
Author(s):  
Ippei Kuwahara ◽  
Erik P. Lillehoj ◽  
Akinori Hisatsune ◽  
Wenju Lu ◽  
Yoichiro Isohama ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Neutrophil Elastase ◽  
Transcription Initiation ◽  
De Novo ◽  
A549 Cells ◽  
Cell Surface Receptor ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Muc1 Gene

We previously reported MUC1 was a cell surface receptor for Pseudomonas aeruginosa, and binding of bacteria to cells was significantly reduced by pretreatment with neutrophil elastase (NE) (Lillehoj EP, Hyun SW, Kim BT, Zhang XG, Lee DI, Rowland S, and Kim KC. Am J Physiol Lung Cell Mol Physiol 280: L181–L187, 2001). The current study was conducted to ascertain NE effects on MUC1 gene transcription, and MUC1 protein synthesis and degradation. A549 human lung carcinoma cells treated with NE exhibited significantly higher MUC1 protein levels in detergent lysates compared with cells treated with vehicle alone. Also, MUC1 protein shed into cell-conditioned medium was rapidly and completely degraded by NE. Actinomycin D blocked NE-stimulated increase in MUC1 protein expression, suggesting a mechanism of increased gene transcription that was confirmed by measurement of quantitatively greater MUC1 mRNA levels in NE-treated cells compared with controls. However, NE did not alter MUC1 mRNA stability, implying increased de novo transcription induced by the protease. NE increased promoter activity in A549 cells transfected with MUC1 gene promoter-luciferase reporter plasmid. This effect of NE was completely blocked by mithramycin A, an inhibitor of Sp1, as well as mutation of one of the putative Sp1 binding sites in MUC1 promoter located at −99/−90 relative to transcription initiation site. EMSA revealed NE enhanced binding of Sp1 to this 10-bp segment in a time-dependent manner. These results indicate the increase in MUC1 gene transcription by NE is mediated through increase in Sp1 binding to −99/−90 segment of MUC1 promoter.

PM10-exposed macrophages stimulate a proinflammatory response in lung epithelial cells via TNF-α

2002 ◽  
Vol 282 (2) ◽  
pp. L237-L248 ◽  
Author(s):  
L. A. Jiménez ◽  
E. M. Drost ◽  
P. S. Gilmour ◽  
I. Rahman ◽  
F. Antonicelli ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Inflammatory Response ◽  
A549 Cells ◽  
Conditioned Media ◽  
Lung Epithelial Cells ◽  
Mrna Levels ◽  
Protein Levels ◽  
Proinflammatory Mediators ◽  
Tnf Α ◽  
Lung Epithelial

There is now considerable evidence for an association between the levels of particulate air pollution [particulate matter <10 μm in aerodynamic diameter (PM10)] and various adverse health endpoints. The release of proinflammatory mediators from PM10-exposed macrophages may be important in stimulating cytokine release from lung epithelial cells, thus amplifying the inflammatory response. A549 cells were treated with conditioned media from monocyte-derived macrophages stimulated with PM10, titanium dioxide (TiO2), or ultrafine TiO2. We demonstrate that only conditioned media from PM10-stimulated macrophages significantly increased nuclear factor-κB and activator protein-1 DNA binding, enhanced interleukin-8 (IL-8) mRNA levels as assessed by RT-PCR, and augmented IL-8 protein levels, over untreated controls. Furthermore, PM10-conditioned media also caused transactivation of IL-8 as determined by an IL-8-chloramphenicol acetyl transferase reporter. Analysis of these conditioned media revealed marked increases in tumor necrosis factor-α (TNF-α) and protein levels and enhanced chemotactic activity for neutrophils. Preincubation of conditioned media with TNF-α-neutralizing antibodies significantly reduced IL-8 production. These data suggest that PM10-activated macrophages may amplify the inflammatory response by enhancing IL-8 release from lung epithelial cells, in part, via elaboration of TNF-α.

scholarly journals Let-7a-5p regulates the inflammatory response in chronic rhinosinusitis with nasal polyps

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Jianwei Zhang ◽  
Lei Han ◽  
Feng Chen
Keyword(s):  
Inflammatory Response ◽  
Chronic Rhinosinusitis ◽  
Nasal Polyps ◽  
Mapk Pathway ◽  
Inhibitory Effect ◽  
Luciferase Reporter ◽  
Western Blot Assay ◽  
Protein Levels ◽  
Tnf Α

Abstract Background Let-7a-5p is demonstrated to be a tumor inhibitor in nasopharyngeal carcinoma. However, the role of let-7a-5p in chronic rhinosinusitis with nasal polyps (CRSwNP) has not been reported. This study is designed to determine the pattern of expression and role of let-7a-5p in CRSwNP. Methods The expression level of let-7a-5p, TNF-α, IL-1β, and IL-6 in CRSwNP tissues and cells were detected by RT-qPCR. Western blot assay was carried out to measure the protein expression of the Ras-MAPK pathway. Dual luciferase reporter assay and RNA pull-down assay were used to explore the relationship between let-7a-5p and IL-6. Results Let-7a-5p was significantly downregulated in CRSwNP tissues and cells. Moreover, the mRNA expression of TNF-α, IL-1β and IL-6 was increased in CRSwNP tissues, while let-7a-5p mimic inhibited the expression of TNF-α, IL-1β and IL-6. Besides that, let-7a-5p was negatively correlated with TNF-α, IL-1β and IL-6 in CRSwNP tissues. In our study, IL-6 was found to be a target gene of let-7a-5p. Additionally, let-7-5p mimic obviously reduced the protein levels of Ras, p-Raf1, p-MEK1 and p-ERK1/2, while IL-6 overexpression destroyed the inhibitory effect of let-7a-5p on the Ras-MAPK pathway in CRSwNP. Conclusion We demonstrated that let-7a-5p/IL-6 interaction regulated the inflammatory response through the Ras-MAPK pathway in CRSwNP.

scholarly journals Application of Humanized Zebrafish Model in the Suppression of SARS-CoV-2 Spike Protein Induced Pathology by Tri-Herbal Medicine Coronil via Cytokine Modulation

Molecules ◽  
2020 ◽  
Vol 25 (21) ◽  
pp. 5091
Author(s):  
Acharya Balkrishna ◽  
Siva Kumar Solleti ◽  
Sudeep Verma ◽  
Anurag Varshney
Keyword(s):  
Herbal Medicine ◽  
High Performance ◽  
Normal Skin ◽  
A549 Cells ◽  
Lung Epithelial Cells ◽  
Spike Protein ◽  
Protective Effects ◽  
Cell Degeneration ◽  
Zebrafish Model ◽  
Tnf Α

Zebrafish has been a reliable model system for studying human viral pathologies. SARS-CoV-2 viral infection has become a global chaos, affecting millions of people. There is an urgent need to contain the pandemic and develop reliable therapies. We report the use of a humanized zebrafish model, xeno-transplanted with human lung epithelial cells, A549, for studying the protective effects of a tri-herbal medicine Coronil. At human relevant doses of 12 and 58 µg/kg, Coronil inhibited SARS-CoV-2 spike protein, induced humanized zebrafish mortality, and rescued from behavioral fever. Morphological and cellular abnormalities along with granulocyte and macrophage accumulation in the swim bladder were restored to normal. Skin hemorrhage, renal cell degeneration, and necrosis were also significantly attenuated by Coronil treatment. Ultra-high-performance liquid chromatography (UHPLC) analysis identified ursolic acid, betulinic acid, withanone, withaferine A, withanoside IV–V, cordifolioside A, magnoflorine, rosmarinic acid, and palmatine as phyto-metabolites present in Coronil. In A549 cells, Coronil attenuated the IL-1β induced IL-6 and TNF-α cytokine secretions, and decreased TNF-α induced NF-κB/AP-1 transcriptional activity. Taken together, we show the disease modifying immunomodulatory properties of Coronil, at human equivalent doses, in rescuing the pathological features induced by the SARS-CoV-2 spike protein, suggesting its potential use in SARS-CoV-2 infectivity.

scholarly journals PPARγ inhibits airway epithelial cell inflammatory response through a MUC1-dependent mechanism

2012 ◽  
Vol 302 (7) ◽  
pp. L679-L687 ◽  
Author(s):  
Yong Sung Park ◽  
Erik P. Lillehoj ◽  
Kosuke Kato ◽  
Choon Sik Park ◽  
Kwang Chul Kim
Keyword(s):  
Epithelial Cells ◽  
Culture Media ◽  
A549 Cells ◽  
Luciferase Reporter ◽  
Muc1 Expression ◽  
Mrna Levels ◽  
Airway Epithelial ◽  
Pparγ Agonist ◽  
Tnf Α ◽  
Primary Mouse

This study was conducted to examine the relationship between the peroxisome proliferator-associated receptor-γ (PPARγ) and MUC1 mucin, two anti-inflammatory molecules expressed in the airways. Treatment of A549 lung epithelial cells or primary mouse tracheal surface epithelial (MTSE) cells with phorbol 12-myristate 13-acetate (PMA) increased the levels of tumor necrosis factor (TNF)-α in cell culture media compared with cells treated with vehicle alone. Overexpression of MUC1 in A549 cells decreased PMA-stimulated TNF-α levels, whereas deficiency of Muc1 expression in MTSE cells from Muc1 null mice increased PMA-induced TNF-α levels. Treatment of A549 or MTSE cells with the PPARγ agonist troglitazone (TGN) blocked the ability of PMA to stimulate TNF-α levels. However, the effect of TGN required the presence of MUC1/Muc1, since no differences in TNF-α levels were seen between PMA and PMA plus TGN in MUC1/Muc1-deficient cells. Similarly, whereas TGN decreased interleukin-8 (IL-8) levels in culture media of MUC1-expressing A549 cells treated with Pseudomonas aeruginosa strain K (PAK), no differences in IL-8 levels were seen between PAK and PAK plus TGN in MUC1-nonexpressing cells. EMSA confirmed the presence of a PPARγ-binding element in the MUC1 gene promoter. Finally, TGN treatment of A549 cells increased MUC1 promoter activity measured using a MUC1-luciferase reporter gene, augmented MUC1 mRNA levels by quantitative RT-PCR, and enhanced MUC1 protein expression by Western blot analysis. These combined data are consistent with the hypothesis that PPARγ stimulates MUC1/Muc1 expression, thereby blocking PMA/PAK-induced TNF-α/IL-8 production by airway epithelial cells.

Dual mechanism forMycobacterium tuberculosiscytotoxicity on lung epithelial cells

2012 ◽  
Vol 58 (7) ◽  
pp. 909-916 ◽  
Author(s):  
Jorge Castro-Garza ◽  
W. Edward Swords ◽  
Russell K. Karls ◽  
Frederick D. Quinn
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Neutralizing Antibodies ◽  
Cytotoxic Effect ◽  
Alveolar Epithelial Cell ◽  
A549 Cells ◽  
Lung Epithelial Cells ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Lung Epithelial

Mycobacterium tuberculosis strains CDC1551 and Erdman were used to assess cytotoxicity in infected A549 human alveolar epithelial cell monolayers. Strain CDC1551 was found to induce qualitatively greater disruption of A549 monolayers than was strain Erdman, although total intracellular and cell-associated bacterial growth rates over the course of the infections were not significantly different. Cell-free culture supernatants from human monocytic cells infected with either of the 2 M. tuberculosis strains produced a cytotoxic effect on A549 cells, correlating with the amount of tumor necrosis factor alpha (TNF-α) released by the infected monocytes. The addition of TNF-α-neutralizing antibodies to the supernatants from infected monocyte cultures did prevent the induction of a cytotoxic effect on A549 cells overlaid with this mixture but did not prevent the death of epithelial cells when added prior to infection with M. tuberculosis bacilli. Thus, these data agree with previous observations that lung epithelial cells infected with M. tuberculosis bacilli are rapidly killed in vitro. In addition, the data indicate that some of the observed epithelial cell killing may be collateral damage; the result of TNF-α released from M. tuberculosis-infected monocytes.

Effects of TNF-α and IL-1β on iron metabolism by A549 cells and influence on cytotoxicity

1999 ◽  
Vol 277 (2) ◽  
pp. L257-L263 ◽  
Author(s):  
Igor M. Smirnov ◽  
Kirstin Bailey ◽  
Carol H. Flowers ◽  
Ned W. Garrigues ◽  
Lewis J. Wesselius
Keyword(s):  
Cell Death ◽  
Lactate Dehydrogenase ◽  
Iron Metabolism ◽  
Iron Uptake ◽  
A549 Cells ◽  
Receptor Expression ◽  
Alveolar Cells ◽  
Lactate Dehydrogenase Release ◽  
Ferritin Synthesis ◽  
Tnf Α

Extracellular iron, which is predominantly bound by transferrin, is present in low concentrations within alveolar structures, and concentrations are increased in various pulmonary disorders. Iron accumulation by cells can promote oxidative injury. However, the synthesis of ferritin stimulated by metal exposure for intracellular iron storage is normally protective. The cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β may alter iron metabolism by alveolar cells. In this study, we assessed the effects of TNF-α and IL-1β on iron metabolism with a cell line with properties of type 2 alveolar epithelial cells (A549) exposed to non-transferrin-bound (NTBI; FeSO4) or transferrin-bound (TBI) iron. In addition, we assessed the cytotoxicity of these exposures by measuring the cell accumulation of malondialdehyde (MDA), a product of lipid peroxidation, and cell death (MTT assay and lactate dehydrogenase release). A549 cells treated with NTBI or TBI in concentrations up to 40 μM accumulated iron and synthesized predominantly L-type ferritin without accumulation of MDA or cell death. Treatment of A549 cells with TNF-α (20 ng) or IL-1β (20 ng) decreased cell transferrin-receptor expression and induced synthesis of H-type ferritin. TNF-α and IL-1β decreased the uptake of TBI; however, the uptake of NTBI was increased. Both cytokines enhanced total ferritin synthesis (H plus L types) in response to iron treatments due to enhanced synthesis of H-type ferritin. Coexposure to TNF-α and NTBI, but not to TBI, induced MDA accumulation and greater cytotoxicity (MTT and lactate dehydrogenase release) than TNF-α alone. These findings indicate that TNF-α and IL-1β modulate iron uptake by A549 cells, with differing effects on TBI and NTBI, as well as on H-ferritin synthesis. Enhanced iron uptake induced by TNF-α and NTBI was also associated with increased cytotoxicity to A549 cells.

NF-κB inhibition by ω-3 fatty acids modulates LPS-stimulated macrophage TNF-α transcription

2003 ◽  
Vol 284 (1) ◽  
pp. L84-L89 ◽  
Author(s):  
Todd E. Novak ◽  
Tricia A. Babcock ◽  
David H. Jho ◽  
W. Scott Helton ◽  
N. Joseph Espat
Keyword(s):  
Signal Transduction Pathway ◽  
Raw 264.7 Cells ◽  
Luciferase Reporter ◽  
Transduction Pathway ◽  
Rt Pcr ◽  
Protein Levels ◽  
Cytokine Inhibition ◽  
Tnf Α ◽  
Mrna And Protein Expression ◽  
Luciferase Reporter Vector

ω-3 Fatty acid (FA) emulsions reduce LPS-stimulated murine macrophage TNF-α production, but the exact mechanism has yet to be defined. The purpose of this study was to determine the mechanism for ω-3 FA inhibition of macrophage TNF-α production following LPS stimulation. RAW 264.7 cells were pretreated with isocaloric emulsions of ω-3 FA (Omegaven), ω-6 FA (Lipovenos), or DMEM and subsequently exposed to LPS. IκB-α and phospho-IκB-α were determined by Western blotting. NF-κB binding was assessed using the electromobility shift assay, and activity was measured using a luciferase reporter vector. RT-PCR and ELISA quantified TNF-α mRNA and protein levels, respectively. Pretreatment with ω-3 FA inhibited IκB phosphorylation and significantly decreased NF-κB activity. Moreover, ω-3-treated cells demonstrated significant decreases in both TNF-α mRNA and protein expression by 47 and 46%, respectively. These experiments demonstrate that a mechanism for proinflammatory cytokine inhibition in murine macrophages by ω-3 FA is mediated, in part, through inactivation of the NF-κB signal transduction pathway secondary to inhibition of IκB phosphorylation.

scholarly journals Thyroid transcription factor-1 (TTF-1) gene: identification of ZBP-89, Sp1, and TTF-1 sites in the promoter and regulation by TNF-α in lung epithelial cells

2011 ◽  
Vol 301 (4) ◽  
pp. L427-L440 ◽  
Author(s):  
Aparajita Das ◽  
Sunil Acharya ◽  
Koteswara Rao Gottipati ◽  
James B. McKnight ◽  
Hemakumar Chandru ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Surfactant Protein ◽  
Serine Phosphorylation ◽  
Lung Epithelial Cells ◽  
Proximal Promoter ◽  
Protein Levels ◽  
Thyroid Transcription Factor 1 ◽  
Thyroid Transcription Factor ◽  
Tnf Α ◽  
Transcription Factor 1

Thyroid transcription factor-1 (TTF-1/Nkx2.1/TITF1) is a homeodomain-containing transcription factor essential for the morphogenesis and differentiation of the lung. In the lung, TTF-1 controls the expression of surfactant proteins that are essential for lung stability and lung host defense. In this study, we identified functionally important transcription factor binding sites in the TTF-1 proximal promoter and studied tumor necrosis factor-α (TNF-α) regulation of TTF-1 expression. TNF-α, a proinflammatory cytokine, has been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) and inhibits surfactant protein levels. Deletion analysis of TTF-1 5′-flanking DNA indicated that the TTF-1 proximal promoter retained high-level activity. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and mutational analysis experiments identified functional ZBP-89, Sp1, Sp3, and TTF-1 sites in the TTF-1 proximal promoter. TNF-α inhibited TTF-1 protein levels in H441 and primary alveolar type II cells. TNF-α inhibited TTF-1 gene transcription and promoter activity, indicating that transcriptional mechanisms play important roles in the inhibition of TTF-1 levels. TNF-α inhibited TTF-1 but not Sp1 or hepatocyte nuclear factor-3 DNA binding to TTF-1 promoter. Transactivation experiments in A549 cells indicated that TNF-α inhibited TTF-1 promoter activation by exogenous Sp1 and TTF-1 without altering their levels, suggesting inhibition of transcriptional activities of these proteins. TNF-α inhibition of TTF-1 expression was associated with increased threonine, but not serine, phosphorylation of Sp1. Because TTF-1 serves as a positive regulator for surfactant protein gene expression, TNF-α inhibition of TTF-1 expression could have important implications for the reduction of surfactant protein levels in diseases such as ARDS.

scholarly journals Induction of Proinflammatory Cytokines in Human Lung Epithelial Cells during Chlamydia pneumoniae Infection

2003 ◽  
Vol 71 (2) ◽  
pp. 614-620 ◽  
Author(s):  
Jun Yang ◽  
W. Craig Hooper ◽  
Donald J. Phillips ◽  
Maria L. Tondella ◽  
Deborah F. Talkington
Keyword(s):  
Epithelial Cells ◽  
Chlamydia Pneumoniae ◽  
Human Lung ◽  
A549 Cells ◽  
Cytokine Response ◽  
Lung Epithelial Cells ◽  
Cytokine Gene Expression ◽  
Cytokine Release ◽  
Tnf Α ◽  
Respiratory Epithelial

ABSTRACT Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute respiratory diseases such as pneumonia and bronchitis. Previous studies have established that C. pneumoniae can induce cytokines in mouse and/or human cells, but little information is available on the cytokine response of respiratory epithelial cells, a first line of infection. In this study, heparin treatment of C. pneumoniae significantly reduced its ability to induce interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) mRNA in human lung carcinoma cells, indicating that cytadherence is an important early stimulus for induction of proinflammatory mediators. Although the IL-8, gamma interferon, and TNF-α message was consistently induced by infection of A549 cells not treated with heparin, only an elevation of IL-8 protein was detected in A549 supernatants. A549 IL-β and IL-6 mRNA and supernatant protein profiles were not significantly changed by infection. Heat or UV inactivation of C. pneumoniae only partially reduced the cytokine response, and inhibition of C. pneumoniae protein or DNA synthesis did not affect its ability to induce cytokine gene expression. To prevent stress-induced cytokine release by the A549 cells, centrifugation was not utilized for infection experiments. These experiments establish the importance of cytadherence in cytokine release by cells of respiratory epithelial origin and suggest that further work in the area of cytokine mediators is warranted to gain valuable pathogenic and therapeutic insights.

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