Asbestos induction of nuclear transcription factors and interleukin 8 gene regulation

Our plain-English summary of Asbestos induction of nuclear transcription factors and interleukin 8 gene regulation, by P.P. Simeonova, M.I. Luster (American Journal of Respiratory Cell and Molecular Biology, 1996).

The study

• Authors: P.P. Simeonova, M.I. Luster
• Journal: American Journal of Respiratory Cell and Molecular Biology
• Published: 1996;15(6):787-95
• Cited by: 75 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Proinflammatory cytokines and chemotactic peptides are strongly implicated as mediators of the pathophysiologic responses of asbestosis and other chronic inflammatory lung diseases. Recent studies in our laboratory have demonstrated that asbestos fibers stimulate lung epithelial cells to produce interleukin-8 (IL-8), the major neutrophil chemoattractant in the lung. The mechanisms by which asbestos regulates IL-8 expression were studied using the pulmonary type II-like epithelial cell line A549. Membrane permeable hydroxyl scavengers inhibited asbestos induced IL-8 expression. Using A549 cells transfected with the -546 IL-8 construct linked to a chloramphenicol acetyl transferase reporter gene, we have shown that these antioxidants directly inhibited asbestos-stimulated IL-8 promoter-dependent transcription. Asbestos fibers as well as reactive oxygen species generating systems hypoxanthine-xanthine oxidase and hydrogen peroxide stimulated DNA binding activity to the regulatory elements in the IL-8 promoter, binding sites of nuclear factor (NF)-kappaB- and NF-IL-6-like transcription factors. Asbestos-inducible DNA binding activity was partially inhibited by tetramethylthiourea, a hydroxyl radical scavenger. IL-8 secretion was also suppressed by staurosporine, an inhibitor of protein kinase C, and by inhibitors of tyrosine kinase such as herbimycin A and genistein. The suppression paralleled the effect of these inhibitors on asbestos-induced DNA binding to the NF-kappaB- and NF-IL-6-like binding sites of the IL-8 promoter. Taken together, the results suggest that asbestos-induced redox changes and phosphorylation events, mediated by staurosporine-sensitive and tyrosine kinase(s), activate nuclear proteins which recognize the NF-kappaB/NF-IL-6 binding sites of the IL-8 promoter and contribute to the regulation of IL-8 gene expression.

In plain English

This was a laboratory study of human lung cells grown in culture, not of people. The authors exposed A549 cells, a cell line resembling the lung's type II epithelial cells and originally derived from a person with alveolar cell carcinoma, to crocidolite asbestos from South Africa. They studied how the fibers switch on interleukin-8 (IL-8), a chemical signal that attracts white blood cells called neutrophils. Chemicals that neutralize hydroxyl radicals (DMTU, TMTU and DMSO) reduced asbestos-induced IL-8 secretion without changing cell damage as measured by LDH release. Hydrogen peroxide and a hypoxanthine-xanthine oxidase system, both of which generate reactive oxygen species, also significantly increased IL-8 secretion. Asbestos raised the activity of the IL-8 gene promoter about 3-fold, and TMTU prevented this completely. Asbestos increased binding of NF-kappaB-like and NF-IL-6-like nuclear proteins to the IL-8 promoter, and TMTU cut this asbestos-induced binding by 50%. The kinase inhibitors staurosporine, herbimycin A and genistein suppressed IL-8 secretion. The authors concluded that the IL-8 response is mediated by asbestos-activated oxidative stress, with phosphorylation events also involved.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) cell culture experiment
• Who was studied: A549 human pulmonary type II-like epithelial cell line, originally derived from an individual with alveolar cell carcinoma, purchased from the American Type Culture Collection; no human subjects
• Exposure: Crocidolite asbestos (sample ML-6) from a mine in the Kuruman Hills, South Africa, with a mean fiber length of 19 micrometers and a mean width of 1 micrometer. Most experiments used 25 micrograms per ml, and the promoter (transfection) experiments used 12.5 micrograms per ml. The reactive oxygen generating systems hydrogen peroxide and hypoxanthine-xanthine oxidase were also tested.
• Main results:
  • In asbestos-stimulated A549 cells, the hydroxyl radical scavengers DMTU, TMTU and DMSO decreased IL-8 secretion. With TMTU the inhibition was dose-dependent, and it occurred without changes in cytotoxicity as measured by LDH release.
  • Asbestos treatment was estimated to increase IL-8 mRNA by approximately several orders of magnitude. In the presence of TMTU the response was similar to control levels.
  • Hydrogen peroxide or hypoxanthine-xanthine oxidase significantly increased IL-8 secretion compared with untreated cells.
  • In transfected A549 cells, IL-8 promoter-driven CAT expression increased approximately 3-fold with asbestos. This increase was prevented completely by TMTU and reduced significantly by DMTU.
  • Nuclear protein binding to the IL-8 promoter region containing the NF-kappaB-like and NF-IL-6-like sites was present in untreated A549 cells and increased markedly with asbestos. The binding involved the p50 and p65 subunits of NF-kappaB and C/EBP-beta (NF-IL-6).
  • Hydrogen peroxide increased this DNA binding 2-fold and hypoxanthine-xanthine oxidase increased it 2.5-fold. TMTU reduced asbestos-inducible binding by 50%.
  • Staurosporine inhibited asbestos-induced IL-8 secretion with an IC50 (the concentration giving 50% inhibition) of 0.05 micromolar. The table gives IC50 values for asbestos-induced IL-8 printed as greater than 2.0 micromolar for herbimycin A, greater than 33.5 for genistein and greater than 23.4 for H89. Staurosporine prevented asbestos-inducible DNA binding, while herbimycin A and genistein were less effective, reducing it by over 50%.
• The authors' conclusion: The authors concluded that asbestos fibers induced IL-8 gene expression and secretion in A549 cells, and that this response is mediated by asbestos-activated oxidative stress. In their view, phosphorylation events driven by tyrosine kinases and staurosporine-sensitive kinases are also involved in regulating asbestos-induced IL-8 expression.
• Limitations the authors state:
  • The authors noted that the staurosporine concentrations needed to prevent IL-8 secretion would also inhibit other kinases besides PKC, indicating a lack of specificity at these concentrations.
  • The authors noted that the relatively high H89 concentration needed to reduce IL-8 suggests its effect may not be specific for PKA.

Topics

Medical terms in this study, explained in our Lexicon: Cytokine, A549 Cells, Reactive Oxygen Species (ROS), Hydroxyl Radical.

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