A promoter recruitment mechanism for tumor necrosis factor-α-induced interleukin-8 transcription in type II pulmonary epithelial cells. Dependence on nuclear abundance of Rel A, NF-κB1, and c-Rel transcription factors

Allan R. Brasier, Mohammad Jamaluddin, Antonella Casola, Weili Duan, Qing Shen, Roberto Garofalo

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Abstract

The alveolar macrophage-derived peptide tumor necrosis factor-α (TNFα) initiates pulmonary inflammation through its ability to stimulate interleukin-8 (IL-8) synthesis in alveolar epithelial cells through an incompletely described transcriptional mechanism. In this study, we use the technique of ligation-mediated polymerase chain reaction (LMPCR) to record changes in transcription factor occupancy of the IL-8 promoter after TNFα stimulation of A549 human alveolar cells. Using dimethylsulfate/LMPCR, no detectable proteins bind the TATA box in unstimulated cells. By contrast, TNFα rapidly induces protection of G residues at -79 and -80 coincident with endogenous IL-8 gene transcription. Using DNase I/LMPCR, we observe inducible protection of nucleotides -60 to -99 (the TNF response element) and nucleotides -3 to -32 (containing the TATA box). Surprisingly, extensive TATA box protection is only seen after TNFα stimulation. Using a two-step microaffinity isolation/Western immunoblot DNA binding assay, we observe that the NF-κB subunits Rel A, NF-κB1, and c-Rel inducibly bind the TNF response element; these proteins undergo rapid TNFα-inducible increases in nuclear abundance as a consequence of IκBα proteolysis. Furthermore, the peptide aldehyde N-acetyl-Leu-Leu-norleucinal, an agent that blocks both IκBα proteolysis and NF-κB subunit translocation, abrogates recombinant human TNFα-inducible IL-8 gene transcription. These studies demonstrate that IL-8 is activated by a promoter recruitment mechanism in alveolar epithelial cells, where NF-κB subunit translocation is required for (and coincident with) binding of the constitutively active TATA box-binding proteins.

Original languageEnglish
Pages (from-to)3551-3561
Number of pages11
JournalJournal of Biological Chemistry
Volume273
Issue number6
DOIs
StatePublished - Feb 6 1998

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Transcription
Interleukin-8
Transcription Factors
Tumor Necrosis Factor-alpha
Epithelial Cells
Lung
Alveolar Epithelial Cells
TATA Box
Polymerase chain reaction
Proteolysis
Ligation
Response Elements
Polymerase Chain Reaction
leucylleucine
Nucleotides
Genes
TATA-Box Binding Protein
Peptides
Deoxyribonuclease I
Alveolar Macrophages

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "A promoter recruitment mechanism for tumor necrosis factor-α-induced interleukin-8 transcription in type II pulmonary epithelial cells. Dependence on nuclear abundance of Rel A, NF-κB1, and c-Rel transcription factors",
abstract = "The alveolar macrophage-derived peptide tumor necrosis factor-α (TNFα) initiates pulmonary inflammation through its ability to stimulate interleukin-8 (IL-8) synthesis in alveolar epithelial cells through an incompletely described transcriptional mechanism. In this study, we use the technique of ligation-mediated polymerase chain reaction (LMPCR) to record changes in transcription factor occupancy of the IL-8 promoter after TNFα stimulation of A549 human alveolar cells. Using dimethylsulfate/LMPCR, no detectable proteins bind the TATA box in unstimulated cells. By contrast, TNFα rapidly induces protection of G residues at -79 and -80 coincident with endogenous IL-8 gene transcription. Using DNase I/LMPCR, we observe inducible protection of nucleotides -60 to -99 (the TNF response element) and nucleotides -3 to -32 (containing the TATA box). Surprisingly, extensive TATA box protection is only seen after TNFα stimulation. Using a two-step microaffinity isolation/Western immunoblot DNA binding assay, we observe that the NF-κB subunits Rel A, NF-κB1, and c-Rel inducibly bind the TNF response element; these proteins undergo rapid TNFα-inducible increases in nuclear abundance as a consequence of IκBα proteolysis. Furthermore, the peptide aldehyde N-acetyl-Leu-Leu-norleucinal, an agent that blocks both IκBα proteolysis and NF-κB subunit translocation, abrogates recombinant human TNFα-inducible IL-8 gene transcription. These studies demonstrate that IL-8 is activated by a promoter recruitment mechanism in alveolar epithelial cells, where NF-κB subunit translocation is required for (and coincident with) binding of the constitutively active TATA box-binding proteins.",
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T1 - A promoter recruitment mechanism for tumor necrosis factor-α-induced interleukin-8 transcription in type II pulmonary epithelial cells. Dependence on nuclear abundance of Rel A, NF-κB1, and c-Rel transcription factors

AU - Brasier, Allan R.

AU - Jamaluddin, Mohammad

AU - Casola, Antonella

AU - Duan, Weili

AU - Shen, Qing

AU - Garofalo, Roberto

PY - 1998/2/6

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N2 - The alveolar macrophage-derived peptide tumor necrosis factor-α (TNFα) initiates pulmonary inflammation through its ability to stimulate interleukin-8 (IL-8) synthesis in alveolar epithelial cells through an incompletely described transcriptional mechanism. In this study, we use the technique of ligation-mediated polymerase chain reaction (LMPCR) to record changes in transcription factor occupancy of the IL-8 promoter after TNFα stimulation of A549 human alveolar cells. Using dimethylsulfate/LMPCR, no detectable proteins bind the TATA box in unstimulated cells. By contrast, TNFα rapidly induces protection of G residues at -79 and -80 coincident with endogenous IL-8 gene transcription. Using DNase I/LMPCR, we observe inducible protection of nucleotides -60 to -99 (the TNF response element) and nucleotides -3 to -32 (containing the TATA box). Surprisingly, extensive TATA box protection is only seen after TNFα stimulation. Using a two-step microaffinity isolation/Western immunoblot DNA binding assay, we observe that the NF-κB subunits Rel A, NF-κB1, and c-Rel inducibly bind the TNF response element; these proteins undergo rapid TNFα-inducible increases in nuclear abundance as a consequence of IκBα proteolysis. Furthermore, the peptide aldehyde N-acetyl-Leu-Leu-norleucinal, an agent that blocks both IκBα proteolysis and NF-κB subunit translocation, abrogates recombinant human TNFα-inducible IL-8 gene transcription. These studies demonstrate that IL-8 is activated by a promoter recruitment mechanism in alveolar epithelial cells, where NF-κB subunit translocation is required for (and coincident with) binding of the constitutively active TATA box-binding proteins.

AB - The alveolar macrophage-derived peptide tumor necrosis factor-α (TNFα) initiates pulmonary inflammation through its ability to stimulate interleukin-8 (IL-8) synthesis in alveolar epithelial cells through an incompletely described transcriptional mechanism. In this study, we use the technique of ligation-mediated polymerase chain reaction (LMPCR) to record changes in transcription factor occupancy of the IL-8 promoter after TNFα stimulation of A549 human alveolar cells. Using dimethylsulfate/LMPCR, no detectable proteins bind the TATA box in unstimulated cells. By contrast, TNFα rapidly induces protection of G residues at -79 and -80 coincident with endogenous IL-8 gene transcription. Using DNase I/LMPCR, we observe inducible protection of nucleotides -60 to -99 (the TNF response element) and nucleotides -3 to -32 (containing the TATA box). Surprisingly, extensive TATA box protection is only seen after TNFα stimulation. Using a two-step microaffinity isolation/Western immunoblot DNA binding assay, we observe that the NF-κB subunits Rel A, NF-κB1, and c-Rel inducibly bind the TNF response element; these proteins undergo rapid TNFα-inducible increases in nuclear abundance as a consequence of IκBα proteolysis. Furthermore, the peptide aldehyde N-acetyl-Leu-Leu-norleucinal, an agent that blocks both IκBα proteolysis and NF-κB subunit translocation, abrogates recombinant human TNFα-inducible IL-8 gene transcription. These studies demonstrate that IL-8 is activated by a promoter recruitment mechanism in alveolar epithelial cells, where NF-κB subunit translocation is required for (and coincident with) binding of the constitutively active TATA box-binding proteins.

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