A promoter recruitment mechanism for tumor necrosis factor-alpha-induced interleukin-8 transcription in type II pulmonary epithelial cells. Dependence on nuclear abundance of Rel A, NF-kappaB1, and c-Rel transcription factors.

The alveolar macrophage-derived peptide tumor necrosis factor-alpha (TNFalpha) 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 TNFalpha stimulation of A549 human alveolar cells. Using dimethylsulfate/LMPCR, no detectable proteins bind the TATA box in unstimulated cells. By contrast, TNFalpha 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 TNFalpha stimulation. Using a two-step microaffinity isolation/Western immunoblot DNA binding assay, we observe that the NF-kappaB subunits Rel A, NF-kappaB1, and c-Rel inducibly bind the TNF response element; these proteins undergo rapid TNFalpha-inducible increases in nuclear abundance as a consequence of IkappaBalpha proteolysis. Furthermore, the peptide aldehyde N-acetyl-Leu-Leu-norleucinal, an agent that blocks both IkappaBalpha proteolysis and NF-kappaB subunit translocation, abrogates recombinant human TNFalpha-inducible IL-8 gene transcription. These studies demonstrate that IL-8 is activated by a promoter recruitment mechanism in alveolar epithelial cells, where NF-kappaB subunit translocation is required for (and coincident with) binding of the constitutively active TATA box-binding proteins.