Cloning and characterization of the human neutrophil-activating peptide (ENA-78) gene

The neutrophil-activating peptide (ENA-78) is an inflammatory chemokine which is produced concomitantly with interleukin-8 (IL-8) in response to stimulation with either interleukin-1 (IL-1β) or tumor necrosis factor-α (TNF-α). We have identified a full-length ENA-78 cDNA and isolated its genomic clone. The gene was found to consist of four exons and three introns and its structure resembles the IL-8 gene. The human ENA-78 gene was mapped to chromosome 4q13-q21, the same locus as several other inflammatory cytokine genes. The transcription initiation site was mapped to a position 96 base pairs (bp) upstream from the translation initiation site. A fusion gene containing 125 bp upstream of exon I linked to a luciferase reporter gene was expressed in the human embryonic 293 cell line. The expression of the reporter gene was induced by TNF-α, IL-1β, or phorbol 12-myristate 13- acetate. The 125-bp promoter region contained the cis-regulatory elements for enhancer binding protein-like factor (C/EBP) and the nuclear factor (NF- κB). Transfection of 293 cells with deletion mutants demonstrated that the NF-κB element, but not the C/EBP site, is sufficient for expression and induction by either TNF-α or IL-1β. In contrast, the IL-8 gene requires both elements. This report demonstrates that ENA-78 and IL-8 genes shared great similarity in genomic structure and chromosome location. However, these two genes may be regulated by distinct mechanisms.