ZBP-89, Sp1, and Nuclear Factor-κB Regulate Epithelial Neutrophil-activating Peptide-78 Gene Expression in Caco-2 Human Colonic Epithelial Cells

• Published in: The Journal of biological chemistry Vol. 276; no. 47; pp. 43713 - 43722
• Main Authors: Keates, Andrew C., Keates, Sarah, Kwon, John H., Arseneau, Kristen O., Law, David J., Bai, Longchuan, Merchant, Juanita L., Wang, Timothy C., Kelly, Ciarán P.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 23.11.2001
• Subjects: Animals; Binding Sites; Caco-2 Cells; Chemokine CXCL5; Chemokines, CXC; Colon - metabolism; DNA-Binding Proteins - physiology; Drosophila; ENA-78 gene; Epithelial Cells - metabolism; epithelial neutrophil-activating peptide 78; Gene Expression Regulation - genetics; Gene Expression Regulation - physiology; Genes, Reporter; Humans; Interleukin-8 - analogs & derivatives; Interleukin-8 - genetics; Luciferases - genetics; NF-kappa B - physiology; Promoter Regions, Genetic; Sp1 Transcription Factor - genetics; Sp1 Transcription Factor - physiology; Transcription Factors - physiology; Transcription, Genetic - physiology; ulcerative colitis; ZBP-89 protein; Zinc - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M107838200

We reported previously that human colonic epithelial cells produce the C-X-C chemokine epithelial neutrophil-activating peptide-78 (ENA-78) and that its expression is up-regulated in ulcerative colitis. The aim of this study was to investigate the transcriptional regulation of ENA-78 gene expression in Caco-2 intestinal epithelial cells. Reporter gene transfection and electrophoretic mobility shift assay studies demonstrated that cooperation between two regions of the ENA-78 promoter were required for maximal gene expression in interleukin-1β-stimulated Caco-2 cells. Binding of activated p50/p65 nuclear factor-κB to nucleotides −82 to −91 was essential for interleukin-1β-dependent gene transcription, whereas binding of constitutively expressed zinc-requiring nuclear factors to nucleotides −125 to −134 (site A) was required for basal gene expression. Scanning mutagenesis of site A demonstrated overlapping binding elements at this locus. One site (CTCCCCC) bound Sp1 and Sp3, and overexpression of Sp1 (but not Sp3) up-regulated basal ENA-78 transcription. Another site (CCCCTCCCCC) was found to bind the zinc finger nuclear factor ZBP-89, and overexpression of this protein significantly repressed ENA-78 reporter gene activity. This study demonstrates that ENA-78 gene expression in Caco-2 intestinal epithelial cells is subject to complex regulation involving the coordinate binding of ZBP-89, Sp1, and nuclear factor-κB to the ENA-78 promoter.