Abstract
Interleukin-8 (IL-8), a potent neutrophil chemoattractant, can be expressed at high levels by many different cell types after immune stimulation. In contrast, expression of IL-8 in these same cells is virtually absent in the unstimulated state, demonstrating the tight regulation of the IL-8 gene. Although much is known about how this gene is transcriptionally activated after immune stimulation, little is known about the regulation of the IL-8 promoter in the absence of immune activation. In this study we examine how the IL-8 promoter is transcriptionally regulated in the uninduced state and how these mechanisms are altered in response to immune stimulation by IL-1beta. Electrophoretic mobility shift assay and transfection studies show that the IL-8 promoter is transcriptionally regulated by both positive and negative elements. Although the nuclear factor-kappaB (NFkappaB) element regulates only inducible activity of the IL-8 promoter in response to stimulation with IL-1beta, the AP-1 and CCAAT/Enhancer-binding Protein (C/EBP) elements influence both basal and inducible activities. In contrast to these three positive regulatory elements, the binding of the ubiquitously expressed POU-homeodomain transcription factor, Oct-1, strongly represses transcriptional activity of the IL-8 promoter by binding independently to an element overlapping that of C/EBP.
Highlights
Interleukin-8 (IL-8), a potent neutrophil chemoattractant, can be expressed at high levels by many different cell types after immune stimulation
Binding of Nuclear Proteins to the IL-8 nuclear factor-B (NFB) Element Are Necessary but Not Sufficient for Full Transcriptional Activity of the IL-8 Promoter in Response to IL-1—In order to determine the functional effect of previously characterized DNA binding elements in the IL-8 promoter in the colon cancer cell line, Caco-2, transcriptional activation of the wild-type IL-8 promoter ((wt)LUC) or the promoter with point mutations in the NFB ((mNFB)LUC), CCAAT/Enhancer-binding Protein (C/EBP) ((mC/EBP)LUC), or AP-1 ((mAP-1)LUC) binding elements were determined by transient transfection into Caco-2 cells as described under “Experimental Procedures” (Fig. 2)
These results demonstrate that, protein binding to the NFB element of the IL-8 promoter is critical for transcriptional induction in response to IL-1, the AP-1 and C/EBP binding elements are required for the full expression of the IL-8 promoter
Summary
Interleukin-8 (IL-8), a potent neutrophil chemoattractant, can be expressed at high levels by many different cell types after immune stimulation. In order to demonstrate this interaction in Caco-2 cells and to determine if Oct-1 binding occurs within the context of this interaction, EMSAs of nuclear extracts isolated from both control (C) and IL-1-stimulated (S) Caco-2 cells were performed using a double-stranded oligonucleotide probe, named IL-8wt, which spans both the C/EBP and NFB elements of the IL-8 promoter (Fig. 5, lanes 3 through 13).
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