Lipopolysaccharide-Mediated Induction of the Bovine Interleukin-6 Gene in Monocytes Requires Both NF-κB and C/EBP Binding Sites

Abstract

The interleukin-6 (IL-6) gene expression in bovine monocytes is highly induced following bacterial lipopolysaccharide (LPS) stimulation. To identify the promoter element(s) involved in the inducible transcription of IL-6, a 5′-flanking region containing 230 bp of the bovine IL-6 gene was linked to a reporter gene coding for bacterial chloramphenicol acetyltransferase (CAT) and analyzed for its ability to confer LPS-responsiveness to the reporter CAT gene in monocytic cells. Using mutant reporter genes, we demonstrate that although mutation in the NF-κB element produces the major loss of induction, both NF-κB and C/EBP elements are necessary for maximal transcriptional activation of the bovine IL-6 gene. Gel electrophoretic mobility-shift assays have detected induced DNA-binding activities in the LPS-stimulated monocytes. Further characterization has revealed the activation and interaction of C/EBP-α, C/EBP-β (NF-IL6), NFKB1 (p50), and RelA (p65) to their specific binding elements present in the bovine IL-6 gene. These results suggest a model in which induction of C/EBP-α in differentiating monocytes contributes and synergizes with induced C/EBP-β and NF-κB, which are activated following LPS stimulation, to mediate a high rate of IL-6 transcription under inflammatory conditions.