IL-1b induces eotaxin gene transcription in A549 airway epithelial cells through NF-kB

Jedrzkiewicz, Sean, Hidetoshi Nakamura, Eric S. Silverman, Andrew D. Luster, Naresh Mansharamani, Kwang Ho In, Gen Tamura, and Craig M. Lilly. IL-1b induces eotaxin gene transcription in A549 airway epithelial cells through NF-kB. Am J Physiol Lung Cell Mol Physiol 279: L1058–L1065, 2000.—Eotaxin is an asthma-related C-C chemokine that is produced in response to interleukin-1b (IL-1b). We detected an increase in newly transcribed eotaxin mRNA in IL-1b-stimulated airway epithelial cells. Transient transfection assays using promoter-reporter constructs identified a region as essential for IL-1b-induced increases in eotaxin transcription. Using site-directed mutagenesis, we found that a nuclear factor-kB (NF-kB) site located 46 bp upstream from the transcriptional start site was both necessary and sufficient for IL-1b induction of reporter construct activity. Electrophoretic mobility shift assay demonstrated that IL-1b-stimulated airway epithelial cells produced p50 and p65 protein that bound this site in a sequence-specific manner. The functional importance of the NF-kB site was demonstrated by coexpression experiments in which increasing doses of p65 expression vector were directly associated with reporter activity exclusively in constructs with an intact NF-kB site (r 5 0.97, P 5 0.002). Moreover, IL-1b-induced increases in eotaxin mRNA expression are inhibited by inhibitors of NF-kB. Our findings implicate NF-kB and its binding sequence in IL-1b-induced transcriptional activation of the eotaxin gene.