IL-1beta induces eotaxin gene transcription in A549 airway epithelial cells through NF-kappaB.
Eotaxin is an asthma-related C-C chemokine that is produced in response to interleukin-1beta (IL-1beta). We detected an increase in newly transcribed eotaxin mRNA in IL-1beta-stimulated airway epithelial cells. Transient transfection assays using promoter-reporter constructs identified a region as essential for IL-1beta-induced increases in eotaxin transcription. Using site-directed mutagenesis, we found that a nuclear factor-kappaB (NF-kappaB) site located 46 bp upstream from the transcriptional start site was both necessary and sufficient for IL-1beta induction of reporter construct activity. Electrophoretic mobility shift assay demonstrated that IL-1beta-stimulated airway epithelial cells produced p50 and p65 protein that bound this site in a sequence-specific manner. The functional importance of the NF-kappaB 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-kappaB site (r(2) = 0.97, P = 0.002). Moreover, IL-1beta-induced increases in eotaxin mRNA expression are inhibited by inhibitors of NF-kappaB. Our findings implicate NF-kappaB and its binding sequence in IL-1beta-induced transcriptional activation of the eotaxin gene.
Jedrzkiewicz
S
S
Combined Program in Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston 02115, Massachusetts, USA.
Nakamura
H
H
Silverman
E S
ES
Luster
A D
AD
Mansharamani
N
N
In
K H
KH
Tamura
G
G
Lilly
C M
CM
D002282:Adenocarcinoma, Bronchiolo-Alveolar; D054413:Chemokine CCL11; D019742:Chemokines, CC; D016207:Cytokines / Q000235:genetics*; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D008175:Lung Neoplasms; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology / Q000502:physiology*; D015533:Transcriptional Activation / Q000187:drug effects*; D014407:Tumor Cells, Cultured
C515456:CCL11 protein, human; D054413:Chemokine CCL11; D019742:Chemokines, CC; D016207:Cytokines; D007375:Interleukin-1; D016328:NF-kappa B; D012333:RNA, Messenger