Proinflammatory cytokines induce liver and activation-regulated chemokine/macrophage inflammatory protein-3alpha/CCL20 in mucosal epithelial cells through NF-kappaB [correction of NK-kappaB].
Liver and activation-regulated chemokine (LARC)/CCL20 is expressed by surface-lining epithelial and epidermal cells, and is likely to link innate and acquired immunity by attracting immature dendritic cells, effector memory T cells and B cells via CCR6. Here we examined the mechanism of LARC expression in epithelial-type cells. Either IL-1beta or tumor necrosis factor (TNF)-alpha strongly induced LARC mRNA in intestinal cell lines Caco-2 and T84, while both were effective on HEK 293T cells. Induction of LARC was also demonstrated in the intestinal epithelium of BALB/c mice upon treatment with IL-1alpha or TNF-alpha. Transient transfection assays using murine LARC promoter-reporter constructs identified a region essential for IL-1beta- or TNF-alpha-induced promoter activation in Caco-2 and 293T cells. Using site-directed mutagenesis, we demonstrated that an NF-kappaB site located between -96 and -87 bp upstream from the transcriptional start site was both necessary and sufficient for IL-1beta- or TNF-alpha-induced promoter activation in Caco-2 and 293T cells. Electrophoretic mobility shift assays demonstrated that p50/p65 heterodimer and p65 homodimer of NF-kappaB bound to this site in 293T cells upon treatment with IL-1beta and TNF-alpha, and p50/p65 heterodimer bound to this site in Caco-2 cells upon treatment with IL-1beta. Co-expression of constitutively active p65 strongly activated the promoter construct carrying the intact NF-kappaB site in 293T and Caco-2 cells. Collectively, LARC expression in intestinal epithelial-type cells is induced by proinflammatory cytokines such as IL-1 and TNF-alpha primarily through activation of NF-kappaB.
Fujiie
S
S
Department of Surgery II, Kinki University School of Medicine, Osaka-Sayama, Osaka 589-8511, Japan.
Hieshima
K
K
Izawa
D
D
Nakayama
T
T
Fujisawa
R
R
Ohyanagi
H
H
Yoshie
O
O
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D054418:Chemokine CCL20; D019742:Chemokines, CC / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000276:immunology*; D019402:Macrophage Inflammatory Proteins / Q000096:biosynthesis*; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D054399:Receptors, CCR6; D019707:Receptors, Chemokine; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
C108355:CCL20 protein, human; C106311:CCR6 protein, human; D054418:Chemokine CCL20; D019742:Chemokines, CC; D007375:Interleukin-1; D019402:Macrophage Inflammatory Proteins; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D054399:Receptors, CCR6; D019707:Receptors, Chemokine; D014409:Tumor Necrosis Factor-alpha