Activation of eotaxin gene transcription by NF-kappa B and STAT6 in human airway epithelial cells

Abstract

The C-C chemokine eotaxin is a potent chemoattractant for eosinophils and probably plays an important role in the pathogenesis of asthma, although the mechanisms of its regulation are not well known. Airway epithelial cells express eotaxin mRNA and protein after stimulation with a variety of cytokines. We focused on the molecular mechanisms of eotaxin gene regulation by TNF-alpha and IL-4 in the airway epithelial cell line, BEAS-2B. Cells were transfected with luciferase reporter plasmids, which contained up to 1363 bp of the eotaxin promoter. Eotaxin promoter activity was increased by TNF-alpha (2.5-fold) and IL-4 (1.5-fold), respectively. The combination of TNF-alpha and IL-4 produced 3.6-fold activation of the eotaxin promoter. The eotaxin promoter contains overlapping consensus binding sites for transcription factors, NF-kappa B and STAT6, which are known to mediate responses to TNF-alpha and IL-4, respectively. Electrophoretic mobility shift assays revealed NF-kappa B binding after TNF-alpha stimulation and STAT6 binding after IL-4 stimulation using a DNA probe derived from the eotaxin promoter. Mutant plasmids were generated to define the roles of these transcription factors in eotaxin promoter activity. TNF-alpha stimulation, but not IL-4 stimulation, was lost in plasmids mutated at the NF-kappa B binding site, whereas IL-4 stimulation, but not TNF-alpha stimulation, was lost in plasmids mutated at the STAT6 binding site. When both sites were mutated, all transcriptional activation was lost. These results imply that TNF-alpha and IL-4 stimulate expression of the eotaxin gene by activating NF-kappa B and STAT6.