Among the antimicrobial molecules produced by epithelial cells, DEFB4 is inducible in response to pro-inflammatory signals such as cytokines and bacterial molecules. Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen that exacerbates chronic obstructive pulmonary disease in adult and causes otitis media and sinusitis in children. Previously, we have demonstrated that DEFB4 effectively kills NTHi and is induced by NTHi via TLR2 signaling. The 5'-flanking region of DEFB4 contains several NF-kappa B binding motifs, but their NTHi-specific activity remains unclear. In this study, we aimed to elucidate molecular mechanism involved in DEFB4 regulation, focusing on the role of the distal NF-kappa B binding motif of DEFB4 responding to NTHi. Here, we show that the human middle ear epithelial cells up-regulate DEFB4 expression in response to NTHi via NF-kappa B activation mediated by I kappa K alpha/beta-I kappa B alpha, signaling. Deletion of the distal NF-kappa B binding motif led to a significant reduction in NTHi-induced DEFB4 up-regulation. A heterologous construct containing the distal NF-kappa B binding motif was found to increase the promoter activity in response to NTHi, indicating a NTHi-responding enhancer activity of the distal NF-kappa B binding motif. Furthermore, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that the p65 domain of NF-kappa B(13 binds to the distal NF-kappa B binding motif in response to NTHi. Taken together, our results suggest that NTHi-induced binding of p65 NF-kappa B to the distal NF-kappa B binding motif of DEFB4 enhances NTHi-induced DEFB4 regulation in epithelial cells. (C) 2013 Elsevier Inc. All rights reserved.