Pseudomonas Aeruginosa- and IL-1β-Mediated Induction of Human β-Defensin-2 in Keratinocytes Is Controlled by NF-κB and AP-1

Human β-defensin-2 (hBD-2) is an inducible epithelial peptide antibiotic involved in cutaneous defense. Expression of hBD-2 in keratinocytes is strongly induced by IL-1β and culture supernatants of Pseudomonas aeruginosa (PA). The use of an IL-1 receptor antagonist revealed that PA-mediated induction of hBD-2 is not dependent on IL-1. Luciferase gene reporter experiments, demonstrated that a 2,338 bp promoter fragment of hBD-2 containing three putative NF-κB as well as one activator protein-1 (AP-1) binding site was strongly activated by IL-1β and PA. Mutation of all NF-κB binding sites together with mutation of the AP-1 binding site completely abolished hBD-2 promoter activation by IL-1β and PA. Treatment with the NF-κB inhibitor Helenalin as well as with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the p38 mitogen-activated protein kinase inhibitor SB 202190 blocked hBD-2 induction by IL-1β and PA. PD 98059, a selective inhibitor of extracellular signal-regulated kinase 1/2 demonstrated no significant influence. Transcription factor ELISAs indicated that the NF-κB heterodimer p50–p65 binds to all three NF-κB sites in the hBD-2 promoter upon stimulation of primary keratinocytes with IL-1β and PA. We conclude that the activation of NF-κB (p50–p65) and AP-1 are crucial events for induction of hBD-2 in keratinocytes upon IL-1β and PA stimulation.