NF-κB factors are essential, but not the switch, for pathogen-related induction of the bovine β-defensin 5-encoding gene in mammary epithelial cells

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Expression of the bactericidal peptide β-defensin 5 (BNBD5) is strongly induced by bacterial infections of the udder (mastitis). In situ hybridizations showed that bacteria elicit a strong, locally restricted expression of BNBD5 in mammary epithelial cells (MEC). We defined the BNBD5 promoter by primer extension and showed with reporter gene assays in murine HC-11 and primary bovine mammary epithelial cell (pbMEC) cultures that a 1 kb segment of the promoter is induced about 3-fold by heat-killed bacteria, LPS, IL-1β and TNFα. Deletion series and point mutations of the promoter showed that NF-IL6 augments the induction, but that NF-κB must be bound in cis for pathogen-related stimulation of BNBD5 gene expression. EMSA analyses revealed that both un-stimulated MEC models as well as extracts from healthy udders already display considerable levels of binding competent NF-κB. The bacterial stimulus increased this level about 3-fold, as measured with a NF-κB driven reporter gene in pbMEC, matching quantitatively the extent of the BNBD5-reporter gene induction. In contrast, expression of the endogenous BNBD5-gene is stimulated much more (>30-fold) in udders and pbMEC indicating that factors other than elevated levels of binding-competent NF-κB factors determine the induction of the native gene. Supporting this conclusion, we found that expression of bovine TLR2 or TLR4 in HEK293 cells can reconstitute the bacterial activation of the NF-κB expression construct, but not that of the BNBD5-reporter gene. Our data suggest that elevated levels of binding competent NF-κB factors mediated via TLR pathogen recognitions mechanisms are not the key switch for pathogen related induction of the BNBD5-encoding gene in MEC.