Induction of Neutrophil Gelatinase-associated Lipocalin Expression by Co-stimulation with Interleukin-17 and Tumor Necrosis Factor-α Is Controlled by IκB-ζ but neither by C/EBP-β nor C/EBP-δ

Abstract

Neutrophil gelatinase-associated lipocalin (NGAL) is a siderophore-binding antimicrobial protein that is up-regulated in epithelial tissues during inflammation. We demonstrated previously that the gene encoding NGAL (LCN2) is strongly up-regulated by interleukin (IL)-1β in an NF-κB-dependent manner but not by tumor necrosis factor (TNF)-α, another potent activator of NF-κB. This is due to an IL-1β-specific synthesis of the NF-κB-binding co-factor IκB-ζ, which is essential for NGAL induction. We demonstrate here that NGAL is strongly induced by stimulation with TNF-α in the presence of IL-17, a pro-inflammatory cytokine produced by the newly discovered subset of CD4+ T helper cells, TH-17. In contrast to the murine NGAL orthologue, 24p3/lipocalin 2, we found no requirement for C/EBP-β or C/EBP-δ for NGAL induction by IL-17 and TNF-α as neither small interfering RNAs against the two C/EBP mRNAs nor mutation of the C/EBP sites in the LCN2 promoter abolished IL-17- and TNF-α-induced up-regulation of NGAL. NGAL induction is governed solely by NF-κB and its co-factor IκB-ζ. This was demonstrated by a pronounced reduction in the amount of NGAL mRNA and NGAL protein synthesized in cells treated with small interfering RNA against IκB-ζ and a total lack of activation of an LCN2 promoter construct with a mutated NF-κB site. As IL-17 stimulation stabilizes the IκB-ζ transcript, we propose a model where TNF-α induces activation and binding of NF-κB to the promoters of both NFKBIZ and LCN2 genes but induce only transcription of IκB-ζ. Co-stimulation with IL-17 leads to accumulation of IκB-ζ mRNA and IκB-ζ protein, which can bind to NF-κB on the LCN2 promoter and thus induce NGAL expression.