Crucial roles of binding sites for NF-kappaB and C/EBPs in IkappaB-zeta-mediated transcriptional activation.
IkappaB-zeta [inhibitor of NF-kappaB (nuclear factor kappaB) zeta] is a nuclear protein that is induced upon stimulation of TLRs (Toll-like receptors) and IL (interleukin)-1 receptor. IkappaB-zeta harbours C-terminal ankyrin repeats that interact with NF-kappaB. Our recent studies have shown that, upon stimulation, IkappaB-zeta is essential for the induction of a subset of inflammatory genes, represented by IL-6, whereas it inhibits the expression of TNF (tumour necrosis factor)-alpha. In the present study, we investigated mechanisms that determine the different functions of IkappaB-zeta. We found that co-expression of IkappaB-zeta and the NF-kappaB subunits synergistically activates transcription of the hBD-2 (human beta-defensin 2) and NGAL (neutrophil gelatinase-associated lipocalin) genes, whereas it inhibits transcription of E-selectin. Reporter analyses indicated that, in addition to an NF-kappaB-binding site, a flanking C/EBP (CCAAT/enhancer-binding protein)-binding site in the promoters is essential for the IkappaB-zeta-mediated transcriptional activation. Using an artificial promoter consisting of the NF-kappaB- and C/EBP-binding sites, transcriptional activation was observed upon co-transfection with IkappaB-zeta and NF-kappaB, indicating that these sequences are minimal elements that confer the IkappaB-zeta-mediated transcriptional activation. Chromatin immunoprecipitation assays and knockdown experiments showed that both IkappaB-zeta and the NF-kappaB subunits were recruited to the NGAL promoter and were essential for the transcriptional activation of the hBD-2 and NGAL promoters on stimulation with IL-1beta. The activation of the NGAL promoter by transfection of IkappaB-zeta and NF-kappaB was suppressed in C/EBPbeta-depleted cells. Thus IkappaB-zeta acts as an essential transcriptional activator by forming a complex with NF-kappaB on promoters harbouring the NF-kappaB- and C/EBP-binding sites, upon stimulation of TLRs or IL-1 receptor.
Matsuo
Susumu
S
Department of Molecular and Cellular Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Yamazaki
Soh
S
Takeshige
Koichiro
K
Muta
Tatsushi
T
D000209:Acute-Phase Proteins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D020672:I-kappa B Proteins; D000071068:Lipocalin-2; D054834:Lipocalins; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D000209:Acute-Phase Proteins; D048868:Adaptor Proteins, Signal Transducing; D022762:CCAAT-Enhancer-Binding Proteins; D020672:I-kappa B Proteins; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D016328:NF-kappa B; C417234:NFKBIZ protein, human; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger