title: Neutrophil gelatinase-associated lipocalin is up-regulated in human epithelial cells by IL-1 beta, but not by TNF-alpha.
abstract: Synthesis of the antimicrobial protein neutrophil gelatinase-associated lipocalin (NGAL) increases dramatically in bronchial epithelial cells and alveolear type II pneumocytes during lung inflammation. IL-1beta induces a >10-fold up-regulation of NGAL expression in the type II pneumocyte-derived cell line A549 cells, whereas TNF-alpha, IL-6, and LPS had no effect. Similar IL-1beta selectivity was demonstrated in primary bronchial epithelial cells and epidermal keratinocytes and for an NGAL promoter fragment transfected into A549 cells. By deletion and substitution analysis of the NGAL promoter, a 40-bp region containing an NF-kappaB consensus site was found to control the IL-1beta-specific up-regulation. Involvement of the NF-kappaB site was demonstrated by site-directed mutagenesis, by transfection with a dominant-negative inhibitor of the NF-kappaB pathway, and by EMSA. TNF-alpha activation of NF-kappaB, in contrast, did not increase NGAL synthesis, even though induced binding of NF-kappaB to the NGAL promoter was observed in vitro. IL-1beta specificity was not contained within the NF-kappaB site of the NGAL promoter, as determined by exchanging the NGAL promoter's NF-kappaB-binding sequence with that of the IL-8 promoter or with the NF-kappaB consensus sequence and by testing the NF-kappaB-binding sequence of the NGAL promoter against the heterologous SV40 promoter. Selectivity for the IL-1 pathway was substantiated by demonstrating that NGAL promoter activity could be induced by LPS stimulation of A549 cells transiently expressing Toll-like receptor 4, which use the same intracellular signaling pathway as the IL-1R. Together, this demonstrates a selective up-regulation of NGAL by the IL-1 pathway.
authors.affiliation: Department of Hematology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark. jcowland@rh.dk
authors.forename: Jack B
authors.initials: JB
authors.lastname: Cowland
authors.forename: Ole E
authors.initials: OE
authors.lastname: Sørensen
authors.forename: Maxwell
authors.initials: M
authors.lastname: Sehested
authors.forename: Niels
authors.initials: N
authors.lastname: Borregaard
mesh_terms: D020121:5' Untranslated Regions / Q000502:physiology; D000209:Acute-Phase Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D004847:Epithelial Cells / Q000276:immunology / Q000378:metabolism*; D005786:Gene Expression Regulation; D020868:Gene Silencing; D017930:Genes, Reporter; D018922:HL-60 Cells; D006801:Humans; D007375:Interleukin-1 / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D000071068:Lipocalin-2; D054834:Lipocalins; D008070:Lipopolysaccharides / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology; D015513:Oncogene Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000096:biosynthesis; D012097:Repressor Proteins / Q000494:pharmacology; D020545:Respiratory Mucosa / Q000378:metabolism; D017384:Sequence Deletion; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation / Q000276:immunology*
chemical_list: D020121:5' Untranslated Regions; D000209:Acute-Phase Proteins; D007375:Interleukin-1; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D012097:Repressor Proteins; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D015500:Chloramphenicol O-Acetyltransferase