Multiple NF-kappaB enhancer elements regulate cytokine induction of the human inducible nitric oxide synthase gene.
The human inducible nitric oxide synthase (iNOS) gene is overexpressed in a number of human inflammatory diseases. Previously, we observed that the human iNOS gene is transcriptionally regulated by cytokines and demonstrated that the cytokine-responsive regions are upstream of -3.8 kilobase pairs (kb). Therefore, the purpose of this study was to further localize the functional enhancer elements and to assess the role of the transcription factor NF-kappaB in both human liver (AKN-1) and human lung (A549) epithelial cell lines. The addition of NF-kappaB inhibitors significantly suppressed cytokine-stimulated iNOS mRNA expression and NO synthesis, indicating that NF-kappaB is involved in the induction of the human iNOS gene. Analysis of the first 4.7 kb of the 5'-flanking region demonstrated basal promoter activity and failed to show any cytokine-inducible activity. However, promoter constructs extending to -5.8 and -7.2 kb revealed 2-3-fold and 4-5-fold induction, respectively, in the presence of cytokines. DNA sequence analysis from -3.8 to -7.2 kb identified five putative NF-kappaB cis-regulatory transcription factor binding sites upstream of -4.7 kb. Site-directed mutagenesis of these sites revealed that the NF-kappaB motif at -5.8 kb is required for cytokine-induced promoter activity, while the sites at -5.2, -5.5, and -6.1 kb elicit a cooperative effect. Electromobility shift assays using a site-specific oligonucleotide and nuclear extracts from cells stimulated with cytokine-mixture, tumor necrosis factor-alpha or interleukin-1beta, but not interferon-gamma, exhibited inducible DNA binding activity for NF-kappaB. These data indicate that NF-kappaB activation is required for cytokine induction of the human iNOS gene and identifies four NF-kappaB enhancer elements upstream in the human iNOS promoter that confer inducibility to tumor necrosis factor-alpha and interleukin-1beta.
Taylor
B S
BS
Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA. bsta@med.pitt.edu
de Vera
M E
ME
Ganster
R W
RW
Wang
Q
Q
Shapiro
R A
RA
Morris
S M
SM
Billiar
T R
TR
Geller
D A
DA
D001483:Base Sequence; D002460:Cell Line; D016207:Cytokines / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000032:analysis; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D016328:NF-kappa B / Q000235:genetics*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D009687:Nuclear Proteins / Q000032:analysis; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013859:Thiocarbamates / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016207:Cytokines; D004268:DNA-Binding Proteins; D007375:Interleukin-1; D016328:NF-kappa B; D009687:Nuclear Proteins; D012333:RNA, Messenger; D013859:Thiocarbamates; D014409:Tumor Necrosis Factor-alpha; C066229:prolinedithiocarbamate; D011392:Proline; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II