Glucocorticoids inhibit the induction of nitric oxide synthase II by down-regulating cytokine-induced activity of transcription factor nuclear factor-kappa B.
Incubation of human A549/8 cells with human interleukin-1 beta (50 units/ml), interferon-gamma (100 units/ml), and tumor necrosis factor-alpha (10 ng/ml) (cytomix) resulted in a marked expression of the mRNA of the inducible nitric oxide synthase (NOS II). This induction was prevented by cycloheximide. Dexamethasone markedly reduced cytokine-induced NOS II mRNA concentrations; this reduction was prevented by RU 38486 (mifepristone). Pyrrolidine dithiocarbamate, an inhibitor of nuclear factor-kappa B (NF-kappa B) activation, also significantly decreased cytomix-induced NOS II mRNA levels. When A549/8 cells were transfected with a construct containing 1570-bp 5'-flanking sequence of the murine NOS II gene cloned before a reporter gene, the murine NOS II promoter was induced up to 20-fold with cytomix but not with bacterial lipopolysaccharide. Dexamethasone as well as pyrrolidine dithiocarbamate inhibited this induction. In electrophoretic mobility shift assays, nuclear protein extracts from cytomix-induced, but not from unstimulated cells, significantly slowed the migration of an oligonucleotide containing the NF-kappa B-binding site. This band shift was markedly reduced by dexamethasone. On the other hand, cytomix-induced nuclear protein content of NF-kappa B p65 and NF-kappa B p50 was not reduced by dexamethasone (as analyzed by Western blot). Dexamethasone also did not reduce cytomix-induced expression of NF-kappa B p65 mRNA or enhance the expression of NF-kappa B inhibitor mRNA. The human and murine NOS II promoters also contain consensus sequences for activating protein-1 (AP-1) binding. However, AP-1 binding activity of nuclear extracts of A549/8 cells was not enhanced by cytomix or inhibited by dexamethasone. These data suggest that the activated glucocorticoid receptor prevents (by a protein/protein interaction) the binding of transcription factor NF-kappa B, but not AP-1, to the NOS II promoter, thereby inhibiting the induction of NOS II transcription.
Kleinert
H
H
Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany.
Euchenhofer
C
C
Ihrig-Biedert
I
I
Förstermann
U
U
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D016207:Cytokines / Q000494:pharmacology*; D017931:DNA Primers; D003907:Dexamethasone / Q000494:pharmacology*; D015536:Down-Regulation; D004790:Enzyme Induction; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D051841:Transcription Factor RelB; D014157:Transcription Factors; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016207:Cytokines; D017931:DNA Primers; D007375:Interleukin-1; D007527:Isoenzymes; D016328:NF-kappa B; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; C497593:RELB protein, human; D012333:RNA, Messenger; C497594:Relb protein, mouse; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D051841:Transcription Factor RelB; D003907:Dexamethasone; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase