Modulators of inflammation use nuclear factor-kappa B and activator protein-1 sites to induce the caspase-1 and granzyme B inhibitor, proteinase inhibitor 9.
Proteinase inhibitor 9 (PI-9) inhibits caspase-1 (interleukin (IL)-1beta-converting enzyme) and granzyme B, thereby regulating production of the pro-inflammatory cytokine IL-1beta and susceptibility to granzyme B-induced apoptosis. We show that cellular PI-9 mRNA and protein are induced by IL-1beta, lipopolysaccharide, and 12-O-tetradecanoylphorbol-13-acetate. We identified functional imperfect nuclear factor-kappaB (NF-kappaB) sites at -135 and -88 and a consensus activator protein-1 (AP-1) site at -308 in the PI-9 promoter region. Using transient transfections in HepG2 cells to assay PI-9 promoter mutations, we find that mutational ablation of the AP-1 site or of either NF-kappaB site reduces IL-1beta-induced expression of PI-9 by approximately 60%. Mutational ablation of the two NF-kappaB sites and of the AP-1 site nearly abolishes both basal and IL-1beta-induced expression of PI-9. Nuclear extracts from IL-1beta-treated HepG2 cells exhibited strong, IL-1beta-inducible binding to the NF-kappaB sites and to the AP-1 site. Electrophoretic mobility shift assays show that after IL-1beta treatment c-Jun/c-Fos and JunD bind to the AP-1 site, whereas the p50/p65 heterodimer binds to the two NF-kappaB sites. Estrogens induce PI-9, but induction of PI-9 by estrogens and IL-1beta is not synergistic. In transiently transfected, estrogen receptor-positive HepG2ER7 cells, estrogens do not interfere with IL-1beta induction, whereas IL-1beta exhibits dose-dependent repression of estrogen-inducible PI-9 expression. Our surprising finding that the pro-inflammatory cytokine IL-1beta strongly induces PI-9 suggests a novel mechanism for regulating inflammation and apoptosis through a negative feedback loop controlling expression of the anti-inflammatory and anti-apoptotic protein, PI-9.
Kannan-Thulasiraman
Padma
P
Department of Biochemistry, University of Illinois, 600 S Matthews Avenue, Urbana, IL 61801-3602, USA.
Shapiro
David J
DJ
D001483:Base Sequence; D001665:Binding Sites; D061945:Caspase Inhibitors; D002460:Cell Line; D017931:DNA Primers; D004997:Ethinyl Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D053804:Granzymes; D006801:Humans; D007375:Interleukin-1 / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology; D016328:NF-kappa B / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000187:drug effects*; D015843:Serpins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D061945:Caspase Inhibitors; D017931:DNA Primers; D007375:Interleukin-1; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; C102316:SERPINB9 protein, human; D015843:Serpins; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D004997:Ethinyl Estradiol; C011310:moxestrol; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; D013755:Tetradecanoylphorbol Acetate