Modulators of inflammation use nuclear factor-κB and activator protein-1 sites to induce the caspase-1 and granzyme B inhibitor, proteinase inhibitor 9

Abstract

Proteinase inhibitor 9 (PI-9) inhibits caspase-1 (interleukin (IL)-1β-converting enzyme) and granzyme B, thereby regulating production of the pro-inflammatory cytokine IL-1β and susceptibility to granzyme B-induced apoptosis. We show that cellular PI-9 mRNA and protein are induced by IL-1β, lipopolysaccharide, and 12-O-tetradecanoylphorbol-13-acetate. We identified functional imperfect nuclear factor-κB (NF-κB) 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-κB site reduces IL-1β-induced expression of PI-9 by ∼60%. Mutational ablation of the two NF-κB sites and of the AP-1 site nearly abolishes both basal and IL-1β-induced expression of PI-9. Nuclear extracts from IL-1β-treated HepG2 cells exhibited strong, IL-1β-inducible binding to the NF-κB sites and to the AP-1 site. Electrophoretic mobility shift assays show that after IL-1β treatment c-Jun/c-Fos and JunD bind to the AP-1 site, whereas the p50/p65 heterodimer binds to the two NF-κB sites. Estrogens induce PI-9, but induction of PI-9 by estrogens and IL-1β is not synergistic. In transiently transfected, estrogen receptor-positive HepG2ER7 cells, estrogens do not interfere with IL-1β induction, whereas IL-1β exhibits dose-dependent repression of estrogen-inducible PI-9 expression. Our surprising finding that the pro-inflammatory cytokine IL-1β 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.