Signaling role of intracellular iron in NF-κB activation

Iron chelators inhibit endotoxin-induced NF-kappaB activation in hepatic macrophages (HMs), suggesting a role for the intracellular chelatable pool of iron in NF-kappaB activation. The present study tested this hypothesis. Analysis of Fe-59-loaded HMs stimulated with lipopolysaccharide (LPS), revealed a previously unreported, transient rise in intracellular low molecular weight (LMW).Fe-59 complex ([LMW.Fe](i)) at less than or equal to2 min returning to the basal level within 15 min. The [LMW.Fe](i) response preceded IkappaB kinase (IKK) (greater than or equal to15 min) and NF-kappaB (greater than or equal to30 min) activation. Iron chelators (1,2-dimethyl-3-hydroxypyridin-4-one and N,N'-bis-2-hydroxybenzylethylenediamine-N, N'-diacetic acid) abrogated the [LMW.Fe](i) response and IKK and NF-kappaB activation. The [LMW.Fe](i) response was also observed in tumor necrosis factor alpha (TNFalpha)-stimulated HMs and RAW264.7 cells treated with LPS and interferon-gamma but not in primary rat hepatocytes or myofibroblastic cells exposed to LPS or TNFalpha. Both [LMW.Fe](i) response and IKK activation in LPS-stimulated HMs were inhibited by diphenylene iodonium (nonspecific inhibitor for flavin-containing oxidases), L-N-6-(1-iminoethyl)lysine (selective iNOS inhibitor), and adenoviral-mediated expression of a dominant negative mutant of Rac1 or Cu, Zn-superoxide dismutase, suggesting the role of (NO)-N-. and O-2(-). in mediating the iron signaling. In fact, this inhibition was recapitulated by a cell-permeable scavenger of ONOO-, 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron (III) chloride. Conversely, ONOO- alone induced both [LMW.Fe](i) response and IKK activation. Finally, direct addition of ferrous iron to cultured HMs activated IKK and NF-kappaB. These results support a novel signaling role for [LMW.Fe](i) in IKK activation, which appears to be induced by ONOO- and selectively operative in macrophages.