Signaling Role of Intracellular Iron in NF-κB Activation*

Iron chelators inhibit endotoxin-induced NF-κB activation in hepatic macrophages (HMs), suggesting a role for the intracellular chelatable pool of iron in NF-κB activation. The present study tested this hypothesis. Analysis of Fe⁵⁹-loaded HMs stimulated with lipopolysaccharide (LPS), revealed a previously unreported, transient rise in intracellular low molecular weight (LMW)·Fe⁵⁹complex ([LMW·Fe]_i) at ≤2 min returning to the basal level within 15 min. The [LMW·Fe]_i response preceded IκB kinase (IKK) (≥15 min) and NF-κB (≥30 min) activation. Iron chelators (1,2-dimethyl-3-hydroxypyridin-4-one andN,N′-bis-2-hydroxybenzylethylenediamine-N,N′-diacetic acid) abrogated the [LMW·Fe]_i response and IKK and NF-κB activation. The [LMW·Fe]_i response was also observed in tumor necrosis factor α (TNFα)-stimulated HMs and RAW264.7 cells treated with LPS and interferon-γ but not in primary rat hepatocytes or myofibroblastic cells exposed to LPS or TNFα. Both [LMW·Fe]_i response and IKK activation in LPS-stimulated HMs were inhibited by diphenylene iodonium (nonspecific inhibitor for flavin-containing oxidases),l-N⁶-(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 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-κB. These results support a novel signaling role for [LMW·Fe]_iin IKK activation, which appears to be induced by ONOO⁻and selectively operative in macrophages.