Iron accumulates in alcoholic liver disease (ALD) and an increment in hepatic iron content aggravates the disease. However, the precise molecular mechanisms underlying the cause and effect relationship between iron and ALD, are still elusive. Our research has identified an increased chelatable pool of iron in hepatic macrophages (HM) as a critica priming event for accentuated activation of NF-kappaB and expression of NF-kappaB responsive genes such as TNFalpha in experimental ALD. Our attempt to dissect the mechanistic link between iron and IkappaB kinase (IKK) activation, has led to a demonstration of a novel signaling phenomenon in LPS or TNFalpha treated HM: a rapid and transient rise in the intracellular level of low molecular weight-iron complexes ([LMW-Fe]i) at equal to or <2min after agonist treatment. This [LMW-Fe]i response precedes IKK activation (15-30 min) and increased NF-kappaB binding (30-45 min). It is induced by ONOO- and serves as a downstream signaling molecule for IKK activation. Abrogation of this response with iron chelators, a ONOO- decomposition catalyst, an iNOS inhibitor, a dominant negative mutant of Racl, Cu/ZnSOD, all inhibit LPS-induced IKK activation in HM. Macrophages but not hepatocytes or other cell types exhibit this [LMW-Fe]i signaling. However, a direct treatment with ferrous iron activates IKK with similar kinetics in both macrophages and other cell types, indicating exogenous ferrous iron can substitute for the [LMW-Fe]i response. Further, the size of the chelatable iron pool, directly modulates the [LMW-Fe]i response and subsequent IKK activation as exemplified by an enhanced [LMW-Fe]i response in HM from experimental ALD or RAW264.7 murine macrophages with a mutation in Nrampl, a putative protein that regulates iron homeostasis in macrophages. Based on these novel findings, the proposed study is aimed at elucidating the mechanisms underlying elicitation of the [LMW-Fe]i rise and its signaling to activate IKK. The study will further investigate the mechanisms for the increased chelatable iron pool in HM in experimental ALD and the potential roles of Nramp1 and other iron transport proteins in this defect. To this end, we address the following specific aims: 1) to determine the intracellular source of LPS-stimulated [LMW-Fe]i in HM; 2) to investigate whether iron-mediated IKK activation requires upstream kinases, IKKgamma subunit, or iron acceptor proteins; 3) to determine the regulatory role of Nramp1 in [LMW-Fe]i-mediated IKK activation and expression of TNFalpha.; 4) to examine changes in expression of proteins involved in iron homeostasis (DMT1, Nramp1, ferroportin, HFE) and iron handling in HM in experimental ALD for better understanding of iron accumulation. In summary, the proposed research pursues an original hypothesis based on the novel signaling role of [LMW-Fe]i in IKK activation. This finding has far-reaching implications for not only understanding how iron primes the liver for ALD but also disclosing a fundamental mechanism of regulation of IKK by iron.
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