IRP1 activation by extracellular oxidative stress in the perfused rat liver

The expression of several proteins with critical functions in iron metaboli sm is regulated post-transcriptionally by the binding of iron regulatory pr oteins, IRP1 and IRP2, to mRNA iron responsive elements (IREs). In iron def icient tissues and cultured cells, both IRP1 and IRP2 are activated for hig h affinity IRE binding. Previous work showed that IRP1 is also activated wh en cultured cells are exposed to H2O2. The well established role of iron an d H2O2 in tissue injury (based on Fenton chemistry) suggests that this resp onse may have important pathophysiological implications. This is particular ly relevant in inflammation, where cytotoxic immune cells release large amo unts of reactive oxygen species. Here, we describe a rat liver perfusion mo del to study IRP1 activation under H2O2 generation conditions that mimic a physiological inflammatory response, using steady-state concentrations of H 2O2 produced by a glucose/ glucose oxidase/catalase system. We show first t hat stimulated neutrophils are able to increase serum levels of H2O2 by a f actor of 10, even in the presence of H2O2-degrading erythrocytes. We furthe r show that perfusion of rat liver with glucose oxidase leads to a rapid ac tivation of IRE binding activity in the intact organ. Mobility shift assays with liver extracts and IRP1 or IRP2-specific probes indicate that only IR P1 responds to H2O2. Our study demonstrates a principal existence of iron r egulation by oxidative stress at the intact organ level. It also provides a link between iron metabolism and the inflammatory response, as H2O2 is a m ajor product of the oxidative burst of neutrophils and macrophages.