Excess Capacity of the Iron Regulatory Protein System

Abstract

Iron regulatory proteins (IRP1 and IRP2) are master regulators of cellular iron metabolism. IRPs bind to iron-responsive elements (IREs) present in the untranslated regions of mRNAs encoding proteins of iron storage, uptake, transport, and export. Because simultaneous knockout of IRP1 and IRP2 is embryonically lethal, it has not been possible to use dual knockouts to explore the consequences of loss of both IRP1 and IRP2 in mammalian cells. In this report, we describe the use of small interfering RNA to assess the relative contributions of IRP1 and IRP2 in epithelial cells. Iron is an essential element required for the function of numerous critical enzymes and is essential for cell survival. However, because of the ability of iron to catalyze the formation of reactive oxygen species, excess iron is harmful. Consequently, levels of intracellular iron must be tightly controlled (1). Iron-responsive elements (IREs) 2 play a central role in the regulation of iron homeostasis. IREs are mRNA hairpin elements present in the untranslated region of mRNAs encoding proteins of iron storage (ferritin) (2-4), iron uptake (TfR1) (5, 6), iron transport (DMT1) (7,8), and iron export (ferroportin) (9 -11).The role of IRE elements in ferritin and TfR1 has been particularly well studied. In ferritin H and L, an IRE is present in the 5Ј-UTR of the mRNA, whereas in TfR1, five IRE elements are present in the 3Ј-UTR. In both cases, the IRE can be bound by IRE-binding proteins. When IRE-binding proteins bind to the IRE in the 5Ј-UTR of ferritin, they inhibit translation (3, 4). When IRE-binding proteins bind to the 3Ј-UTR of TfR1, they stabilize the mRNA (6).Two IRE-binding proteins have been identified, IRP1 (12, 13) and IRP2 (14). They are highly homologous but respond to iron challenge via different mechanisms. IRP1 is a bifunctional protein; its conformational and functional changes depend on cellular iron status. When iron levels are high, IRP1 forms a 4Fe-4S cluster and functions as a cytosolic aconitase. Under these conditions, IRP1 cannot bind the IRE. Conversely, when iron levels are low, the Fe-S cluster disassembles, and IRP1 binds to IREs. Recently, it has been suggested that iron-dependent regulation of IRP1 abundance represents an additional mechanism of control of IRP1 (15). IRP2 differs from IRP1 by the presence of an additional 73-amino acids in the N terminus. When iron levels are high, IRP2 undergoes iron-dependent degradation (16,17). The consequence of this coordinate regulation of IRP proteins is that in iron-replete conditions, there is less IRP1 and IRP2 available to bind to IREs, leading to an increase in ferritin and a decrease in TfR1. Conversely, under conditions of iron limitation, increases in the activity and level of IRP1 and IRP2 lead to decreased ferritin and increased TfR1. Because increased ferritin increases iron storage, whereas decreased TfR1 decreases iron transport, these IRP-dependent events act in concert to maintain iron homeostasis. IRPs are also regulated by other physiological stimuli, inc...

Iron Regulatory Protein 1 Outcompetes Iron Regulatory Protein 2 in Regulating Cellular Iron Homeostasis in Response to Nitric Oxide

“…Animal and cellular models of IRP deficiency have proven useful for defining the specific as well as redundant functions of IRP1 and IRP2 under basal conditions (4,5) or in response to iron fluctuation (6), local inflammation (36), and oxidative stress (30,37). In our study, primary cultures of BMMs derived from IRP1-and IRP2-null mice were exposed to the chemical NO donor DETA/NO that mimics NO release from NOS2 and recapitulates the aconitase/IRP1 switch induced by IFN-␥/LPSstimulated macrophages (28).…”

Post-transcriptional Modulation of Iron Homeostasis during p53-dependent Growth Arrest

“…IRE Binding Assays-RNA band shift assays were performed as previously described (36). Briefly, the cells (2 ϫ 10 5 ) were lysed in 50 l of extraction buffer (10 mM HEPES, 10 mM KCl, 2 mM MgCl 2, 40 mM NaCl, 5% glycerol, 1 mM dithiothreitol, and 0.2% Nonidet P-40).…”

“…Real Time RT-PCR-Real time RT-PCR was performed as described previously (36). The following primers were used: for hepcidin, forward, 5Ј-CTGCAACCCCAGGACAGAG-3Ј, and reverse, 5Ј-GGAATAAATAAGGAAGGGAGGGG-3Ј (37); and for ferritin H, forward, 5Ј-CTTTGACCGCGATGATGT-GGCTTT-3Ј, and reverse, 5Ј-TTTGTCAGTGGCCAGTTTG-TGCAG-3Ј.…”

Curcumin, a cancer chemopreventive and chemotherapeutic agent, is a biologically active iron chelator

“…Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) was performed essentially as described. 33 The following primers were used: hepcidin forward primer: 5Ј-CTG AGC AGC ACC ACC TAT CTC-3Ј, reverse primer: 5Ј-TGG CTC TAG GCT ATG TTT TGC-3Ј. Samples were normalized using ␤-actin, forward primer: 5Ј-AGA GGG AAA TCG TGC GTG AC-3Ј, and reverse primer: 5Ј-CAA TAG TGA TGA CCT GGC CGT-3Ј.…”