Emerging Regulatory Role of Nrf2 in Iron, Heme, and Hemoglobin Metabolism in Physiology and Disease

Abstract

Iron has played an important role in energy production since the beginning of life, as iron-catalyzed redox reactions are required for energy production. Oxygen, a highly efficient electron acceptor with high reduction potential, facilitates highly efficient energy production in eukaryotic cells. However, the increasing atmospheric oxygen concentration produces new threats to the organism, as oxygen reacts with iron and produces reactive oxygen species unless its levels are strictly regulated. As the size of multicellular organisms increases, these organisms must transport oxygen to the peripheral tissues and begin to employ red blood cells containing hemoglobin. This system is potentially a double-edged sword, as hemoglobin autoxidation occurs at a certain speed and releases free iron into the cytoplasm. Nrf2 belongs to the CNC transcription factor family, in which NF-E2p45 is the founding member. NF-E2p45 was first identified as a transcription factor that binds to the erythroid gene regulatory element NF-E2 located in the promoter region of the heme biosynthetic porphobilinogen deaminase gene. Human Nrf2 was also identified as a transcription factor that binds to the regulatory region of the β-globin gene. Despite these original findings, NF-E2p45 and Nrf2 knockout mice exhibit few erythroid phenotypes. Nrf2 regulates the expression of a wide range of antioxidant and detoxification enzymes. In this review article, we describe and discuss the roles of Nrf2 in various iron-mediated bioreactions and its possible coevolution with iron and oxygen.

Keywords: Nrf2; heme; hemoglobin; iron; oxidative stress.

Figure 1

Members of the Maf and CNC transcription factor families in mammals. Maf transcription factors are classified into large Maf proteins, consisting of c-Maf, MafA, MafB, and NRL, and small Maf proteins (sMafs), comprising MafF, MafG, and MafK. The CNC family comprises NF-E2p45, Nrf1-3, Bach1, and Bach2. Only mammalian family members are shown in this figure. CLS, cytoplasmic localization signal. This figure is modified from reference (4).

Figure 2

Role of Nrf2 in heme and iron metabolism. Nrf2 is a master regulator of the antioxidant response induced by thiol-reactive substances, such as electrophilic reagents and heavy metals. In unstressed cells, Nrf2 is constitutively ubiquitinated by the Keap1-Cullin 3 ubiquitination complex and degraded by the 26S proteasome. Keap1 oxidation induces Nrf2 stabilization, nuclear translocation, heterodimerization with sMafs, and the transcriptional activation of target genes by binding to antioxidant response elements (AREs). An Nrf2 target gene, HO-1, degrades free heme into biliverdin, carbon monoxide and iron. HO-1 transcription is repressed by Bach1. Heme binding induces the nuclear export and degradation of Bach1. Free iron released from heme also regulates ROS production. Nrf2 induces the transcription of ferritin heavy chain (FTH1) and light chain (FTL) to store iron in the oxidized state and of ferroportin 1 (FPN1) to export intracellular labile iron.

Figure 3

Nrf2/HO-1 counteracts heme toxicity. In sickle cell disease, erythrocytes are deformed by the polymerization of hemoglobin S, and oxidative stress subsequently develops, inducing intravascular hemolysis and vaso-occlusion. An increase in the free heme concentration activates Nrf2 and induces HO-1 expression. HO-1 degrades heme into biliverdin, which is subsequently reduced into the antioxidant bilirubin. Nrf2 activation increases fetal hemoglobin levels by inducing the expression of the γ-globin gene and alleviates hemoglobin S production in erythroid progenitor cells. Nrf2 activation attenuates damage to multiple tissues, as well as the production of inflammatory cytokines.

Figure 4

Hemoglobin production and redox homeostasis in erythrocytes. (A) NF-E2p45 participates in the transcriptional activation of α- and β-hemoglobin, as well as heme-synthetic enzymes, such as porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UROS), and ferrochelatase (FECH), in erythroblasts. After enucleation, erythrocytes combat oxidative stress inducined by hemoglobin autoxidation through a mechanism that is not regulated by gene transcription. (B) Glucose metabolism supplies NADH, which is utilized for methemoglobin reduction. Superoxide anions (${\text{O}}_2^{•-}$) are eliminated by superoxide dismutase 1 (SOD1), which converts these ions into hydrogen peroxide (H₂O₂), or are converted into oxygen through heme degradation. Ferric iron reacts with ${\text{O}}_2^{•-}$ by Haber-Weiss reaction to generate highly reactive hydroxyl radical (HO^•) and oxygen molecule. H₂O₂ is further reduced to water by the consumption of NADPH by glutathione peroxidase (GPX) and peroxiredoxin 2 (PRX2); NADPH is produced by the pentose phosphate pathway. FPN1 exports cellular labile iron to prevent ROS production.

Figure 5

Hemoglobin oxidation and redox catastrophe. (A) Oxy-hemoglobin [HbFe(II)-O₂] is partially converted into methemoglobin [HbFe(III)] and the superoxide anion (${\text{O}}_2^{•-}$) by autoxidation. Methemoglobin is then reduced by cytochrome b5 reductase (B5R) using NADH or by glutathione. Superoxide is detoxified by SOD1 and GPX activities (direction illustrated with blue arrows); otherwise hydrogen peroxide (H₂O₂) is converted into the hydroxyl radical (HO^•) by the Fe²⁺ ion or is involved in series of hemoglobin degradation cascades (illustrated with brown arrows). (B) Aniline induces methemoglobinemia and hemolysis by repetitive redox cycles of hemoglobin oxidation. Aniline is oxidized by hepatic mixed-function oxidase into bioactive phenylhydroxylamine, which reacts with oxy-hemoglobin and generates nitrosobenzene and methemoglobin. Nitrosobenzene is reduced again by NADPH flavin reductase (FR). Although glycolytic metabolism supplies NADH and NADPH, the phenylhydroxylamine/nitrosobenzene redox cycle produces methemoglobin production and depletes cellular reductants.