A Red Carpet for Iron Metabolism

Abstract

200 billion red blood cells (RBCs) are produced every day, requiring more than 2 × 10¹⁵ iron atoms every second to maintain adequate erythropoiesis. These numbers translate into 20 mL of blood being produced each day, containing 6 g of hemoglobin and 20 mg of iron. These impressive numbers illustrate why the making and breaking of RBCs is at the heart of iron physiology, providing an ideal context to discuss recent progress in understanding the systemic and cellular mechanisms that underlie the regulation of iron homeostasis and its disorders.

Figure 1. Iron Metabolism in Erythroid Cells

Top: In the bone marrow, macrophages are surrounded by erythroid cells at different stages of differentiation (erythroblastic islands). These macrophages support erythroid cell proliferation and maturation. The erythropoietic (EPO)- and iron-dependent phases of erythropoiesis are indicated. At steady state, most of the daily amount of iron needed for erythropoiesis is recycled by specialized macrophages in the spleen and liver. Bottom: Iron acquisition in erythroid cells is dependent on endocytosis of diferric transferrin (Tf-Fe₂) via the transferrin receptor (TFR1). In acidified endosomes, iron is freed from Tf and exported into the cytoplasm by DMT1 after reduction of the metal by STEAP3 (six-transmembrane epithelial antigen of prostate 3). The recycling of Tf and TFR1 is crucial for optimal iron uptake and requires SNX3 (sortin nexin 3) and EXOC6 (exocyst complex component 6). Pinocytosis of extracellular ferritin may also contribute iron but cannot substitute for TFR1-mediated Tf-Fe₂ uptake. The binding of Tf-Fe₂ and/or pIgA1 (polymeric immunoglobulin A1) to TFR1 triggers signaling events independent of iron uptake. TFR2 is involved in sensing plasma Tf-Fe₂ concentration and in turn modulates erythropoietin receptor (EPOR) signaling. The metabolically active labile iron pool is used directly for incorporation into iron proteins or transported into mitochondria via mitoferrin 1 (MFRN1, MFRN2 in non-erythroid cells), which forms a complex with ABCB10. In mitochondria, iron is inserted into protoporphyrin IX (PPIX) by ferrochelatase (FECH) to produce heme. Heme is transported outside of mitochondria via the 1b isoform of FLVCR (feline leukemia virus subgroup C cellular receptor) for incorporation into hemoproteins, mostly hemoglobin; the FLVCR1a isoform exports heme. Iron is also used for Fe-S cluster synthesis, which among other factors involves frataxin (FXN) and GLRX5 (Glutaredoxin-related protein 5). Fe-S clusters are incorporated into client proteins throughout the cell. Maturation of cytosolic Fe-S proteins by the CIA (cytosolic Fe-S cluster assembly) system depends on an unknown compound (designated X) generated during mitochondrial Fe-S cluster biosynthesis. Its export into the cytosol requires the ATP-binding cassette protein ABCB7. Fe-S proteins play key roles in the regulation of iron metabolism. For instance, under conditions of iron or GLRX5 deficiency, iron regulatory protein 1 (IRP1) is devoid of a Fe-S cluster and inhibits translation of ALAS2, the first and rate-limiting enzyme of heme biogenesis, thereby preventing the accumulation of toxic heme intermediates. In the mitochondria, excess iron can be stored in the organelle-specific form of ferritin (FTMT). In the cytosol, excess iron is sequestered within heteropolymers of ferritin H and L chains. Cellular iron efflux is mediated by ferroportin (FPN) and requires iron oxidation on the extracellular side.

Figure 2. Regulation of Cellular Iron Metabolism

(A) In iron-deficient cells (right), IRP1 or IRP2 bind to cis-regulatory iron-responsive elements (IREs) present in the untranslated regions (UTRs) of target mRNAs. The binding of IRPs to single IREs in the 5′ UTRs of ferritin and FPN mRNAs inhibits their translation, whereas IRP interaction with multiple 3′ UTR IREs in the TFR1 mRNA inhibits its decay; ferritin protein is targeted to autolysomal degradation by NCOA4 (Nuclear receptor coactivator 4) to release iron. The ensuing increase in TFR1-mediated iron uptake and decrease in iron storage in ferritin and export via FPN augment the labile iron pool. In iron-loaded cells (left), IRP binding to IREs is suppressed. IRP1 assembles a cubane Fe-S cluster with the assistance of FAM96A and adopts a conformation that precludes IRE binding while conferring aconitase activity to interconvert citrate and isocitrate. IRP2 lacks a Fe-S cluster and is instead regulated by interaction with FBXL5, which bears an iron- and oxygen-sensing domain. FBXL5 accumulates in iron-replete cells and triggers the proteasomal degradation of IRP2 by recruiting a SKP1-CUL1 E3 ubiquitin ligase complex. In parallel, NCOA4 is destabilized and ferritin accumulates. IRPs also modulate the translation of the mRNAs that encode the erythroid-specific ALAS2 enzyme involved in heme biosynthesis, mitochondrial aconitase (ACO2), and of the transcription factor HIF2α. A single 3′ UTR IRE motif is present in the DMT1 mRNA, but its mechanism of action is not fully resolved. (B) FPN is subject to multilayered regulation in macrophages. FPN transcription is suppressed by BACH1 (Btb and Cnc Homology 1) and stimulated by NFE2L2 (Nuclear Factor Erythroid 2-like). BACH1 and NFE2L2, respectively, bind MARE (Maf recognition elements) and ARE (Antioxidant Response Elements) cassettes in the FPN promoter. Heme derived from erythrophagocytosis stimulates FPN transcription by alleviating the inhibitory effect of BACH1 and by promoting the dissociation of NFE2L2 from KEAP1 (Kelch-like ECH-associated protein 1). The binding of specific pathogen-associated molecular patterns (PAMPs) to toll-like receptors (TLRs) leads to suppression of FPN transcription; the exact signaling events between TLRs and FPN are not known. Once transcribed, the FPN mRNA is regulated by the IRPs (see above) and the mIR-485-3p and miR-20a microRNAs; mIR-485-3p is induced by iron deficiency. At the plasma membrane FPN exports iron, possibly by switching between cytoplasmic-versus extracellular space-facing conformations. Hepcidin inhibits cellular iron efflux by binding to FPN, which leads to ubiquitination and degradation of the iron transporter.

Figure 3. Systemic Iron Homeostasis

Enterocytes take up elemental iron from the intestinal lumen via DMT1 after reduction of the metal by e.g., Dcytb at the apical membrane. Dietary heme is taken up via an unknown transport system and degraded inside the cell by heme oxygenase 1 (HMOX1). The export of iron into the bloodstream via FPN and its loading onto Tf requires oxidation of the metal by hephaestin (HEPH) at the basolateral side. Iron passage across the enterocyte is transcriptionally stimulated by the hypoxia-inducible factor 2α (HIF2α) and inhibited through iron sequestration in ferritin. Whether PCBP is involved as an iron-binding chaperone during iron transport is unclear. Diferric transferrin (Tf-Fe₂) is delivered to tissues via the bloodstream and is mostly used for the hemoglobinization of new red blood cells (RBCs). Specialized tissue macrophages engulf and degrade senescent RBCs to release heme into the phagolysosome. Heme is then exported into the cytoplasm by the heme transporter HRG-1 (SLC48A1) and processed by HMOX1 to release iron, which is either stored in ferritin or exported into the circulation via FPN with the assistance of the ceruloplasmin (CP) ferroxidase. Iron utilization by the erythroid marrow and its recycling by RES macrophages account for the major iron fluxes in the body. If not used, iron can be stored in hepatocytes. Hepatocytes can acquire Tf-Fe₂ and non-transferrin-bound iron, respectively, via TFR1 and ZIP14. Hepatocytes store iron in ferritin and they also express FPN and CP for iron export. Heme and hemoglobin (HB) present in the circulation interact with hemopexin (HPX) and haptoglobin (HP), respectively, and are scavenged via the CD163 and CD91 receptors. Peritubular fibroblasts of the kidney sense iron and oxygen deficiency and release EPO to enhance erythropoiesis. When bound to iron and oxygen, prolyl hydroxylase 2 (PHD2) normally triggers the degradation of the HIF2α; iron supply to PHD2 may be facilitated by Poly(rC)-binding proteins (PCBP). Low iron and/or oxygen conditions inactivate PHD2, leading to HIF2α accumulation and stimulation of EPO transcription. In addition to PHD2, HIF2α is also translationally inhibited by IRP1.

Figure 4. Pathways Involved in Hepcidin Regulation

Hepcidin expression is regulated by iron signals (middle), erythropoiesis (left), and inflammation (right). High iron stores augment BMP6 (bone morphogenetic protein 6), which together with its co-receptor hemojuvelin (HJV) activates type 1 (Alk2/3) and type 2 (BMPR2, ACVR2A) BMP serine threonine kinase receptors, leading to phosphorylation of receptor-activated SMAD (R-SMAD) proteins and formation of active transcriptional complexes with SMAD4. The HJV-BMP interaction may be facilitated by neogenin. High concentrations of Tf-Fe₂ displace HFE from TFR1, which then forms a complex with TFR2 and HJV to promote BMP/SMAD signaling to Hepcidin. HFE additionally interacts with ALK3 and prevents its ubiquitination (Ub) and degradation. BMP/SMAD signaling to Hepcidin is suppressed by matriptase 2 (TMPRSS6), a serine protease that cleaves and generates a soluble form of HJV (sHJV). The pathway is also feedback inhibited by SMAD7 and dampened by the miR-122 microRNA, which lowers HJV and HFE expression. In inflammation, the interaction of interleukin 6 (IL-6) with its receptor (IL-6R) activates JAK tyrosine kinases, triggering the formation of STAT3 (Signal transducer and transcription activator 3) complexes that bind to the Hepcidin promoter in the nucleus. Hepcidin stimulation by activin B is dependent on the BMP/SMAD signaling pathway. High erythropoietic activity increases plasma levels of ERFE, which suppresses Hepcidin via as yet unknown signaling pathways. Additional signals modulate Hepcidin expression. These include protein misfolding (ER stress), which activates Hepcidin transcription through CREBH (cyclic AMP-responsive element-binding protein 3-like protein 3, also known as CREB3L3), as well as hormones/growth factors and various metabolic cues. The STAT3- and BMP-response elements (REs) in the Hepcidin promoter are depicted.

Figure 5. Therapeutic Strategies in Abnormal Erythropoiesis

(A) Under baseline conditions, iron flows mostly through the erythron and iron-recycling macrophages. EPO controls the rate of erythropoiesis, which in turn inhibits Hepcidin expression through erythroferrone (ERFE) to adjust iron efflux from macrophages and enterocytes. (B) A situation of ineffective erythropoiesis is depicted. Hypoxia stimulates erythropoiesis through EPO in an attempt to compensate for decreased oxygen transport. The ensuing increase in ERFE production suppresses Hepcidin and results in iron overload due to enhanced dietary iron absorption and iron release from macrophages. Therapeutic strategies to prevent iron overload while improving erythropoiesis can be envisaged. Those include engineered minihepcidins, which limit dietary iron absorption by inhibiting FPN iron export. Similarly, siRNAs, antisense oligonucleotides (ASO), and inhibitors of TMPRSS6 aim at enhancing endogenous hepcidin activity. Targeting ERFE also seems a logical choice to alleviate Hepcidin suppression, but ERFE deficiency is insufficient to correct the anemia of thalassemic mice. Thus, this approach may require combination with other therapies. If selectively delivered to intestinal enterocytes, HIF2 inhibitors may also have the potential to limit dietary iron assimilation. Injection of apo-transferrin was shown to prevent hemichrome formation and ameliorate RBC survival in mice.