Regulators of Iron Homeostasis- New Players in Metabolism, Cell Death, and Disease.pdf

Regulators of Iron Homeostasis:New Players in Metabolism,Cell Death,and DiseaseAlexander R.Bogdan1,2,Masaki Miyazawa1,2,Kazunori Hashimoto1,and Yoshiaki Tsuji1,*1Department of Biological Sciences,North Carolina State University,Campus Box 7633,Raleigh,NC,27695-7633,USAAbstractIron is necessary for life,but can also cause cell death.Accordingly,cells evolved a robust,tightly regulated suite of genes for maintaining iron homeostasis.Previous mechanistic studies on iron homeostasis have granted insight into the role of iron in human health and disease.We highlight new regulators of iron metabolism,including iron-trafficking proteins solute carrier family 39,SLC39,also known as ZRT/IRT-like protein,ZIP;and poly-(rC)-binding protein,PCBP and a cargo receptor(NCOA4)that is crucial for release of ferritin-bound iron.We also discuss emerging roles of iron in apoptosis and a novel iron-dependent cell death pathway termed ferroptosis,the dysregulation of iron metabolism in human pathologies,and the use of iron chelators in cancer therapy.Iron Homeostasis Is a Complex,Highly Regulated ProcessIron is required in a variety of important biological processes including oxygen transport(as heme in hemoglobin),DNA biosynthesis(as a cofactor of ribonucleotide reductase),and ATP generation(as a cofactor for many proteins in the citric acid cycle and electron transport chain);therefore,cells must maintain a sufficient amount of iron.However,iron is redox-active and can generate reactive oxygen species(ROS),leading to oxidative stress and initiation of signaling pathways crucial for cell survival and cell death 1.To maintain adequate and safe amounts of iron,cells require the coordination of a wide variety of genes,which tightly control both intracellular(reviewed in 2,3)and systemic(reviewed in 4)iron metabolism.Extensive research by many groups has revealed key mechanisms in iron homeostasis(Box 1),as well as links between aberrations in iron homeostasis and human disease.The study of iron metabolism continues to be a dynamic field,with many breakthroughs and novel insights in the past several years.In this review we discuss recent advances in the function and regulation of key iron metabolism genes,including:ferritin(FTH1 and FTL),a protein complex that safely concentrates intracellular iron in a mineralized,redox-inactive form for later use;transferrin(TF),an iron-binding serum protein;transferrin receptor 1(TfR1,TFRC),a plasma membrane protein that allows cellular uptake of iron-loaded transferrin;divalent metal transporter 1(DMT1,SLC11A2),a metal transporter that is important for TfR1-mediated iron uptake and dietary iron*Correspondence:ytsujincsu.edu(Y.Tsuji).2These authors contributed equally to this workHHS Public AccessAuthor manuscriptTrends Biochem Sci.Author manuscript;available in PMC 2017 March 01.Published in final edited form as:Trends Biochem Sci.2016 March;41(3):274286.doi:10.1016/j.tibs.2015.11.012.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptabsorption;ferroportin(Fpn,SLC40A1),the only known cellular iron efflux pump;and hepcidin(HAMP),a circulating peptide hormone that regulates serum iron levels by causing ferroportin degradation.We also examine newly identified regulators in iron metabolism,including new membrane iron transporters and cytosolic iron trafficking proteins.In addition,we review new roles for iron in cell death pathways,as well as the importance of aberrant iron metabolism in human diseases such as cancer.New Proteins Are Involved in Iron Trafficking and UtilizationIn recent years several new proteins have been identified as key players in intracellular iron trafficking and utilization.This section discusses novel iron transporters,iron chaperones,and ferritin-shuttling proteins.SLC39/ZIP Family Transporters Are a New Class of Iron-Trafficking ProteinsThe solute carrier family 39(SLC39),also known as the ZRT/IRT-like protein(ZIP)family,contains 14 members and has classically been understood to comprise transmembrane zinc transporters that pump extracellular Zn2+into the cell 5.However,recent studies have uncovered a role for two ZIP family proteins in iron transport 6.ZIP14 was found to mediate the uptake of non-transferrin-bound iron(NTBI)7 by directly transporting NTBI across the cell membrane 8(Figure 1,left,Uptake).ZIP14-mediated NTBI transport is enhanced by the Fe3+to Fe2+ferrireductase activity of the prion protein PRNP 9.ZIP14 is also capable of transporting transferrin-bound iron from the endosome to the cytoplasm,similarly to DMT1 10,although subsequent studies have revealed that this is likely not the major function of ZIP14 11.ZIP8,the closest relative to ZIP14 6,was also found to transport NTBI 12(Figure 1,left,Uptake).Interestingly,both ZIP8 and ZIP14 show maximal iron transport above pH 7,near physiological pH 8,12,whereas DMT1 is most efficient at pH 5.5 13,which corresponds to the pH of acidified endosome.The widely disparate pH activities suggest that ZIP8/14 and DMT1 may have different biological roles.Indeed,a recent report utilizing primary rat hippocampal neurons showed that ZIP8 is the major NTBI transporter whereas DMT1 was principally responsible for transferrin-bound iron transport from the endosome 11.ZIP8 and ZIP14 appear to have nonredundant functions because they have distinct tissue expression profiles and knockout mouse models show different phenotypes.Knockout of ZIP8 in mice causes failures in hematopoiesis and organogenesis of the spleen,liver,kidney,and lung,with associated perinatal lethality 14.Zip8/mice exhibited profound anemia and have significantly lowered amounts of iron and zinc in various tissues,with the hematopoietic defect seemingly being caused by the combination of iron and zinc deficiency.By contrast,Zip14/mice are viable and have adequate iron stores,but exhibit dwarfism owing to aberrant bone growth.The dwarfism was found to primarily be the result of altered zinc,rather than iron,metabolism,indicating that zinc transport is more important than iron transport by ZIP14 for normal growth 15.However,a more recent study uncovered a crucial role for ZIP14 in iron transport by knocking out ZIP14 in two mouse models for hereditary hemochromatosis,a family of iron overload diseases 16.Hemochromatosis results in a progressive accumulation of iron primarily in the liver,resulting in hepatic fibrosis and potentially hepatocellular carcinoma.Knocking out ZIP14 Bogdan et al.Page 2Trends Biochem Sci.Author manuscript;available in PMC 2017 March 01.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptin Hfe/(hereditary hemochromatosis protein)or Hfe2/(hemojuvelin)mice modeling type 1 and type 2(juvenile)hemochromatosis,respectively prevented iron accumulation in the liver.ZIP14 knockout also prevented iron overload in Hfe2/mouse pancreas,but not heart 16,which could be attributable to the high pancreatic but low cardiac expression of ZIP14 17.Uncovering the importance of ZIP14 in iron loading provides a new target in the treatment of iron overload diseases.Namely,inhibition of ZIP14 could potentially block hepatic and pancreatic iron overloading,and prevent subsequent organ damage.The expression of ZIP8 and ZIP14 is regulated differently than other iron transporter genes such as TfR1 and DMT1,both of which are controlled post-transcriptionally by changes of mRNA stability through the 3 untranslated region(3-UTR)iron-responsive element(IRE)iron regulatory protein(IRP)interaction(Box 1)2,3.By contrast,ZIP8 and ZIP14 mRNA stability is unaffected by iron,and ZIP protein levels are positively correlated with iron status,in that iron loading increases ZIP8/14 protein amount whereas iron deficiency decreases protein amount 12,18.In summary,ZIP8 and ZIP14 are newly recognized iron transporters with important roles in the uptake of NTBI and in iron overload diseases.Further investigation will be necessary to determine the importance of ZIP iron transport in other contexts,such as disease states with disordered iron homeostasis(anemia,cancer,etc.).PCBP Family Proteins Are Important Iron Chaperones for Cytosolic Iron DistributionPCBPs are a family of four proteins that were originally identified for their high affinity binding to tracts of polycytosine and have been studied as regulators of gene expression(reviewed in 19).Each member of the PCBP family contains three highly conserved K homology(KH)domains and can form complexes with other family members.Recently,PCBP1 and PCBP2 were identified as cytosolic iron chaperones that are responsible for delivering iron to ferritin,the protein that is primarily responsible for storing intracellular iron in a non-toxic,mineralized state 20,21(Figure 1,center,Distribution).PCBP3 and PCBP4 are expressed at much lower levels,with a limited tissue distribution and an unclear biological role,but there is some evidence that they can also act as iron chaperones and that PCBP3 can bind to ferritin 21.To date,the majority of the research on the iron chaperone function of the PCBP family has focused on PCBP1 and PCBP2.Both PCBP1 and PCBP2 are necessary to form a stable ternary complex with ferritin,and ferritinPCBP interactions were dependent upon iron-loaded PCBP1/2 21.In addition to ferritin,PCBP1 and PCBP2 can also deliver iron to proteins that require non-heme iron as a cofactor(metallation,Figure 1,center,Distribution),such as hypoxia-inducible factor(HIF)prolyl and asparginyl hydoxylases 22,and deoxyhypusine hydroxylase 23.PCBP2 is loaded with iron through binding to the iron transporter DMT1.Iron transfer to PCBP2 is mediated through interaction of the second KH domain of PCBP2 with the N-terminal cytoplasmic domain of DMT1 24.After transfer of Fe2+,PCBP2 subsequently dissociates from DMT1(Figure 1,center,Distribution).By contrast,PCPB1 does not interact with DMT1,and the mechanism of apo-PCBP1 iron acquisition is still undetermined.A comprehensive screen of both iron-loaded and apo-PCBP1 and-2 binding to other iron metabolism genes(ZIP transporters,Bogdan et al.Page 3Trends Biochem Sci.Author manuscript;available in PMC 2017 March 01.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptetc.)is crucial for understanding overall cytoplasmic iron shuttling and may shed light on PCBP1 iron loading.Many questions about the roles and mechanisms of the PCBP proteins in iron homeostasis remain unanswered.For instance,it was noted that PCBP2 can also bind to the iron exporter ferroportin(Fpn)24(Figure 1,center,Distribution),but that association remains to be characterized in depth,including what effects,if any,PCBP2 has on Fpn iron export.In addition,the question of what roles,if any,PCBP3 and PCBP4 have in intracellular iron homeostasis remains to be answered.Lysosomal Ferritin Degradation Involves both Autophagy and Non-Autophagic PathwaysFerritin stores ferric iron in a mineralized,non-toxic state.However,ferritin-bound iron cannot be utilized by the cell.Consequently,iron must be released from ferritin to be biologically useful,usually through lysosomal degradation of ferritin 2528.Ferritin protein turnover is a constant process in cells.While the rate of ferritin degradation appears to be the same in iron-deficient and iron-replete conditions,the delivery mechanism of ferritin to lysosomes seems to be different:autophagy is responsible for delivering ferritin to the lysosome during iron deficiency,whereas a non-autophagic pathway dominates during iron sufficiency 26.Recently,nuclear receptor coactivator 4(NCOA4)was identified as the protein responsible for mediating ferritin autophagy 29,30.NCOA4 was originally identified as a coactivator of several nuclear receptors but was later found to primarily be localized in the cytoplasm and have roles in various biological processes independent of its coactivator function(reviewed in 31).Two groups found that NCOA4 is greatly enriched in the autophagosome,and identified ferritin as an NCOA4 binding partner 29,30(Figure 1,right,Utilization).Knockdown of NCOA4 in various cell types prevented ferritin degradation in iron-deficient conditions 30,suggesting the importance of NCOA4 in ferritin autophagy.By contrast,autophagy-deficient mouse embryonic fibroblasts(MEF)produced by knockout of the key autophagy mediators Atg5 or Atg7 degraded ferritin in the lysosome only in iron-replete conditions and not during iron deficiency 26.Collectively,lysosomal ferritin degradation appears to involve autophagy in iron deficiency but a non-autophagic pathway dominates during iron sufficiency.NCOA4 knockdown also resulted in an increase in IRP2 and TfR1 30,likely from a decrease in labile iron owing to ferritin protein accumulation.Furthermore,Ncoa4/mice exhibit profound iron deposits within spleen macrophages,suggesting that NCOA4-mediated ferritin autophagy is crucial for splenic iron homeostasis 29.Ferritin stores iron in a 24-subunit multimer that is a combination of ferritin heavy(FTH1)and light(FTL)chains.NCOA4 was found to interact with the 24-subunit ferritin lattice rather than ferritin monomers in a FTH1-dependent manner 29,but several important aspects of the interaction remain to be characterized,including identification of the NCOA4 and FTH1 interacting domains.In addition,the ratio of FTH1 and FTL protein expression is plastic in various physiological conditions and tissue types(reviewed in 32,33),and the ability of NCOA4 to mediate autophagy of different ferritin lattice compositions needs to be tested.Bogdan et al.Page 4Trends Biochem Sci.Author manuscript;available in PMC 2017 March 01.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptAberrations of ferritin degradation in cancer are an ongoing area of research.The non-autophagic ferritin degradation pathway in iron-replete cells is deficient in various tumor-derived cells(HeLa,MCF-7,and Hepa16)compared to normal cells,allowing cancer cells to maintain a higher level of ferritin protein during times of iron sufficiency/excess and potentially explaining why some cancer-derived cells are resistant to iron toxicity 26.Reactivation of non-autophagic ferritin degradation may increase iron toxicity in cancer,and requires further investigation to determine if this is a viable therapeutic strategy.Modulating ferritin autophagy has provided some interesting avenues for cancer treatment.The anti-malaria drug artesunate was found to accumulate within lysosomes of cancer cells and enhance lysosomal activity,accelerating autophagic degradation of ferritin and increasing lysosomal iron stores 34.Artesunate reacts with the increased lysosomal iron to generate ROS,leading to apoptosis in cervical and liver cancer cells 34,35.As expected,treatment with an iron chelator or knockdown of NCOA4 could prevent artesunate-induced apoptosis 34,indicating that lysosomal iron derived from the ferritinNCOA4 autophagic pathway is necessary for artesunate-mediated apoptosis.Iron Is an Important Mediator