Managing Iron Overload: A Gut Check.

Divalent metal transporter 1 (DMT1) is the major importer of ferrous iron at the apical surface of enterocytes in the duodenum. Multiple groups have tried to design specific inhibitors for DMT1 both to study its contributions to iron (and metal ion) homeostasis and to provide a pharmacological means to treat iron overload disorders like hereditary hemochromatosis and thalassemias. This task faces challenges because many tissues express DMT1 and DMT1 transports other metals adding to standard risks in making specific inhibitors. Xenon Pharmaceuticals have published several papers on their efforts. Their latest paper in this issue of the journal culminates their efforts with compounds named XEN601 and XEN602 but implies that these very effective inhibitors have sufficient toxicity for them to halt development. This Viewpoint evaluates their efforts and briefly considers alternative routes to the goal. SIGNIFICANCE STATEMENT: This Viewpoint briefly reviews the paper on inhibitors of DMT1 that appears in this issue of the journal and commends the effort and research utility of those developed by Xenon. The inhibitors have proven to be valuable research tools for studying metal ion homeostasis particularly for iron. If Xenon is ceasing to try to develop them for treatment of iron overload disorders, then new alternatives need to come to the fore.

Fetal factors disrupt placental and maternal iron homeostasis in murine ß-thalassemia.

Pregnancy rates in ß-thalassemia are increasing but the risk of complications is higher; thus, better understanding of maternal and fetal iron homeostasis in this disorder is needed. HbbTh3/+ (Th3/+) mice model human ß-thalassemia. Both the murine and human diseases are characterized by low hepcidin, high iron absorption, and tissue iron overload, with concurrent anemia. We hypothesized that disordered iron metabolism in pregnant Th3/+ mice would negatively affect their unborn offspring. The experimental design included these groups: wild-type (WT) dams carrying WT fetuses (WT1); WT dams carrying WT and Th3/+ fetuses (WT2); Th3/+ dams carrying WT and Th3/+ fetuses (Th3/+); and age-matched, nonpregnant adult females. Serum hepcidin was low, and mobilization of splenic and hepatic storage iron was enhanced in all 3 groups of experimental dams. Intestinal 59Fe absorption was lower in Th3/+ dams (as compared with WT1/2 dams) but splenic 59Fe uptake was higher. Th3/+ dams had hyperferremia, which led to fetal and placenta iron loading, fetal growth restriction, and placentomegaly. Notably, Th3/+ dams loaded Th3/+ and WT fetuses, with the latter situation more closely mirroring human circumstances when mothers with thalassemia have relatively unaffected (thalassemia trait) offspring. Iron-related oxidative stress likely contributed to fetal growth impairment; enhanced placental erythropoiesis is a probable cause of placental enlargement. Moreover, high fetal liver iron transactivated Hamp; fetal hepcidin downregulated placental ferroportin expression, limiting placental iron flux and thus mitigating fetal iron loading. Whether gestational iron loading occurs in human thalassemic pregnancy, when blood transfusion can further elevate serum iron, is worth consideration.

Corrigendum: Iron and Cadmium Entry Into Renal Mitochondria: Physiological and Toxicological Implications.

[This corrects the article DOI: 10.3389/fcell.2020.00848.].

Iron and Cadmium Entry Into Renal Mitochondria: Physiological and Toxicological Implications.

Regulation of body fluid homeostasis is a major renal function, occurring largely through epithelial solute transport in various nephron segments driven by Na+/K+-ATPase activity. Energy demands are greatest in the proximal tubule and thick ascending limb where mitochondrial ATP production occurs through oxidative phosphorylation. Mitochondria contain 20-80% of the cell's iron, copper, and manganese that are imported for their redox properties, primarily for electron transport. Redox reactions, however, also lead to reactive, toxic compounds, hence careful control of redox-active metal import into mitochondria is necessary. Current dogma claims the outer mitochondrial membrane (OMM) is freely permeable to metal ions, while the inner mitochondrial membrane (IMM) is selectively permeable. Yet we recently showed iron and manganese import at the OMM involves divalent metal transporter 1 (DMT1), an H+-coupled metal ion transporter. Thus, iron import is not only regulated by IMM mitoferrins, but also depends on the OMM to intermembrane space H+ gradient. We discuss how these mitochondrial transport processes contribute to renal injury in systemic (e.g., hemochromatosis) and local (e.g., hemoglobinuria) iron overload. Furthermore, the environmental toxicant cadmium selectively damages kidney mitochondria by "ionic mimicry" utilizing iron and calcium transporters, such as OMM DMT1 or IMM calcium uniporter, and by disrupting the electron transport chain. Consequently, unraveling mitochondrial metal ion transport may help develop new strategies to prevent kidney injury induced by metals.

Animal Models of Normal and Disturbed Iron and Copper Metabolism.

Research on the interplay between iron and copper metabolism in humans began to flourish in the mid-20th century, and diseases associated with dysregulated homeostasis of these essential trace minerals are common even today. Iron deficiency is the most frequent cause of anemia worldwide, leading to significant morbidity, particularly in developing countries. Iron overload is also quite common, usually being the result of genetic mutations which lead to inappropriate expression of the iron-regulatory hormone hepcidin. Perturbations of copper homeostasis in humans have also been described, including rare genetic conditions which lead to severe copper deficiency (Menkes disease) or copper overload (Wilson disease). Historically, the common laboratory rat (Rattus norvegicus) was the most frequently utilized species to model human physiology and pathophysiology. Recently, however, the development of genetic-engineering technology combined with the worldwide availability of numerous genetically homogenous (i.e., inbred) mouse strains shifted most research on iron and copper metabolism to laboratory mice. This created new opportunities to understand the function of individual genes in the context of a living animal, but thoughtful consideration of whether mice are the most appropriate models of human pathophysiology was not necessarily involved. Given this background, this review is intended to provide a guide for future research on iron- and copper-related disorders in humans. Generation of complementary experimental models in rats, swine, and other mammals is now facile given the advent of newer genetic technologies, thus providing the opportunity to accelerate the identification of pathogenic mechanisms and expedite the development of new treatments to mitigate these important human disorders.

A direct comparison of divalent metal-ion transporter (DMT1) and hinokitiol, a potential small molecule replacement.

Hinokitiol, a natural lipophilic chelator, appears capable of replacing several iron transporters after they have been genetically ablated. Divalent metal-ion transporter (DMT1) is the major iron importer in enterocytes and erythroblasts. We have compared DMT1 and hinokitiol in multiple fashions to learn if the smaller molecule is a suitable substitute using two HEK293 cell lines engineered to overexpress different isoforms of DMT1. Both the macromolecule and the lipophilic chelator enable import of ferrous ions into HEK293 cells. Hinokitiol also mediates ferric ion import but DMT1 cannot do so. While DMT1 can also import Mn2+ ions, hinokitiol lacks this ability. The Michaelis-Menten analysis for kinetics of macromolecular catalysis is also suitable for hinokitiol-supported iron import. To compare hinokitiol to DMT1 relative to other metal ions that DMT1 can transport, we employed an organic extraction procedure with which we initially matched the results obtained for Fe2+, Fe3+ and Mn2+, and then showed that multiple other cations were unlikely to enter via hinokitiol. The small chelator thus shares some functional properties with DMT1, but distinct difference were also noted.

Oral Gavage of Ginger Nanoparticle-Derived Lipid Vectors Carrying Dmt1 siRNA Blunts Iron Loading in Murine Hereditary Hemochromatosis.

Nanoparticles (NPs) have been utilized to deliver drugs to the intestinal epithelium in vivo. Moreover, NPs derived from edible plants are less toxic than synthetic NPs. Here, we utilized ginger NP-derived lipid vectors (GDLVs) in a proof-of-concept investigation to test the hypothesis that inhibiting expression of divalent metal-ion transporter 1 (Dmt1) would attenuate iron loading in a mouse model of hereditary hemochromatosis (HH). Initial experiments using duodenal epithelial organ cultures from intestine-specific Dmt1 knockout (KO) (Dmt1int/int) mice in the Ussing chamber established that Dmt1 is the only active iron importer during iron-deficiency anemia. Further, when Dmt1int/int mice were crossed with mice lacking the iron-regulatory hormone, hepcidin (Hepc-/-), iron loading was abolished. Hence, intestinal Dmt1 is required for the excessive iron absorption that typifies HH. Additional experiments established a protocol to produce GDLVs carrying functional Dmt1 small interfering RNAs (siRNAs) and to target these gene delivery vehicles to the duodenal epithelium in vivo (by incorporating folic acid [FA]). When FA-GDLVs carrying Dmt1 siRNA were administered to weanling Hepc-/- mice for 16 days, intestinal Dmt1 mRNA expression was attenuated and tissue iron accumulation was blunted. Oral delivery of functional siRNAs by FA-GDLVs is a suitable therapeutic approach to mitigate iron loading in murine HH.

A role for divalent metal transporter (DMT1) in mitochondrial uptake of iron and manganese.

Much of iron and manganese metabolism occurs in mitochondria. Uptake of redox-active iron must be tightly controlled, but little is known about how metal ions enter mitochondria. Recently, we established that the divalent metal transporter 1 (DMT1) is present in the outer mitochondrial membrane (OMM). Therefore we asked if it mediates Fe2+ and Mn2+ influx. Mitochondria were isolated from HEK293 cells permanently transfected with inducible rat DMT1 isoform 1 A/+IRE (HEK293-rDMT1). Fe2+-induced quenching of the dye PhenGreen™SK (PGSK) occurred in two phases, one of which reflected OMM DMT1 with stronger Fe2+ uptake after DMT1 overexpression. DMT1-specific quenching showed an apparent affinity of ~1.5 µM for Fe2+and was blocked by the DMT1 inhibitor CISMBI. Fe2+ influx reflected an imposed proton gradient, a response that was also observed in purified rat kidney cortex (rKC) mitochondria. Non-heme Fe accumulation assayed by ICPOES and stable 57Fe isotope incorporation by ICPMS were increased in HEK293-rDMT1 mitochondria. HEK293-rDMT1 mitochondria displayed higher 59Fe2+ and 54Mn2+ uptake relative to controls with 54Mn2+ uptake blocked by the DMT1 inhibitor XEN602. Such transport was defective in rKC mitochondria with the Belgrade (G185R) mutation. Thus, these results support a role for DMT1 in mitochondrial Fe2+ and Mn2+ acquisition.

Prion protein functions as a ferrireductase partner for ZIP14 and DMT1.

Excess circulating iron is stored in the liver, and requires reduction of non-Tf-bound iron (NTBI) and transferrin (Tf) iron at the plasma membrane and endosomes, respectively, by ferrireductase (FR) proteins for transport across biological membranes through divalent metal transporters. Here, we report that prion protein (PrP(C)), a ubiquitously expressed glycoprotein most abundant on neuronal cells, functions as a FR partner for divalent-metal transporter-1 (DMT1) and ZIP14. Thus, absence of PrP(C) in PrP-knock-out (PrP(-/-)) mice resulted in markedly reduced liver iron stores, a deficiency that was not corrected by chronic or acute administration of iron by the oral or intraperitoneal routes. Likewise, preferential radiolabeling of circulating NTBI with (59)Fe revealed significantly reduced uptake and storage of NTBI by the liver of PrP(-/-) mice relative to matched PrP(+/+) controls. However, uptake, storage, and utilization of ferritin-bound iron that does not require reduction for uptake were increased in PrP(-/-) mice, indicating a compensatory response to the iron deficiency. Expression of exogenous PrP(C) in HepG2 cells increased uptake and storage of ferric iron (Fe(3+)), not ferrous iron (Fe(2+)), from the medium, supporting the function of PrP(C) as a plasma membrane FR. Coexpression of PrP(C) with ZIP14 and DMT1 in HepG2 cells increased uptake of Fe(3+) significantly, and surprisingly, increased the ratio of N-terminally truncated PrP(C) forms lacking the FR domain relative to full-length PrP(C). Together, these observations indicate that PrP(C) promotes, and possibly regulates, the uptake of NTBI through DMT1 and Zip14 via its FR activity. Implications of these observations for neuronal iron homeostasis under physiological and pathological conditions are discussed.

Mitochondria represent another locale for the divalent metal transporter 1 (DMT1).

The divalent metal transporter (DMT1) is well known for its roles in duodenal iron absorption across the apical enterocyte membrane, in iron efflux from the endosome during transferrin-dependent cellular iron acquisition, as well as in uptake of non-transferrin bound iron in many cells. Recently, using multiple approaches, we have obtained evidence that the mitochondrial outer membrane is another subcellular locale of DMT1 expression. While iron is of vital importance for mitochondrial energy metabolism, its delivery is likely to be tightly controlled due to iron's damaging redox properties. Here we provide additional support for a role of DMT1 in mitochondrial iron acquisition by immunofluorescence colocalization with mitochondrial markers in cells and isolated mitochondria, as well as flow cytometric quantification of DMT1-positive mitochondria from an inducible expression system. Physiological consequences of mitochondrial DMT1 expression are discussed also in consideration of other DMT1 substrates, such as manganese, relevant to mitochondrial antioxidant defense.

Evidence for mitochondrial localization of divalent metal transporter 1 (DMT1).

In mammalian cells, mitochondria receive most incoming iron, yet no entry pathway for iron at the outer mitochondrial membrane (OMM) has been characterized. Our results show that the divalent metal transporter 1 (DMT1) occurs in the OMM. Immunoblots detected DMT1 in mitochondria from a pneumocyte cell model in their OMM. Using the split-ubiquitin yeast 2-hybrid system, we found that cytochrome c oxidase subunit II (COXII) and the translocase of OMM 6-kDa subunit (Tom6) homologue interact with DMT1. COXII coimmunoprecipitates with DMT1. There are 4 DMT1 isoforms that differ at the N and C termini. Using HEK293 cells that inducibly express all of the 4 ends of DMT1, we found all of them in the OMM, as detected by immunoblots after cell fractionation, and in isolated mitochondria, as detected by immunofluorescence. Immunoblot analysis of purified cell fractions from rat renal cortex confirmed and extended these results to the kidney, which expressed high levels of DMT1. Immunogold labeling detected DMT1 colocalization in mitochondria with the voltage-dependent anion-selective channel protein-1, which is expressed in the OMM. We suggest that DMT1 not only exports iron from endosomes, but also serves to import the metal into the mitochondria.

Mouse divalent metal transporter 1 is a copper transporter in HEK293 cells.

Divalent Metal Transporter 1 (DMT1) is an apical Fe transporter in the duodenum and is involved in endosomal Fe export. Four protein isoforms have been described for DMT1, two from mRNA with an iron responsive element (IRE) and two from mRNA without it. The sets of two begin in exon 1A or 2. We have characterized copper transport using mouse 2/-IRE DMT1 during regulated ectopic expression. HEK293 cells carrying a TetR:Hyg element were stably transfected with pDEST31 containing a 2/-IRE construct. (64)Cu(1+) incorporation in doxycycline treated cells exhibited 18.6 and 30.0-fold increases in Cu content, respectively when were exposed to 10 and 100 µM of extracellular Cu. Cu content was ~4-fold above that of parent cells or cells carrying just the vector. (64)Cu uptake in transfected cells pre-incubated with 5 µM of Cu-His revealed a Vmax and Km of 11.98 ± 0.52 pmol mg protein(-1) min(-1) and 2.03 ± 0.03 µM, respectively. Doxycycline-stimulated Cu uptake was linear with time. The rates of apical Cu uptake decreased and transepithelial transport increased when intracellular Cu increased. The optimal pH for Cu transport was 6.5; uptake of Cu was temperature dependent. Silver does not inhibit Cu uptake in cells carrying the vector. In conclusion, Cu uptake in HEK293 cells that over-expressed the 2/-IRE isoform of DMT1 transporter supports our earlier contention that DMT1 transports Cu as Cu(1+).

Divalent metal transporter 1 (Dmt1) mediates copper transport in the duodenum of iron-deficient rats and when overexpressed in iron-deprived HEK-293 cells.

Intracellular copper-binding proteins (metallothionein I/II) and a copper exporter (Menkes copper-transporting ATPase) are upregulated in duodenal enterocytes from iron-deficient rats, consistent with copper accumulation in the intestinal mucosa. How copper enters enterocytes during iron deficiency is, however, not clear. Divalent metal transporter 1 (Dmt1), the predominant iron importer in the mammalian duodenum, also transports other metal ions, possibly including copper. Given this possibility and that Dmt1 expression is upregulated by iron deprivation, we sought to test the hypothesis that Dmt1 transports copper during iron deficiency. Two model systems were utilized: the Belgrade (b) rat, expressing mutant Dmt1, and an inducible Dmt1-overexpression cell culture system. Mutant rats (b/b) were fed a semipurified, AIN93G-based control diet and phenotypically normal littermates (+/b) were fed control or iron-deficient diets for ~14 wk. An everted gut sleeve technique and a colorimetric copper quantification assay were utilized to assess duodenal copper transport. The control diet-fed +/b rats had normal hematological parameters, whereas iron-deprived +/b and b/b rats were iron deficient and Dmt1 mRNA and protein levels increased. Importantly, duodenal copper transport was similar in the control +/b and b/b rats; however, it significantly increased (~4-fold) in the iron-deprived +/b rats. Additional experiments in Dmt1 overexpressing HEK-293 cells showed that copper ((64)Cu) uptake was stimulated (∼3-fold) in the presence of an iron chelator. Dmt1 transcript stabilization due to a 3' iron-responsive element was also documented, likely contributing to increased transport activity. In summary, these studies suggest that Dmt1 enhances copper uptake into duodenal enterocytes during iron deprivation.

Heme carrier protein 1 transports heme and is involved in heme-Fe metabolism.

Heme-Fe is an important source of dietary iron in humans; however, the mechanism for heme-Fe uptake by enterocytes is poorly understood. Heme carrier protein 1 (HCP1) was originally identified as mediating heme-Fe transport although it later emerged that it was a folate transporter. We asked what happened to heme-Fe and folate uptake and the relative abundance of hcp1 and ho1 mRNA in Caco-2 cells after knockdown by transfection with HCP1-directed short hairpin (sh)RNA. Control Caco-2 cells were cultured in bicameral chambers with 0-80 µM heme-Fe for selected times. Intracellular Fe and heme concentration increased in Caco-2 cells reflecting higher external heme-Fe concentrations. Maximum Fe, heme, and heme oxygenase 1 (HO1) expression and activity were observed between 12 and 24 h of incubation. Quantitative RT-PCR for hcp1 revealed that its mRNA decreased at 20 µM heme-Fe while ho1 mRNA and activity increased. When shRNA knocked down hcp1 mRNA, heme-(55)Fe uptake and [(3)H]folate transport mirrored the mRNA decrease, ho1 mRNA increased, and flvcr mRNA was unchanged. These data argue that HCP1 is involved in low-affinity heme-Fe uptake not just in folate transport.

Isoform specific regulation of divalent metal (ion) transporter (DMT1) by proteasomal degradation.

Divalent metal ion transporter (DMT1) is the major transporter for iron entrance into mammalian cells and iron exit from endosomes during the transferrin cycle. Four major mRNA isoforms correspond to four protein isoforms, differing at 5'/3' and N-/C-termini, respectively. Isoforms are designated 1A versus 1B reflecting where transcription starts or +iron responsive element (+IRE) versus -IRE reflecting the presence/absence of an IRE in the 3' end of the mRNA. These differences imply regulation at transcriptional and posttranscriptional levels. Many proteins are degraded by a ubiquitination-dependent mechanism. Two different ubiquitin ligases (E3s) appear to be involved in DMT1 ubiquitination: Parkin or neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4) family E3s which often utilize Nedd4 family interacting protein-1 and -2 (Ndfip1 and 2) to ubiquitinate their substrate proteins. Prior data suggest that Parkin ubiquitinates 1B DMT1 but not 1A DMT1 while Nedd4/Ndfips ligate ubiquitin to DMT1 in the duodenum where 1A/+IRE DMT1 predominates. Our assay for whether these systems target DMT1 depends on two HEK293 cell lines that express permanently transfected 1A/+IRE DMT1 or 1B/-IRE DMT1 after induction by doxycycline. Transient transfection with a Parkin construct before induction diminishes 1B/-IRE DMT1 detected by immune-blots but not 1A/+IRE DMT1. Mutant Parkin serves as a control that does not affect DMT1 levels. Thus DMT1 regulation in an isoform specific fashion can occur by ubiquitination and the events involved have implications for DMT1 function and disease processes.

Human iron transporters.

Human iron transporters manage iron carefully because tissues need iron for critical functions, but too much iron increases the risk of reactive oxygen species. Iron acquisition occurs in the duodenum via divalent metal transporter (DMT1) and ferroportin. Iron trafficking depends largely on the transferrin cycle. Nevertheless, non-digestive tissues have a variety of other iron transporters that may render DMT1 modestly redundant, and DMT1 levels exceed those needed for the just-mentioned tasks. This review begins to consider why and also describes advances after 2008 that begin to address this challenge.

Ndfip1-deficient mice have impaired DMT1 regulation and iron homeostasis.

The divalent metal ion transporter DMT1 is critical for nonheme iron import. We have previously shown that DMT1 is regulated in vitro by ubiquitination that is facilitated by the adaptor proteins Ndfip1 and Ndfip2. Here we report that in Ndfip1(-/-) mice fed a low- iron diet, DMT1 expression and activity in duodenal enterocytes are significant higher than in the wild-type animals. This correlates with an increase in serum iron levels and transferrin saturation. Liver and spleen iron stores were also increased in Ndfip1(-/-) mice fed a normal diet. Counterintuitive to the increase in iron uptake, Ndfip1(-/-) mice fed a low iron diet develop severe microcytic, hypochromic anemia. We demonstrate that this is due to a combination of iron deficiency and inflammatory disease in Ndfip1(-/-) mice, because Ndfip1(-/-)/Rag1(-/-) immunodeficient mice fed a low iron diet did not develop anemia and showed an iron overload phenotype. These data demonstrate that Ndfip1 is a critical mediator of DMT1 regulation in vivo, particularly under iron restricted conditions.