Patients with iron overload disorders frequently accumulate iron in the pituitary and adrenal glands, leading to endocrine dysfunction. Although this iron loading is well documented, the specific molecular mechanisms of iron accumulation are not well understood. Under normal conditions, most tissues, including pituitary and adrenal gland, acquire iron from circulating transferrin. In iron overload, however, transferrin levels become saturated with iron, resulting in plasma non-transferrin-bound iron (NTBI), which becomes a significant contributor to tissue iron accumulation. Previous studies have shown that the metal-ion transport protein SLC39A14 mediates NTBI uptake in the liver and pancreas. To investigate if SLC39A14 plays a similar role in the pituitary and adrenal glands, we utilized Slc39a14 knockout (Slc39a14-/-) mice and crossed them with hemojuvelin knockout (Hjv-/-) mice, a model of genetic iron overload. Using Perls' staining, we found that loss of SLC39A14 in Hjv-/-;Slc39a14-/- mice prevented iron accumulation in the anterior pituitary, and decreased iron concentrations in pooled pituitary glands by 40% relative to Hjv-/- mice. In contrast, iron accumulation in the posterior pituitary and adrenal gland was unaffected by the absence of SLC39A14, indicating that alternative, tissue-specific mechanisms of iron accumulation exist. We additionally observed that SLC39A14 deficiency led to elevated pituitary manganese levels, consistent with human studies. This study is the first to demonstrate that SLC39A14 is essential for iron loading in the anterior pituitary, providing new insights into the molecular pathways of metal accumulation in endocrine glands. These findings could inform the development of targeted therapies for iron overload-related endocrine complications.

Black rhinoceros (Diceros bicornis) face threats to survival due to poaching in the wild and an incomplete understanding of preventive health monitoring under human care. Black rhinos under human care develop iron overload disorder (IOD) which is associated with predisposition to other disease, compromised immune function, hemolytic crisis, and death. Management of IOD is challenging but has been mitigated in some cases with dietary intervention and phlebotomy and documented through serum biomarker evaluation. Chelation therapy to reduce iron is rare in rhinos partially because of limited product availability and route of administration. An iron-specific chelator HBED (N,N’-Di(2-hydroxybenzyl)ethylenediamine-N,N’-diacetic acid) was investigated for oral use in southern black rhinos (n = 3) after successful testing was performed with equids as a model. Using a cross-over design, we tested the efficacy of short-term HBED administration. HBED was dosed at 40 mg/kg body weight for 10 days and resulted in increased urinary excretion of iron but unaltered fecal iron excretion in rhinos compared to control trials. Two rhinos maintained blood chemistry and cell distribution considered normal for the species. The third rhino experienced a hemolytic event after stopping HBED administration in the same time frame and at the same dose as the two conspecifics but fully recovered. Careful monitoring and tapering the drug at the completion of treatment is warranted, especially if the rhino’s iron load is considered high. HBED’s potential to induce iron excretion safely, as well as prevent excessive dietary iron uptake, may significantly benefit the black rhino population under human care.

Iron deficiency (ID) and iron-deficiency anaemia (IDA) are global public health concerns, most commonly afflicting children, pregnant women and women of childbearing age. Pathological outcomes of ID include delayed cognitive development in children, adverse pregnancy outcomes and decreased work capacity in adults. IDA is usually treated by oral iron supplementation, typically using iron salts (e.g. FeSO4 ); however, dosing at several-fold above the RDA may be required due to less efficient absorption. Excess enteral iron causes adverse gastrointestinal side effects, thus reducing compliance, and negatively impacts the gut microbiome. Recent research has sought to identify new iron formulations with better absorption so that lower effective dosing can be utilized. This article outlines emerging research on oral iron supplementation and focuses on molecular mechanisms by which different supplemental forms of iron are transported across the intestinal epithelium and whether these transport pathways are subject to regulation by the iron-regulatory hormone hepcidin.

In this issue of Blood, Fisher et al1 demonstrate that metal -ion transporter ZIP8 (SLC39A8) in mouse liver endothelial cells (LECs) plays a significant, albeit modest, role in the in vivo expression of bone morphogenetic protein 6 (BMP6) in response to iron overload. This work helps to elucidate how LECs take up and sense iron to regulate body iron balance, which is a central and unanswered question in iron biology.

Manganese (Mn) is an essential nutrient, but is toxic in excess. Whole-body Mn levels are regulated in part by the metal-ion influx transporter SLC39A8, which plays an essential role in the liver by reclaiming Mn from bile. Physiological roles of SLC39A8 in Mn homeostasis in other tissues, however, remain largely unknown. To screen for extrahepatic requirements for SLC39A8 in tissue Mn homeostasis, we crossed Slc39a8-inducible global-KO (Slc39a8 iKO) mice with Slc39a14 KO mice, which display markedly elevated blood and tissue Mn levels. Tissues were then analyzed by inductively coupled plasma-mass spectrometry to determine levels of Mn. Although Slc39a14 KO; Slc39a8 iKO mice exhibited systemic hypermanganesemia and increased Mn loading in the bone and kidney due to Slc39a14 deficiency, we show Mn loading was markedly decreased in the brains of these animals, suggesting a role for SLC39A8 in brain Mn accumulation. Levels of other divalent metals in the brain were unaffected, indicating a specific effect of SLC39A8 on Mn. In vivo radiotracer studies using 54Mn in Slc39a8 iKO mice revealed that SLC39A8 is required for Mn uptake by the brain, but not most other tissues. Furthermore, decreased 54Mn uptake in the brains of Slc39a8 iKO mice was associated with efficient inactivation of Slc39a8 in isolated brain microvessels but not in isolated choroid plexus, suggesting SLC39A8 mediates brain Mn uptake via the blood-brain barrier. These findings establish SLC39A8 as a candidate therapeutic target for mitigating Mn uptake and accumulation in the brain, the primary organ of Mn toxicity.

ZIP8 (SLC39A8) is a transmembrane divalent metal ion importer that is most highly expressed in the lung and is inducible by inflammatory stimuli. In addition to zinc and manganese, ZIP8 can transport iron, but its specific roles in iron regulation during homeostatic and pathologic processes remain poorly understood. Using a novel global inducible ZIP8 knockout (KO) mouse, we analyzed the role of ZIP8 in steady-state iron homeostasis and during inflammation and infection. We observed an unexpected phenotype of elevated spleen iron levels and decreased serum iron in ZIP8 KO mice, suggesting that ZIP8 plays a role in iron recycling. We also showed that ZIP8 is expressed on lung distal airspace epithelial cells and transports iron from the airway into lung tissue. LPS-induced inflammation induced ZIP8 expression in the lung, but ZIP8 deletion had no detrimental effect on the severity of LPS-induced acute lung injury or on the outcomes of Klebsiella pneumoniae lung infection. Thus, ZIP8 plays a role in systemic iron homeostasis but does not modulate the severity of inflammatory lung injury or the host defense against a common bacterial cause of pneumonia.

In this issue of Blood, Charlebois et al(1) report that non-transferrin-bound iron (NTBI) is the primary driver of bone morphogenetic protein 6 (BMP6) expression in liver sinusoidal endothelial cells (LSECs) during iron overload. This finding is important because LSEC-derived BMP6 prompts the liver to produce hepcidin, the chief iron-regulatory hormone that regulates body iron balance.

While iron overload disorder (IOD) and related disease states are not considered a common occurrence in domestic equids, these issues appear prevalent in black rhinoceroses under human care. In addressing IOD in black rhinos, altering dietary iron absorption and excretion may be the most globally practical approach. A main option for treatment used across other species such as humans, is chelation therapy using iron-specific synthetic compounds. As horses may serve as an appropriate digestive model for the endangered rhinoceros, we evaluated the potential use of the oral iron chelator N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid (HBED) in horses for safety and efficacy prior to testing in black rhinoceros. Health and iron digestibility and dynamics were assessed in horses (n = 6) before, and after treatment with HBED (50 mg/kg body weight) for 8 days using a crossover design with serum, faecal and urine collection. A preliminary pharmacokinetic trial was also performed but no trace of HBED was found in serially sampled plasma through 8 h post-oral dosing. HBED increased urinary iron output in horses compared to control by 0.7% of total iron intake (p < 0.01), for an average of 27 mg urinary iron/day, similar to human chelation goals. Blood chemistry, blood cell counts and overall wellness were not affected by treatment. As healthy horses are able to regulate iron absorption, the lack of change in iron balance is unsurprising. Short-term HBED administration appeared to be safely tolerated by horses, therefore it was anticipated it would also be safe to administer to black rhinos for the management of iron overload.

Studies in recent years have significantly expanded, refined, and redefined the repertoire of transporters and other proteins involved in iron and manganese (Mn) transport and homeostasis. In this review, we discuss highlights of the recent literature on iron and Mn transport, focusing on the roles of membrane transporters and related proteins. Studies are considered from the vantage point of main organs, tissues, and cell types that actively control whole-body iron or Mn homeostasis, with emphasis on studies in which in vivo metal transport was measured directly or implicated by using knockout mouse models. Overviews of whole-body and cellular iron and Mn homeostasis are also provided to give physiological context for key transporters and to highlight how they participate in the uptake, intracellular trafficking, and efflux of each metal. Important similarities and differences in iron and Mn transport are noted, and future research opportunities and challenges are identified.

Although iron overload-related cardiomyopathy is a leading cause of morbidity and mortality in iron-overload disorders (e.g., thalassemia major and hemochromatosis), the molecular mechanisms that mediate cardiac iron uptake and accumulation are incompletely understood. Previous studies using Slc39a14 knockout mice have revealed that SLC39A14 is required for the uptake of non-transferrin-bound iron (NTBI) by the liver and pancreas and is essential for iron loading of hepatocytes and pancreatic acinar cells. To investigate the requirement for SLC39A14 in cardiac iron accumulation, we generated cardiomyocyte-specific Slc39a14 knockout (Slc39a14 hrt/hrt) mice and crossed them with iron-loading hemojuvelin (Hjv) knockout mice to generate Hjv -/-;Slc39a14 hrt/hrt animals. At 12 and 24 weeks of age, cardiac nonheme iron levels were ~340% higher in Hjv -/- mice than in controls. By contrast, cardiac nonheme iron levels in Hjv -/-;Slc39a14 hrt/hrt mice at these ages were only ~60% higher than those than in controls, and ~65% less than those in Hjv -/- mice. Moreover, cardiac nonheme iron levels in Hjv -/-;Slc39a14 +/hrt (heterozygous conditional Slc39a14 knockout) mice were between those of Hjv -/- and Hjv -/-;Slc39a14 hrt/hrt mice, suggesting a gene-dosage effect of Slc39a14 on cardiac iron accumulation. A role for voltage-dependent calcium channels in mediating the uptake of NTBI into cardiomyocytes has been proposed based on observations of the effects of L-type calcium-channel blockers on iron uptake and accumulation in vitro and in vivo. We considered the possibility that these observations could be explained if SLC39A14 were reactive with calcium-channel blockers. To test this hypothesis, we examined the effects of blockers on the activity of SLC39A14 by using radiotracer assays in RNA-injected Xenopus oocytes expressing mouse SLC39A14. We found that 100 µM amlodipine (Amld), nifedipine, and nicardipine each afforded modest inhibition of SLC39A14-mediated 55Fe 2+. Inhibition of iron transport by Amld was dose-dependent, EC 50 = 167 µM ± (SEM) 30 µM. Our findings implicate SLC39A14 in mediating cardiomyocyte NTBI uptake in the mouse and raise doubts about the relative importance of calcium channels as a mechanism by which NTBI gains entry to the heart.

Transferrin-bound iron (TBI), the physiological circulating iron form, is acquired by cells through the transferrin receptor (TfR1) by endocytosis. In erythroid cells, most of the acquired iron is incorporated into heme in the mitochondria. Cellular trafficking of heme is indispensable for erythropoiesis and many other essential biological processes. Comprehensive elucidation of molecular pathways governing and regulating cellular iron acquisition and heme trafficking is required to better understand physiological and pathological processes affecting erythropoiesis. Here, we report the first genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens in human erythroid cells to identify determinants of iron and heme uptake, as well as heme-mediated erythroid differentiation. We identified several candidate modulators of TBI acquisition including TfR1, indicating that our approach effectively revealed players mechanistically relevant to the process. Interestingly, components of the endocytic pathway were also revealed as potential determinants of transferrin acquisition. We deciphered a role for the vacuolar-type H+ - ATPase (V- ATPase) assembly factor coiled-coil domain containing 115 (CCDC115) in TBI uptake and validated this role in CCDC115 deficient K562 cells. Our screen in hemin-treated cells revealed perturbations leading to cellular adaptation to heme, including those corresponding to trafficking mechanisms and transcription factors potentiating erythroid differentiation. Pathway analysis indicated that endocytosis and vesicle acidification are key processes for heme trafficking in erythroid precursors. Furthermore, we provided evidence that CCDC115, which we identified as required for TBI uptake, is also involved in cellular heme distribution. This work demonstrates a previously unappreciated common intersection in trafficking of transferrin iron and heme in the endocytic pathway of erythroid cells.

Cigarette smoke exposure is a major cause of chronic obstructive pulmonary disease. Cadmium is a leading toxic component of cigarette smoke. Cadmium and zinc are highly related metals. Whereas, zinc is an essential metal required for normal health, cadmium is highly toxic. Zrt- and Irt-like protein 8 (ZIP8) is an avid transporter of both zinc and cadmium into cells and is abundantly expressed in the lung of smokers compared to nonsmokers. Our objective was to determine whether disturbed zinc homeostasis through diet or the zinc transporter ZIP8 increase susceptibility to lung damage following prolonged cigarette smoke exposure.METHODS:Cigarette smoke exposure was evaluated in the lungs of mice subject to insufficient and sufficient zinc intakes, in transgenic ZIP8 overexpressing mice, and a novel myeloid-specific ZIP8 knockout strain.RESULTS:Moderate depletion of zinc intakes in adult mice resulted in a significant increase in lung cadmium burden and permanent lung tissue loss following prolonged smoke exposure. Overexpression of ZIP8 resulted in increased lung cadmium burden and more extensive lung damage, whereas cigarette smoke exposure in ZIP8 knockout mice resulted in increased lung tissue loss without a change in lung cadmium content, but a decrease in zinc.CONCLUSIONS:Overall, findings were consistent with past human studies. Imbalance in Zn homeostasis increases susceptibility to permanent lung injury following prolonged cigarette smoke exposure. Based on animal studies, both increased and decreased ZIP8 expression enhanced irreversible tissue damage in response to prolonged tobacco smoke exposure. We believe these findings represent an important advancement in our understanding of how imbalance in zinc homeostasis and cadmium exposure via tobacco smoke may increase susceptibility to smoking-induced lung disease.

Most cells in the body acquire iron via receptor-mediated endocytosis of transferrin, the circulating iron transport protein. When cellular iron levels are sufficient, the uptake of transferrin decreases to limit further iron assimilation and prevent excessive iron accumulation. In iron overload conditions, such as hereditary hemochromatosis and thalassemia major, unregulated iron entry into the plasma overwhelms the carrying capacity of transferrin, resulting in non-transferrin-bound iron (NTBI), a redox-active, potentially toxic form of iron. Plasma NTBI is rapidly cleared from the circulation primarily by the liver and other organs (e.g., pancreas, heart, and pituitary) where it contributes significantly to tissue iron overload and related pathology. While NTBI is usually not detectable in the plasma of healthy individuals, it does appear to be a normal constituent of brain interstitial fluid and therefore likely serves as an important source of iron for most cell types in the CNS. A growing body of literature indicates that NTBI uptake is mediated by non-transferrin-bound iron transporters such as ZIP14, L-type and T-type calcium channels, DMT1, ZIP8, and TRPC6. This review provides an overview of NTBI uptake by various tissues and cells and summarizes the evidence for and against the roles of individual transporters in this process.

Significance Manganese (Mn) is an essential nutrient that is toxic in excess. Exposure to excess Mn can result in Mn accumulation in the brain and neurological and motor disturbances resembling Parkinson disease. Here, we demonstrate that the transmembrane metal-ion transporter solute carrier family 39, member 14 (SLC39A14) is essential for Mn homeostasis. We provide evidence that SLC39A14 is required for efficient Mn uptake by the liver and pancreas, two organs that are known to actively participate in Mn excretion from the body. Accordingly, loss of SLC39A14 impairs Mn excretion, leading to Mn accumulation in the brain and most other extrahepatic tissues. Slc39a14 -deficient mice, similar to SLC39A14 -deficient human patients, display motor deficits, and thus offer a convenient model to study Mn/SLC39A14-related neurotoxicity.

Genetic factors predictive of severe adolescent idiopathic scoliosis (AIS) are largely unknown. To identify genetic variation associated with severe AIS, we performed an exome-wide association study of 457 severe AIS cases and 987 controls. We find a missense SNP in SLC39A8 (p.Ala391Thr, rs13107325) associated with severe AIS ( P = 1.60 × 10 −7 , OR = 2.01, CI = 1.54–2.62). This pleiotropic SNP was previously associated with BMI, blood pressure, cholesterol, and blood manganese level. We replicate the association in a second cohort (841 cases and 1095 controls) resulting in a combined P = 7.02 × 10 −14 , OR = 1.94, CI = 1.63–2.34. Clinically, the minor allele of rs13107325 is associated with greater spinal curvature, decreased height, increased BMI and lower plasma manganese in our AIS cohort. Functional studies demonstrate reduced manganese influx mediated by the SLC39A8 p.Ala391Thr variant and vertebral abnormalities, impaired growth, and decreased motor activity in slc39a8 mutant zebrafish. Our results suggest the possibility that scoliosis may be amenable to dietary intervention.

Much evidence indicates that iron stored in ferritin is mobilized through protein degradation in lysosomes, but concerns about this process have lingered, and the mechanistic details of its aspects are lacking. In the studies presented here, 59Fe-labeled ferritin was induced by preloading hepatic (HepG2) cells with radiolabeled Fe. Placing these cells in a medium containing desferrioxamine resulted in the loss of ferritin-59Fe, but adding high concentrations of reducing agents or modulating the internal GSH concentration failed to alter the rates of ferritin-59Fe release. Confocal microscopy showed that Fe deprivation increased the movement of ferritin into lysosomes and hyperaccumulation was observed when lysosomal proteolysis was inhibited. It also resulted in the rapid movement of DMT1 to lysosomes, which was inhibited by bafilomycin. Ferrihydrite crystals isolated from purified rat liver/spleen ferritin were solubilized at pH 5 and 7 by GSH, ascorbate, citrate and lysosomal fluids obtained from livers and J774a.1 macrophages. The inhibition of DMT1/Nramp2 and siRNA knockdown of Nramp1 each reduced the transfer of 59Fe from lysosomes to the cytosol; and hepatocyte-specific knockout of DMT1 in mice prevented the release of Fe from the liver responding to EPO treatment, but did not inhibit lysosomal ferritin degradation. We conclude that ferritin-Fe mobilization does not occur through changes in cellular concentrations of reducing/chelating agents but by the coordinated movement of ferritin and DMT1 to lysosomes, where the ferrihydrite crystals exposed by ferritin degradation dissolve in the lysosomal fluid, and the reduced iron is transported back to the cytosol via DMT1 in hepatocytes, and by both DMT1 and Nramp1 in macrophages, prior to release into the blood or storage in ferritin.

Patients with metastatic cancer experience a severe loss of skeletal muscle mass and function known as cachexia. Cachexia is associated with poor prognosis and accelerated death in patients with cancer, yet its underlying mechanisms remain poorly understood. Here, we identify the metal-ion transporter ZRT- and IRT-like protein 14 (ZIP14) as a critical mediator of cancer-induced cachexia. ZIP14 is upregulated in cachectic muscles of mice and in patients with metastatic cancer and can be induced by TNF-α and TGF-β cytokines. Strikingly, germline ablation or muscle-specific depletion of Zip14 markedly reduces muscle atrophy in metastatic cancer models. We find that ZIP14-mediated zinc uptake in muscle progenitor cells represses the expression of MyoD and Mef2c and blocks muscle-cell differentiation. Importantly, ZIP14-mediated zinc accumulation in differentiated muscle cells induces myosin heavy chain loss. These results highlight a previously unrecognized role for altered zinc homeostasis in metastatic cancer–induced muscle wasting and implicate ZIP14 as a therapeutic target for its treatment. Accumulation of zinc in muscle cells resulting from transcriptional upregulation of metal transporter ZIP14 causes muscle atrophy and promotes cachexia in metastatic cancer.

Loss of p53’s proper function accounts for over half of identified human cancers. We identified the metal transporter ZIP14 (Zinc-regulated transporter (ZRT) and Iron-regulated transporter (IRT)-like Protein 14) as a p53-regulated protein. ZIP14 protein levels were upregulated by lack of p53 and downregulated by increased p53 expression. This regulation did not fully depend on the changes in ZIP14’s mRNA expression. Co-precipitation studies indicated that p53 interacts with ZIP14 and increases its ubiquitination and degradation. Moreover, knockdown of p53 resulted in higher non-transferrin-bound iron uptake, which was mediated by increased ZIP14 levels. Our study highlights a role for p53 in regulating nutrient metabolism and provides insight into how iron and possibly other metals such as zinc and manganese could be regulated in p53-inactivated tumor cells.