Iron potentiates microglial interleukin-1ß secretion induced by amyloid-ß.

Alzheimer's disease (AD) is characterized by accumulation of amyloid-beta (Aß) senile plaques in patients' brain tissues. Elevated levels of interleukin-1beta (IL-1ß) have been identified in cerebrospinal fluid of living AD patients and in animal models of AD. Increased expression of IL-1ß and iron accumulation have been identified in microglial cells that cluster around amyloid plaques in AD mouse models and post-mortem brain tissues of AD patients. The goals of this study were to determine the effects of Aß on the secretion of IL-1ß by microglial cells and whether iron status influences this pro-inflammatory signaling cue. Immortalized microglial (IMG) cells were incubated with Aß with or without iron. qRT-PCR and western blot analyses showed that Aß induces biosynthesis of IL-1ß by IMG cells. IMG cells secrete the mature form of IL-1ß in a caspase 1-dependent manner. Incubation with iron provoked a greater pro-inflammatory response. Inhibition of the iron transporter divalent metal transporter 1 protected IMG cells against Aß-induced inflammation. Potentiation of Aß-elicited IL-1ß induction by iron was also antagonized by ROS inhibitors, supporting the model that divalent metal transporter 1-mediated iron loading and subsequent increase in ROS contribute to the inflammatory effects of Aß in microglia. Immunoblotting and immunofluorescence microscopy indicate that iron enhances Aß activation of NF-κB signaling to promote IL-1ß synthesis. These results support the hypothesis that Aß stimulates IL-1ß expression by activating NF-κB signaling in microglia cells. Most importantly, iron appears to exacerbate the pro-inflammatory effects of Aß to increase IL-1ß levels.

ZIP14 is degraded in response to manganese exposure.

Manganese (Mn) is an essential element necessary for proper development and brain function. Circulating Mn levels are regulated by hepatobiliary clearance to limit toxic levels and prevent tissue deposition. To characterize mechanisms involved in hepatocyte Mn uptake, polarized human HepaRG cells were used for this study. Western blot analysis and immunofluorescence microscopy showed the Mn transporter ZIP14 was expressed and localized to the basolateral surface of polarized HepaRG cells. HepaRG cells took up 54Mn in a time- and temperature-dependent manner but uptake was reduced after exposure to Mn. This loss in transport activity was associated with decreased ZIP14 protein levels in response to Mn exposure. Mn-induced degradation of ZIP14 was blocked by bafilomycin A1, which increased localization of the transporter in Lamp1-positive vesicles. Mn exposure also down-regulated the Golgi proteins TMEM165 and GPP130 while the ER stress marker BiP was induced. These results indicate that Mn exposure decreases ZIP14 protein levels to limit subsequent uptake of Mn as a cytoprotective response. Thus, high levels of Mn may compromise first-pass-hepatic clearance mechanisms.

Brain Iron Homeostasis: A Focus on Microglial Iron.

Iron is an essential trace element required for important brain functions including oxidative metabolism, synaptic plasticity, myelination, and the synthesis of neurotransmitters. Disruptions in brain iron homeostasis underlie many neurodegenerative diseases. Increasing evidence suggests that accumulation of brain iron and chronic neuroinflammation, characterized by microglia activation and secretion of proinflammatory cytokines, are hallmarks of neurodegenerative disorders including Alzheimer' s disease. While substantial efforts have led to an increased understanding of iron metabolism and the role of microglial cells in neuroinflammation, important questions still remain unanswered. Whether or not increased brain iron augments the inflammatory responses of microglial cells, including the molecular cues that guide such responses, is still unclear. How these brain macrophages accumulate, store, and utilize intracellular iron to carry out their various functions under normal and disease conditions is incompletely understood. Here, we describe the known and emerging mechanisms involved in microglial cell iron transport and metabolism as well as inflammatory responses in the brain, with a focus on AD.

Crossing the Iron Gate: Why and How Transferrin Receptors Mediate Viral Entry.

Because both the host and pathogen require iron, the innate immune response carefully orchestrates control over iron metabolism to limit its availability during times of infection. Nutritional iron deficiency can impair host immunity, while iron overload can cause oxidative stress to propagate harmful viral mutations. An emerging enigma is that many viruses use the primary gatekeeper of iron metabolism, the transferrin receptor, as a means to enter cells. Why and how this iron gate is a viral target for infection are the focus of this review.

Manganese transport and toxicity in polarized WIF-B hepatocytes.

Manganese (Mn) toxicity arises from nutritional problems, community and occupational exposures, and genetic risks. Mn blood levels are controlled by hepatobiliary clearance. The goals of this study were to determine the cellular distribution of Mn transporters in polarized hepatocytes, to establish an in vitro assay for hepatocyte Mn efflux, and to examine possible roles the Mn transporters would play in metal import and export. For these experiments, hepatocytoma WIF-B cells were grown for 12-14 days to achieve maximal polarity. Immunoblots showed that Mn transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14 were present. Indirect immunofluorescence microscopy localized Fpn and ZIP14 to WIF-B cell basolateral domains whereas ZnT10 and ZIP8 associated with intracellular vesicular compartments. ZIP8-positive structures were distributed uniformly throughout the cytoplasm, but ZnT10-positive vesicles were adjacent to apical bile compartments. WIF-B cells were sensitive to Mn toxicity, showing decreased viability after 16 h exposure to >250 µM MnCl2. However, the hepatocytes were resistant to 4-h exposures of up to 500 µM MnCl2 despite 50-fold increased Mn content. Washout experiments showed time-dependent efflux with 80% Mn released after a 4 h chase period. Hepcidin reduced levels of Fpn in WIF-B cells, clearing Fpn from the cell surface, but Mn efflux was unaffected. The secretory inhibitor, brefeldin A, did block release of Mn from WIF-B cells, suggesting vesicle fusion may be involved in export. These results point to a possible role of ZnT10 to import Mn into vesicles that subsequently fuse with the apical membrane and empty their contents into bile. NEW & NOTEWORTHY Polarized WIF-B hepatocytes express manganese (Mn) transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14. Fpn and ZIP14 localize to basolateral domains. ZnT10-positive vesicles were adjacent to apical bile compartments, and ZIP8-positive vesicles were distributed uniformly throughout the cytoplasm. WIF-B hepatocyte Mn export was resistant to hepcidin but inhibited by brefeldin A, pointing to an efflux mechanism involving ZnT10-mediated uptake of Mn into vesicles that subsequently fuse with and empty their contents across the apical bile canalicular membrane.

Inflammation-induced iron transport and metabolism by brain microglia.

Microglia are immune cells of the central nervous system and are implicated in brain inflammation. However, how brain microglia modulate transport and metabolism of the essential metal iron in response to pro- and anti-inflammatory environmental cues is unclear. Here, we characterized uptake of transferrin (Tf)-bound iron (TBI) and non-Tf-bound iron (NTBI) by immortalized microglial (IMG) cells. We found that these cells preferentially take up NTBI in response to the proinflammatory stimulus lipopolysaccharide (LPS) or ß-amyloid (Aß). In contrast, the anti-inflammatory cytokine interleukin 4 (IL-4) promoted TBI uptake. Concordant with these functional data, levels of the Tf receptor (TfR) in IMG cells were up-regulated in response to IL-4, whereas divalent metal transporter-1 (DMT1) and ferritin levels increased in response to LPS or Aß. Similar changes in expression were confirmed in isolated primary adult mouse microglia treated with pro- or anti-inflammatory inducers. LPS-induced changes in IMG cell iron metabolism were accompanied by notable metabolic changes, including increased glycolysis and decreased oxidative respiration. Under these conditions, the extracellular acidification rate was increased, compatible with changes in the cellular microenvironment that would support the pH-dependent function of DMT1. Moreover, LPS increased heme oxygenase-1 (HO1) expression in IMG cells, and iron released because of HO1 activity increased the intracellular labile free-iron pool. Together, this evidence indicates that brain microglia preferentially acquire iron from Tf or from non-Tf sources, depending on their polarization state; that NTBI uptake is enhanced by the proinflammatory response; and that under these conditions microglia sequester both extra- and intracellular iron.

Novel founder intronic variant in SLC39A14 in two families causing Manganism and potential treatment strategies.

Congenital disorders of manganese metabolism are rare occurrences in children, and medical management of these disorders is complex and challenging. Homozygous exonic mutations in the manganese transporter SLC39A14 have recently been associated with a pediatric-onset neurodegenerative disorder characterized by brain manganese accumulation and clinical signs of manganese neurotoxicity, including parkinsonism-dystonia. We performed whole exome sequencing on DNA samples from two unrelated female children from the United Arab Emirates with progressive movement disorder and brain mineralization, identified a novel homozygous intronic mutation in SLC39A14 in both children, and demonstrated that the mutation leads to aberrant splicing. Both children had consistently elevated serum manganese levels and were diagnosed with SLC39A14-associated manganism. Over a four-year period, we utilized a multidisciplinary management approach for Patient 1 combining decreased manganese dietary intake and chelation with symptomatic management of dystonia. Our treatment strategy appeared to slow disease progression, but did not lead to a cure or reversal of already established deficits. Clinicians should consider testing for noncoding mutations in the diagnosis of congenital disorders of manganese metabolism and utilizing multidisciplinary approaches in the management of these disorders.

Excess iron: considerations related to development and early growth.

What effects might arise from early life exposures to high iron? This review considers the specific effects of high iron on the brain, stem cells, and the process of erythropoiesis and identifies gaps in our knowledge of what molecular damage may be incurred by oxidative stress that is imparted by high iron status in early life. Specific areas to enhance research on this topic include the following: longitudinal behavioral studies of children to test associations between iron exposures and mood, emotion, cognition, and memory; animal studies to determine epigenetic changes that reprogram brain development and metabolic changes in early life that could be followed through the life course; and the establishment of human epigenetic markers of iron exposures and oxidative stress that could be monitored for early origins of adult chronic diseases. In addition, efforts to understand how iron exposure influences stem cell biology could be enhanced by establishing platforms to collect biological specimens, including umbilical cord blood and amniotic fluid, to be made available to the research community. At the molecular level, there is a need to better understand stress erythropoiesis and changes in iron metabolism during pregnancy and development, especially with respect to regulatory control under high iron conditions that might promote ineffective erythropoiesis and iron-loading anemia. These investigations should focus not only on factors such as hepcidin and erythroferrone but should also include newly identified interactions between transferrin receptor-2 and the erythropoietin receptor. Finally, despite our understanding that several key micronutrients (e.g., vitamin A, copper, manganese, and zinc) support iron's function in erythropoiesis, how these nutrients interact remains, to our knowledge, unknown. It is necessary to consider many factors when formulating recommendations on iron supplementation.

Restored iron transport by a small molecule promotes absorption and hemoglobinization in animals.

Multiple human diseases ensue from a hereditary or acquired deficiency of iron-transporting protein function that diminishes transmembrane iron flux in distinct sites and directions. Because other iron-transport proteins remain active, labile iron gradients build up across the corresponding protein-deficient membranes. Here we report that a small-molecule natural product, hinokitiol, can harness such gradients to restore iron transport into, within, and/or out of cells. The same compound promotes gut iron absorption in DMT1-deficient rats and ferroportin-deficient mice, as well as hemoglobinization in DMT1- and mitoferrin-deficient zebrafish. These findings illuminate a general mechanistic framework for small molecule-mediated site- and direction-selective restoration of iron transport. They also suggest that small molecules that partially mimic the function of missing protein transporters of iron, and possibly other ions, may have potential in treating human diseases.

Regulation of divalent metal transporter-1 by serine phosphorylation.

Divalent metal transporter-1 (DMT1) mediates dietary iron uptake across the intestinal mucosa and facilitates peripheral delivery of iron released by transferrin in the endosome. Here, we report that classical cannabinoids (Δ9-tetrahydrocannabinol, Δ9-THC), nonclassical cannabinoids (CP 55,940), aminoalkylindoles (WIN 55,212-2) and endocannabinoids (anandamide) reduce 55Fe and 54Mn uptake by HEK293T(DMT1) cells stably expressing the transporter. siRNA knockdown of cannabinoid receptor type 2 (CB2) abrogated inhibition. CB2 is a G-protein (GTP-binding protein)-coupled receptor that negatively regulates signal transduction cascades involving serine/threonine kinases. Immunoprecipitation experiments showed that DMT1 is serine-phosphorylated under basal conditions, but that treatment with Δ9-THC reduced phosphorylation. Site-directed mutation of predicted DMT1 phosphosites further showed that substitution of serine with alanine at N-terminal position 43 (S43A) abolished basal phosphorylation. Concordantly, both the rate and extent of 55Fe uptake in cells expressing DMT1(S43A) was reduced compared with those expressing wild-type DMT1. Among kinase inhibitors that affected DMT1-mediated iron uptake, staurosporine also reduced DMT1 phosphorylation confirming a role for serine phosphorylation in iron transport regulation. These combined data indicate that phosphorylation at serine 43 of DMT1 promotes transport activity, whereas dephosphorylation is associated with loss of iron uptake. Since anti-inflammatory actions mediated through CB2 would be associated with reduced DMT1 phosphorylation, we postulate that this pathway provides a means to reduce oxidative stress by limiting iron uptake.

Dietary supplementation with ipriflavone decreases hepatic iron stores in wild type mice.

Hepcidin, a peptide produced in the liver, decreases intestinal iron absorption and macrophage iron release by causing degradation of the iron exporter, ferroportin. Because its levels are inappropriately low in patients with iron overload syndromes, hepcidin is a potential drug target. We previously conducted a chemical screen that revealed ipriflavone, an orally available small molecule, as a potent inducer of hepcidin expression. To evaluate ipriflavone's effect on iron homeostasis, we placed groups of 5-week old wild type or thalassemia intermedia (Hbb(Th3+/-)) mice on a soy-free, iron-sufficient diet, AIN-93G containing 220mg iron and 0-750mgipriflavone/kg of food for 50days. Ipriflavone 500mg/kg significantly reduced liver iron stores and intestinal ferroportin expression in WT mice, while increasing the ratio of hepcidin transcript levels to liver iron stores. Ipriflavone supplementation in Hbb(Th3+/-) mice failed to alleviate iron overload and was associated with a milder reduction in intestinal ferroportin and a failure to alter the ratio of hepcidin transcript levels to liver iron stores or splenic expression of the hepcidin-regulatory hormone, erythroferrone. These data suggest that dietary supplementation with ipriflavone alone would not be sufficient to treat iron overload in thalassemia intermedia.

Characterization of a novel adult murine immortalized microglial cell line and its activation by amyloid-beta.

BACKGROUND: Alzheimer's disease is associated with amyloid-beta (Aß)-induced microglia activation. This pro-inflammatory response promotes neuronal damage, and therapies are sought to limit microglial activation. Screening efforts to develop new pharmacological inhibitors require a robust in vitro cell system. Current models lack significant responses to Aß, and their use in examining age-related neurodegenerative diseases is questionable. For example, the commonly used BV-2 microglial line was derived from embryonic mononuclear cells and its activation by various stimuli is limited. To this end, we have established a new immortalized microglial (IMG) cell line from adult murine brain. The objective of this study was to characterize Aß-induced activation of IMG cells, and here, we demonstrate the ability of cannabinoids to significantly reduce this inflammatory response. METHODS: Microglial cells derived from adult murine brain were immortalized via infection with the v-raf/v-myc retrovirus under conditions that selectively promote microglia growth. The presence or absence of markers CD11b and F4/80 (microglial), NeuN (neuronal), and GFAP (astrocytic) was assessed by immunofluorescence microscopy and western blotting. Using IMG and BV-2 cells, levels of pro- and anti-inflammatory transcripts in response to extracellular stimuli were determined by quantitative PCR (qPCR). Phagocytosis of fluorescent beads and fluorescein isothiocyanate (FITC)-labeled Aß oligomers was assessed using flow cytometry and fluorescence microscopy. FITC-Aß uptake was quantified using a fluorescence plate reader. The ability of cannabinoids to mitigate Aß-induced expression of inducible nitric oxide synthase (iNOS) was evaluated. RESULTS: IMG cells express the microglial markers CD11b and F4/80 but not NeuN or GFAP. Relative to BV-2 cells, IMG cells increased iNOS (>200-fold) and Arg-1 (>100-fold) in response to pro- and anti-inflammatory stimuli. IMG cells phagocytose foreign particles and Aß oligomers, with the latter trafficked to phagolysosomes. Aß-induced activation of IMG cells was suppressed by delta-9-tetrahydrocannabinol and the CB2-selective agonist JWH-015 in a time- and concentration-dependent manner. CONCLUSIONS: IMG cells recapitulate key features of microglial cell activation. As an example of their potential pharmacological use, cannabinoids were shown to reduce activation of Aß-induced iNOS gene expression. IMG cells hold promising potential for drug screening, mechanistic studies, and functional investigations directed towards understanding how Aß interacts with microglia.

Distribution of manganese and other biometals in flatiron mice.

Flatiron (ffe) mice display features of "ferroportin disease" or Type IV hereditary hemochromatosis. While it is known that both Fe and Mn metabolism are impaired in flatiron mice, the effects of ferroportin (Fpn) deficiency on physiological distribution of these and other biometals is unknown. We hypothesized that Fe, Mn, Zn and/or Cu distribution would be altered in ffe/+ compared to wild-type (+/+) mice. ICP-MS analysis showed that Mn, Zn and Cu levels were significantly reduced in femurs from ffe/+ mice. Bone deposits reflect metal accumulation, therefore these data indicate that Mn, Zn and Cu metabolism are affected by Fpn deficiency. The observations that muscle Cu, lung Mn, and kidney Cu and Zn levels were reduced in ffe/+ mice support the idea that metal metabolism is impaired. While all four biometals appeared to accumulate in brains of flatiron mice, significant gender effects were observed for Mn and Zn levels in male ffe/+ mice. Metals were higher in olfactory bulbs of ffe/+ mice regardless of gender. To further study brain metal distribution, (54)MnCl2 was administered by intravenous injection and total brain (54)Mn was measured over time. At 72 h, (54)Mn was significantly greater in brains of ffe/+ mice compared to +/+ mice while blood (54)Mn was cleared to the same levels by 24 h. Taken together, these results indicate that Fpn deficiency decreases Mn trafficking out of the brain, alters body Fe, Mn, Zn and Cu levels, and promotes metal accumulation in olfactory bulbs.

Nramp1 and Other Transporters Involved in Metal Withholding during Infection.

During the course of infection, many natural defenses are set up along the boundaries of the host-pathogen interface. Key among these is the host response to withhold metals to restrict the growth of invading microbes. This simple act of nutritional warfare, starving the invader of an essential element, is an effective means of limiting infection. The physiology of metal withholding is often referred to as "nutritional immunity," and the mechanisms of metal transport that contribute to this host response are the focus of this review.

The small molecule ferristatin II induces hepatic hepcidin expression in vivo and in vitro.

Previous studies have shown that administration of ferristatin II to rats is associated with decreased serum iron, reduced transferrin saturation, and increased hepatic hepcidin expression. BMP and IL-6 signaling act via Smad and Stat3 transcription factors, respectively, to increase expression of hepcidin, the master regulator of iron metabolism. In this study, we aimed to explore the underlying mechanism of ferristatin II action on hepcidin production. We found that ferristatin II greatly increased hepcidin expression both in vivo and in vitro. In the rat liver, ferristatin II treatment decreased expression of Smad downstream targets Smad7 and Id1 and increased expression of Stat3 downstream targets α-2-macroglobulin, α-1-acid glycoprotein, and C-reactive peptide. Ferristatin II also increased Stat3 phosphorylation in the rat liver without affecting serum or hepatic IL-6 levels. It is unclear whether the Stat3 activation observed in vivo is a cause or a consequence to hepcidin induction. Reporter gene expression studies demonstrated that ferristatin II synergized with BMP6 and IL-6 to enhance hepcidin expression in vitro. However, this synergy was not due to activation of either Smad or Stat3 signaling, raising the possibility that ferristatin II may activate a novel pathway for hepcidin regulation.

Ferroportin deficiency impairs manganese metabolism in flatiron mice.

We examined the physiologic role of ferroportin (Fpn) in manganese (Mn) export using flatiron (ffe/+) mice, a genetic model of Fpn deficiency. Blood (0.0123 vs. 0.0107 mg/kg; P = 0.0003), hepatic (1.06 vs. 0.96 mg/kg; P = 0.0125), and bile Mn levels (79 vs. 38 mg/kg; P = 0.0204) were reduced in ffe/+ mice compared to +/+ controls. Erythrocyte Mn-superoxide dismutase was also reduced at 6 (0.154 vs. 0.096, P = 0.0101), 9 (0.131 vs. 0.089, P = 0.0162), and 16 weeks of age (0.170 vs. 0.090 units/mg protein/min; P < 0.0001). (54)Mn uptake after intragastric gavage was markedly reduced in ffe/+ mice (0.0187 vs. 0.0066% dose; P = 0.0243), while clearance of injected isotope was similar in ffe/+ and +/+ mice. These values were compared to intestinal absorption of (59)Fe, which was significantly reduced in ffe/+ mice (8.751 vs. 3.978% dose; P = 0.0458). The influence of the ffe mutation was examined in dopaminergic SH-SY5Y cells and human embryonic HEK293T cells. While expression of wild-type Fpn reversed Mn-induced cytotoxicity, ffe mutant H32R failed to confer protection. These combined results demonstrate that Fpn plays a central role in Mn transport and that flatiron mice provide an excellent genetic model to explore the role of this exporter in Mn homeostasis. -

Iron and mechanisms of emotional behavior.

Iron is required for appropriate behavioral organization. Iron deficiency results in poor brain myelination and impaired monoamine metabolism. Glutamate and γ-aminobutyric acid homeostasis is modified by changes in brain iron status. Such changes produce not only deficits in memory/learning capacity and motor skills, but also emotional and psychological problems. An accumulating body of evidence indicates that both energy metabolism and neurotransmitter homeostasis influence emotional behavior, and both functions are influenced by brain iron status. Like other neurobehavioral aspects, the influence of iron metabolism on mechanisms of emotional behavior is multifactorial: brain region-specific control of behavior, regulation of neurotransmitters and associated proteins, temporal and regional differences in iron requirements, oxidative stress responses to excess iron, sex differences in metabolism, and interactions between iron and other metals. To better understand the role that brain iron plays in emotional behavior and mental health, this review discusses the pathologies associated with anxiety and other emotional disorders with respect to body iron status.

Pathophysiology of the Belgrade rat.

The Belgrade rat is an animal model of divalent metal transporter 1 (DMT1) deficiency. This strain originates from an X-irradiation experiment first reported in 1966. Since then, the Belgrade rat's pathophysiology has helped to reveal the importance of iron balance and the role of DMT1. This review discusses our current understanding of iron transport homeostasis and summarizes molecular details of DMT1 function. We describe how studies of the Belgrade rat have revealed key roles for DMT1 in iron distribution to red blood cells as well as duodenal iron absorption. The Belgrade rat's pathology has extended our knowledge of hepatic iron handling, pulmonary and olfactory iron transport as well as brain iron uptake and renal iron handling. For example, relationships between iron and manganese metabolism have been discerned since both are essential metals transported by DMT1. Pathophysiologic features of the Belgrade rat provide us with a unique and interesting animal model to understand iron homeostasis.

Impaired renal function and development in Belgrade rats.

Belgrade rats carry a disabling mutation in the iron transporter divalent metal transporter 1 (DMT1). Although DMT1 plays a major role in intestinal iron absorption, the transporter is also highly expressed in the kidney, where its function remains unknown. The goal of this study was to characterize renal physiology of Belgrade rats. Male Belgrade rats died prematurely with ∼50% survival at 20 wk of age. Necropsy results indicated marked glomerular nephritis and chronic end-stage renal disease. By 15 wk of age, Belgrade rats displayed altered renal morphology associated with sclerosis and fibrosis. Creatinine clearance was significantly lower compared with heterozygote littermates. Urinary biomarkers of kidney injury, including albumin, fibrinogen, and kidney injury molecule-1, were significantly elevated. Pilot morphological studies suggest that nephrogenesis is delayed in Belgrade rat pups due to their low iron status and fetal growth restriction. Such defects in renal development most likely underlie the compromised renal metabolism observed in adult b/b rats. Belgrade rat kidney nonheme iron levels were not different from controls but urinary iron and transferrin levels were higher. These results further implicate an important role for the transporter in kidney function not only in iron reabsorption but also in glomerular filtration of the serum protein.

Ferristatin II promotes degradation of transferrin receptor-1 in vitro and in vivo.

Previous studies have shown that the small molecule iron transport inhibitor ferristatin (NSC30611) acts by down-regulating transferrin receptor-1 (TfR1) via receptor degradation. In this investigation, we show that another small molecule, ferristatin II (NSC8679), acts in a similar manner to degrade the receptor through a nystatin-sensitive lipid raft pathway. Structural domains of the receptor necessary for interactions with the clathrin pathway do not appear to be necessary for ferristatin II induced degradation of TfR1. While TfR1 constitutively traffics through clathrin-mediated endocytosis, with or without ligand, the presence of Tf blocked ferristatin II induced degradation of TfR1. This effect of Tf was lost in a ligand binding receptor mutant G647A TfR1, suggesting that Tf binding to its receptor interferes with the drug's activity. Rats treated with ferristatin II have lower TfR1 in liver. These effects are associated with reduced intestinal (59)Fe uptake, lower serum iron and transferrin saturation, but no change in liver non-heme iron stores. The observed hypoferremia promoted by degradation of TfR1 by ferristatin II appears to be due to induced hepcidin gene expression.