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In previous studies we demonstrated that zinc stimulates iron uptake in intestinal Caco-2 cells via Zinc-PI3K-IRP2-DMT1 axis. In the current study we investigated the effect of zinc on basolateral iron release and characterized the associated mechanisms. In Caco-2 cells grown on permeable supports, zinc induced iron transport and expression of DMT1, HEPH mRNA and protein, but not that of FPN1. LY294002, an inhibitor of PI3K, inhibited the zinc-induced iron transport, DMT1, HEPH mRNA and protein expression. In addition, LY294002 also inhibited the basal expression of HEPH and FPN1 resulting in blockade of iron egress from cells. In addition, siRNA-silencing of HEPH led to inhibition of both zinc-induced and basal iron transport. Conversely, TPEN, a chelator of zinc, inhibited iron uptake, DMT1, HEPH and FPN1 mRNA and protein expression. These results suggest that intestinal cell zinc status is a critical determinant of iron absorption and effects are mediated via activation of PI3K. Further, PI3K pathway appears to selectively modulate the expression of iron transporters and iron absorption, therefore this might serve as a therapeutic target in iron overload disorders., Competing Interests: Declaration of competing interest The Authors declare that there are no competing interests associated with the manuscript., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

The amyloid precursor protein is so named, because a proteolytic fragment of it was found associated with a neuropathic disorder now known as Alzheimer's disease. This fragment, Aβ, along with tau makes up the plaques and tangles that are the hallmark of AD. Iron (and other first-row transition metals) is found associated with these proteinaceous deposits. Much research has focused on the relationship of the plaques and iron to the etiology of the disease. This commentary asks another question, one only more recently addressed namely, what is the physiologic function of the amyloid precursor protein (APP) and of its secretase-generated soluble species? Overall, the data make clear that APP and its products have neurotrophic functions and some data indicate one of these may be to modulate the trafficking of iron in the brain.

Iron efflux from mammalian cells is supported by the synergistic actions of the ferrous iron efflux transporter, ferroportin (Fpn) and a multicopper ferroxidase, that is, hephaestin (Heph), ceruloplasmin (Cp) or both. The two proteins stabilize Fpn in the plasma membrane and catalyze extracellular Fe 3+ release. The membrane stabilization of Fpn is also stimulated by its interaction with a 22-amino acid synthetic peptide based on a short sequence in the extracellular E2 domain of the amyloid precursor protein (APP). However, whether APP family members interact with Fpn in vivo is unclear. Here, using cyan fluorescent protein (CFP)-tagged Fpn in conjunction with yellow fluorescent protein (YFP) fusions of Heph and APP family members APP, APLP1, and APLP2 in HEK293T cells we used fluorescence and surface biotinylation to quantify Fpn membrane occupancy and also measured 59 Fe efflux. We demonstrate that Fpn and Heph co-localize, and FRET analysis indicated that the two proteins form an iron-efflux complex. In contrast, none of the full-length, cellular APP proteins exhibited Fpn co-localization or FRET. Moreover, iron supplementation increased surface expression of the iron-efflux complex, and copper depletion knocked down Heph activity and decreased Fpn membrane localization. Whereas cellular APP species had no effects on Fpn and Heph localization, addition of soluble E2 elements derived from APP and APLP2, but not APLP1, increased Fpn membrane occupancy. We conclude that a ferroportin-targeting sequence, (K/R)EWEE, present in APP and APLP2, but not APLP1, helps modulate Fpn-dependent iron efflux in the presence of an active multicopper ferroxidase., (© 2019 Dlouhy et al.)

Two of the major cancerous diseases associated with asbestos exposure are malignant pleural mesothelioma (MPM) and lung cancer (LC). In addition to asbestos exposure, genetic factors have been suggested to be associated with asbestos-related carcinogenesis and lung genotoxicity. While genetic factors involved in the susceptibility to MPM were reported, to date the influence of individual genetic variations on asbestos-related lung cancer risk is still poorly understood. Since inflammation and disruption of iron (Fe) homeostasis are hallmarks of asbestos exposure affecting the pulmonary tissue, this study aimed at investigating the association between Fe-metabolism and inflammasome gene variants and susceptibility to develop LC or MPM, by comparing an asbestos-exposed population affected by LC with an "asbestos-resistant exposed population". A retrospective approach similar to our previous autopsy-based pilot study was employed in a novel cohort of autoptic samples, thus giving us the possibility to corroborate previous findings obtained on MPM by repeating the analysis in a novel cohort of autoptic samples. The protective role of HEPH coding SNP was further confirmed. In addition, the two non-coding SNPs, either in FTH1 or in TF , emerged to exert a similar protective role in a new cohort of LC exposed individuals from the same geographic area of MPM subjects. No association was found between NLRP1 and NLRP3 polymorphisms with susceptibility to develop MPM and LC. Further research into a specific MPM and LC "genetic signature" may be needed to broaden our knowledge of the genetic landscape attributed to result in MPM and LC.

Multi-copper ferroxidases (MCFs) play important roles in cellular iron metabolism and homeostasis. In this study, we generated the hephaestin (Heph), ceruloplasmin (Cp) single and Heph/Cp double knockout (KO) mice to investigate the roles of MCFs in iron transport among system and vital organs in mice at 4 weeks and 6 months of age. Compared with wild-type (WT) mice, Heph/Cp mice at both ages presented with severe anemia and significantly lower iron level in the serum and spleen, but with significantly higher iron level in the liver, heart, kidney, and duodenal enterocytes. Furthermore, Heph/Cp mice displayed significantly lower level of hepcidin mRNA and transferrin receptor 1 (TFR1) protein expression, but significantly higher level of ferroportin 1 (FPN1) protein expression in the liver than WT mice at 6 months of age. Liver superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzyme activities were significantly lower in Heph/Cp KO mice than WT mice at 6 months of age. Together, our results suggest that ablation of HEPH and CP could lead to severe systemic iron deficiency and local tissue iron overload, which disrupt the whole body iron homeostasis and impact on tissue functions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Little is known about the iron efflux from the pancreas, but it is likely that multicopper ferroxidases (MCFs) are involved in this process. We thus used hephaestin (Heph) and ceruloplasmin (Cp) single-knockout mice and Heph/Cp double-knockout mice to investigate the roles of MCFs in pancreatic iron homeostasis. We found that both HEPH and CP were expressed in the mouse pancreas, and that ablation of either MCF had limited effect on the pancreatic iron levels. However, ablation of both MCFs together led to extensive pancreatic iron deposition and severe oxidative damage. Perls' Prussian blue staining revealed that this iron deposition was predominantly in the exocrine pancreas, while the islets were spared. Consistent with these results, plasma lipase and trypsin were elevated in Heph/Cp knockout mice, indicating damage to the exocrine pancreas, while insulin secretion was not affected. These data indicate that HEPH and CP play mutually compensatory roles in facilitating iron efflux from the exocrine pancreas, and show that MCFs are able to protect the pancreas against iron-induced oxidative damage., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Intracellular retinal iron accumulation has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of irreversible blindness among individuals over the age of 50. Ceruloplasmin/hephaestin double knockout mice (Cp/Heph DKO) and hepcidin knockout mice (Hepc KO) accumulate retinal iron and model some features of AMD. Two canonical pathways govern cellular iron import - transferrin-bound iron import and non-transferrin bound iron import. In Cp/Heph DKO and Hepc KO iron-loaded retinas, transferrin-bound iron import is downregulated. Despite this effort to reduce cellular iron burden, iron continues to accumulate in these retinas in an age-dependent manner. Quantitative RT-PCR and Western analysis were used to quantify the expression of three ferrous iron importers, Dmt1, Zip8, and Zip14, in wild-type (Wt), Cp/Heph DKO, and Hepc KO retinas. Zip8 and Zip14 protein levels were analyzed using Western analysis in mice injected intravitreally with either apo- or holo-transferrin to elucidate one possible mechanism of Zip14 regulation in the retina. Both zip8 and zip14 were expressed in the mouse retina. Paradoxically, protein levels of non-transferrin bound iron importers were upregulated in both Cp/Heph DKO and Hepc KO retinas. Intravitreal holo-transferrin injection decreased Zip 14 protein levels. These data indicate that Zip8 and Zip14 may take up increasing amounts of non-transferrin bound iron in these two mouse models of retinal iron accumulation. Their upregulation in these already iron-loaded retinas suggests a vicious cycle leading to toxicity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The mechanisms for iron transport through the blood-brain barrier (BBB) remain a controversy. We analyzed for expression of mRNA and proteins involved in oxidation and transport of iron in isolated brain capillaries from dietary normal, iron-deficient, and iron-reverted rats. The expression was also investigated in isolated rat brain endothelial cells (RBECs) and in immortalized rat brain endothelial (RBE4) cells grown as monoculture or in hanging culture inserts with defined BBB properties. Transferrin receptor 1, ferrireductases Steap 2 and 3, divalent metal transporter 1 (DMT1), ferroportin, soluble and glycosylphosphatidylinositol (GPI)-anchored ceruloplasmin, and hephaestin were all expressed in brain capillaries in vivo and in isolated RBECs and RBE4 cells. Gene expression of DMT1, ferroportin, and soluble and GPI-anchored ceruloplasmin were significantly higher in isolated RBECs with induced BBB properties. Primary pericytes and astrocytes both expressed ceruloplasmin and hephaestin, and RBECs, pericytes, and astrocytes all exhibited ferrous oxidase activity. The coherent protein expression of these genes was demonstrated by immunocytochemistry. The data show that brain endothelial cells provide the machinery for receptor-mediated uptake of ferric iron-containing transferrin. Ferric iron can then undergo reduction to ferrous iron by ferrireductases inside endosomes followed by DMT1-mediated pumping into the cytosol and subsequently cellular export by ferroportin. The expression of soluble ceruloplasmin by brain endothelial cells, pericytes, and astrocytes that together form the neurovascular unit (NVU) provides the ferroxidase activity necessary to reoxidize ferrous iron once released inside the brain.

The effect of iron on the progress of atherosclerosis is still controversial. To explore the relationship between atherosclerotic plaques and iron metabolism and how iron is accumulated in plaque macrophages, we performed Oil red O staining to detect the lipid of the atherosclerotic plaques, enzyme-linked immunosorbent assay to detect the intracellular lipids (total cholesterol, free cholesterol) and serum hepcidin, Western-blot to examine the iron-related proteins, immunohistochemical and immunofluorescence assays to localize ferroportin 1 in macrophages. The contents of serum iron and transferrin saturation were measured. The results confrimed that atherosclerotic plaques were all lipid-rich. Compared to normal vessel wall, atherosclerotic plaques had significantly higher levels of ferritin and ferroportin 1. Strikingly, we found the much lower levels of ferroxidases ceruloplasmin and hephaestin in plaque tissue than the normal vessel, while the content of serum hepcidin, iron and transferrin saturation were similar in these two groups. The novel finding suggests that the inability of ferrous iron to be oxidized into ferric iron might be a potential mechanism for iron retention in plaques., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Iron accumulation in the central nervous system (CNS) is a common feature of many neurodegenerative diseases. Multicopper ferroxidases (MCFs) play an important role in cellular iron metabolism. However, the role of MCFs in the CNS in health and disease remains poorly characterized., Objective: The aim was to study the role of hephaestin (HEPH) and ceruloplasmin (CP) in CNS iron metabolism and homeostasis., Methods: Iron concentrations and L-ferritin protein levels of selected brain regions were determined in global hephaestin knockout (Heph KO), global ceruloplasmin knockout (Cp KO), and wild-type (WT) male mice at 6-7 mo of age. Gene expression of divalent metal transporter 1 (Dmt1), ferroportin 1 (Fpn1), Heph, Cp, and transferrin receptor 1 (Tfrc) and HEPH protein level was quantitated in the same brain regions., Results: Iron and L-ferritin protein levels were significantly increased in Heph KO mouse brain cortex (iron: 30%, P < 0.05; L-ferritin: 200%, P < 0.05), hippocampus (iron: 80%, P < 0.05; L-ferritin: 300%, P < 0.05), brainstem (iron: 20%, P < 0.05; L-ferritin: 150%, P < 0.05), and cerebellum (iron: 20%, P < 0.05; L-ferritin: 100%, P < 0.05) regions than in WT and Cp KO mouse brain regions at 6 mo of age. Expression of the Heph gene was significantly increased in the Cp KO mouse cortex (100%; P < 0.01), hippocampus (350%; P < 0.001), brainstem (30%; P < 0.01), and cerebellum (150%; P < 0.001) than in WT controls, and Cp gene expression was significantly decreased in the Heph KO mouse hippocampus (20%; P < 0.05) than in WT control mice at 6 mo of age., Conclusions: Ablation of HEPH or CP results in disordered brain iron homeostasis in mice. Heph KO may provide a novel model for neurodegenerative disorders., (© 2015 American Society for Nutrition.)

Background and Aim: The body's requirement for iron is different at different developmental stages. However, the molecular mechanisms of age-dependent iron metabolism are poorly understood. In the present study, we investigated the expression of iron transport proteins in the duodenum of Sprague-Dawley rats at five different age stages., Methods: Male Sprague-Dawley rats at postnatal week (PNW) 1, 3, 12, 44, and 88 were employed in the study. Serum iron status and tissue non-heme iron concentrations in the spleen, liver, bone marrow, heart, kidney, duodenal epithelium, and gastrocnemius were examined at each age stage. The expression of duodenal cytochrome b (DcytB), divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), hephaestin, and hepcidin were measured by real-time polymerase chain reaction or Western blot., Results: The levels of serum iron and transferrin saturation were higher in the rats at PNW1 and 3 than in those at PNW12, 44, and 88. Non-heme iron contents decreased from PNW1 to PNW3 and then increased thereafter. Duodenal DcytB, DMT1, and FPN1 increased to the highest level at PNW3 and then decreased from PNW12 to 88. The hepatic hepcidin mRNA level decreased to the lowest level at PNW3 and then increased with age., Conclusion: Our findings showed that age had a significant effect on body iron status. The increased duodenal DcytB, DMT1, and FPN1 expression can enhance intestinal iron absorption to meet the high iron requirements in infants. Hepcidin or enterocyte iron levels may be involved in the regulation of age-dependent FPN1, DMT1, and DcytB expression in the duodenum., (© 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.)

Iron is an essential trace mineral that plays a number of important physiological roles in humans, including oxygen transport, energy metabolism, and neurotransmitter synthesis. Iron absorption by the proximal small bowel is a critical checkpoint in the maintenance of whole-body iron levels since, unlike most other essential nutrients, no regulated excretory systems exist for iron in humans. Maintaining proper iron levels is critical to avoid the adverse physiological consequences of either low or high tissue iron concentrations, as commonly occurs in iron-deficiency anemia and hereditary hemochromatosis, respectively. Exquisite regulatory mechanisms have thus evolved to modulate how much iron is acquired from the diet. Systemic sensing of iron levels is accomplished by a network of molecules that regulate transcription of the HAMP gene in hepatocytes, thus modulating levels of the serum-borne, iron-regulatory hormone hepcidin. Hepcidin decreases intestinal iron absorption by binding to the iron exporter ferroportin 1 on the basolateral surface of duodenal enterocytes, causing its internalization and degradation. Mucosal regulation of iron transport also occurs during low-iron states, via transcriptional (by hypoxia-inducible factor 2α) and posttranscriptional (by the iron-sensing iron-regulatory protein/iron-responsive element system) mechanisms. Recent studies demonstrated that these regulatory loops function in tandem to control expression or activity of key modulators of iron homeostasis. In health, body iron levels are maintained at appropriate levels; however, in several inherited disorders and in other pathophysiological states, iron sensing is perturbed and intestinal iron absorption is dysregulated. The iron-related phenotypes of these diseases exemplify the necessity of precisely regulating iron absorption to meet body demands., (Copyright © 2014 the American Physiological Society.)

Objective: To investigate the multiple iron metabolism-related genes expression, its regulation by iron and the expression correlation among the genes in rat tissues., Methods: Two groups (n=30) of Sprague-Dawley female weanling rats were fed with a control diet and an iron deficient diet respectively for 4 weeks. All rats were then sacrificed, and blood and tissue samples were collected. The routine blood examination was performed with a veterinary automatic blood cell analyzer. Elemental iron levels in liver, spleen and serum were determined by atomic absorption spectrophotometry. The mRNA expression of genes was detected by real-time fluorescence quantitative PCR., Results: After 4 weeks, the hemoglobin (Hb) level and red blood cell (RBC) count were significantly lower in the iron deficient group compared with those in the control group. The iron levels in liver, spleen and serum in the iron deficient group were significantly lower than those in the control group. In reference to small intestine, the relative expression of each iron-related gene varied in the different tissues. Under the iron deficiency, the expression of these genes changed in a tissue-specific manner. The expression of most of the genes significantly correlated in intestine, spleen and lung, but few correlated in liver, heart and kidney., Conclusion: Findings from our study provides new understandings about the relative expression, regulation by iron and correlation among the mRNA expressions of transferrin receptors 1 and 2, divalent metal transporter 1, ferritin, iron regulation proteins 1 and 2, hereditary hemochromatosis protein, hepcidin, ferroportin 1 and hephaestin in intestine, liver, spleen, kidney, heart, and lung of rat., (Copyright © 2013 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

The mechanism(s) of iron flux across the brain microvasculature endothelial cells (BMVEC) of the blood-brain barrier remains unknown. Although both hephaestin (Hp) and the ferrous iron permease ferroportin (Fpn) have been identified in BMVEC, their roles in iron efflux have not been examined. Using a human BMVEC line (hBMVEC), we have demonstrated that these proteins are required for iron efflux from these cells. Expression of both Hp and Fpn protein was confirmed in hBMVEC by immunoblot and indirect immunofluorescence; we show that hBMVEC express soluble ceruloplasmin (Cp) transcript as well. Depletion of endogenous Hp and Cp via copper chelation leads to the reduction of hBMVEC Fpn protein levels as well as a complete inhibition of (59)Fe efflux. Both hBMVEC Fpn protein and (59)Fe efflux activity are restored upon incubation with 6.6 nm soluble plasma Cp. These results are independent of the source of cell iron, whether delivered as transferrin- or non-transferrin-bound (59)Fe. Our results demonstrate that iron efflux from hBMVEC Fpn requires the action of an exocytoplasmic ferroxidase, which can be either endogenous Hp or extracellular Cp.

The mammalian multicopper ferroxidases (MCFs) ceruloplasmin (CP), hephaestin (HEPH) and zyklopen (ZP) comprise a family of conserved enzymes that are essential for body iron homeostasis. Each of these enzymes contains six biosynthetically incorporated copper atoms which act as intermediate electron acceptors, and the oxidation of iron is associated with the four electron reduction of dioxygen to generate two water molecules. CP occurs in both a secreted and GPI-linked (membrane-bound) form, while HEPH and ZP each contain a single C-terminal transmembrane domain. These enzymes function to ensure the efficient oxidation of iron so that it can be effectively released from tissues via the iron export protein ferroportin and subsequently bound to the iron carrier protein transferrin in the blood. CP is particularly important in facilitating iron release from the liver and central nervous system, HEPH is the major MCF in the small intestine and is critical for dietary iron absorption, and ZP is important for normal hair development. CP and HEPH (and possibly ZP) function in multiple tissues. These proteins also play other (non-iron-related) physiological roles, but many of these are ill-defined. In addition to disrupting iron homeostasis, MCF dysfunction perturbs neurological and immune function, alters cancer susceptibility, and causes hair loss, but, despite their importance, how MCFs co-ordinately maintain body iron homeostasis and perform other functions remains incompletely understood.

Abstract Background Apo- (iron free) and holo- (iron bound) transferrin (Tf) participate in precise regulation of brain iron uptake at endothelial cells of the blood–brain barrier. Apo-Tf indicates an iron-deficient environment and stimulates iron release, while holo-Tf indicates an iron sufficient environment and suppresses additional iron release. Free iron is exported through ferroportin, with hephaestin as an aid to the process. Until now, the molecular mechanisms of apo- and holo-Tf influence on iron release was largely unknown. Methods Here we use a variety of cell culture techniques, including co-immunoprecipitation and proximity ligation assay, in iPSC-derived endothelial cells and HEK 293 cells to investigate the mechanism by which apo- and holo-Tf influence cellular iron release. Given the established role of hepcidin in regulating cellular iron release, we further explored the relationship of hepcidin to transferrin in this model. Results We demonstrate that holo-Tf induces the internalization of ferroportin through the established ferroportin degradation pathway. Furthermore, holo-Tf directly interacts with ferroportin, whereas apo-Tf directly interacts with hephaestin. Only pathophysiological levels of hepcidin disrupt the interaction between holo-Tf and ferroportin, but similar hepcidin levels are unable to interfere with the interaction between apo-Tf and hephaestin. The disruption of the holo-Tf and ferroportin interaction by hepcidin is due to hepcidin’s ability to more rapidly internalize ferroportin compared to holo-Tf. Conclusions These novel findings provide a molecular mechanism for apo- and holo-Tf regulation of iron release from endothelial cells. They further demonstrate how hepcidin impacts these protein–protein interactions, and offer a model for how holo-Tf and hepcidin cooperate to suppress iron release. These results expand on our previous reports on mechanisms mediating regulation of brain iron uptake to provide a more thorough understanding of the regulatory mechanisms mediating cellular iron release in general.

The skin protects the body against exogenous stressors. Its function is partially achieved by the permanent regeneration of the epidermis, which requires high metabolic activity and the shedding of superficial cells, leading to the loss of metabolites. Iron is involved in a plethora of important epidermal processes, including cellular respiration and detoxification of xenobiotics. Likewise, microorganisms on the surface of the skin depend on iron, which is supplied by the turnover of epithelial cells. Here, we review the metabolism of iron in the skin with a particular focus on the fate of iron in epidermal keratinocytes. The iron metabolism of the epidermis is controlled by genes that are differentially expressed in the inner and outer layers of the epidermis, establishing a system that supports the recycling of iron and counteracts the release of iron from the skin surface. Heme oxygenase-1 (HMOX1), ferroportin (SLC40A1) and hephaestin-like 1 (HEPHL1) are constitutively expressed in terminally differentiated keratinocytes and allow the recycling of iron from heme prior to the cornification of keratinocytes. We discuss the evidence for changes in the epidermal iron metabolism in diseases and explore promising topics of future studies of iron-dependent processes in the skin. [ABSTRACT FROM AUTHOR]

Background: Apo- (iron free) and holo- (iron bound) transferrin (Tf) participate in precise regulation of brain iron uptake at endothelial cells of the blood–brain barrier. Apo-Tf indicates an iron-deficient environment and stimulates iron release, while holo-Tf indicates an iron sufficient environment and suppresses additional iron release. Free iron is exported through ferroportin, with hephaestin as an aid to the process. Until now, the molecular mechanisms of apo- and holo-Tf influence on iron release was largely unknown. Methods: Here we use a variety of cell culture techniques, including co-immunoprecipitation and proximity ligation assay, in iPSC-derived endothelial cells and HEK 293 cells to investigate the mechanism by which apo- and holo-Tf influence cellular iron release. Given the established role of hepcidin in regulating cellular iron release, we further explored the relationship of hepcidin to transferrin in this model. Results: We demonstrate that holo-Tf induces the internalization of ferroportin through the established ferroportin degradation pathway. Furthermore, holo-Tf directly interacts with ferroportin, whereas apo-Tf directly interacts with hephaestin. Only pathophysiological levels of hepcidin disrupt the interaction between holo-Tf and ferroportin, but similar hepcidin levels are unable to interfere with the interaction between apo-Tf and hephaestin. The disruption of the holo-Tf and ferroportin interaction by hepcidin is due to hepcidin's ability to more rapidly internalize ferroportin compared to holo-Tf. Conclusions: These novel findings provide a molecular mechanism for apo- and holo-Tf regulation of iron release from endothelial cells. They further demonstrate how hepcidin impacts these protein–protein interactions, and offer a model for how holo-Tf and hepcidin cooperate to suppress iron release. These results expand on our previous reports on mechanisms mediating regulation of brain iron uptake to provide a more thorough understanding of the regulatory mechanisms mediating cellular iron release in general. [ABSTRACT FROM AUTHOR]

Previous studies demonstrated that quercetin, a polyphenolic compound, inhibits the transport of iron by down-regulation of ferroportin (FPN1), an iron export protein. We have previously demonstrated that activation of the PI3K signaling pathway by zinc stimulates the intestinal iron uptake and transport by stimulating the expression of iron regulatory protein 2 (IRP2) dependent divalent metal iron transporter 1 (DMT1, apical iron transporter) expression and caudal-related homeobox transcription factor 2 (CDX2) dependent hephaestin (HEPH, basolateral ferroxidase required for iron oxidation) expression, respectively. Since polyphenols are antagonists of the PI3K pathway, we hypothesized that quercetin might inhibit basolateral iron transport via the down-regulation of hephaestin (HEPH). Here in we investigated the effect of quercetin on iron uptake, transport, and expression of iron transporters in intestinal cells. In differentiated Caco-2 cells grown on permeable supports, quercetin inhibited the basolateral iron transport while increasing the iron uptake, possibly due to higher cellular retention. Further, quercetin down-regulated the protein and mRNA expression of HEPH and FPN1 but not that of IRP2 or DMT1. In addition, quercetin also abrogated the zinc-induced Akt, CDX2 phosphorylation, and HEPH expression. Together these results suggest that inhibition of iron transport by quercetin is mediated via the down-regulation of CDX2-dependent HEPH expression via inhibition of the PI3K pathway. [ABSTRACT FROM AUTHOR]

Background & aimsMulticopper ferroxidases (MCFs) facilitate intestinal iron absorption and systemic iron recycling, likely by a mechanism involving the oxidization of Fe2+ from the iron exporter ferroportin 1 for delivery to the circulating Fe3+ carrier transferrin. Hephaestin (HEPH), the only MCF known to be expressed in enterocytes, aids in the basolateral transfer of dietary iron to the blood. Mice lacking HEPH in the whole body (Heph-/- ) or intestine alone (Hephint/int ) exhibit defects in dietary iron absorption but still survive and grow. Circulating ceruloplasmin (CP) is the only other known MCF likely to interact with enterocytes. Our aim was to assess the effects of combined deletion of HEPH and CP on intestinal iron absorption and homeostasis in mice.MethodsMice lacking both HEPH and CP (Heph-/-Cp-/- ) and mice with whole-body knockout of CP and intestine-specific deletion of HEPH (Hephint/intCp-/- ) were generated and phenotyped.ResultsHeph-/-Cp-/- mice were severely anemic and had low serum iron, but they exhibited marked iron loading in duodenal enterocytes, the liver, heart, pancreas, and other tissues. Hephint/intCp-/- mice were moderately anemic (similar to Cp-/- mice) but were iron loaded only in the duodenum and liver, as in Hephint/int and Cp-/- mice, respectively. Both double knockout models absorbed iron in radiolabeled intestinal iron absorption studies, but the iron was inappropriately distributed, with an abnormally high percentage retained in the liver.ConclusionsThese studies indicate that HEPH and CP, and likely MCFs in general, are not essential for intestinal iron absorption but are required for proper systemic iron distribution. They also point to important extra-intestinal roles for HEPH in maintaining whole-body iron homeostasis.

Zinc stimulates intestinal iron absorption via induction of divalent metal ion transporter (DMT1) and hephaestin (HEPH). While the increase in DMT1 is mediated via a PI3K/IPR2 axis, the mechanisms of Zn-induced HEPH expression downstream of PI3K remain elusive. In the current study we probed the role of Caudal-related homeobox transcription factor-2 (CDX2) on Zn-induced HEPH expression. Zn treatment of Caco-2 cells increased CDX2 phosphorylation and HEPH protein and mRNA expression. siRNA-silencing of CDX2 inhibited Zn-induced HEPH expression. LY294002, an antagonist of PI3K inhibited Zn-induced phosphorylation of CDX2, and downstream HEPH expression. These results suggest that increased expression of HEPH in intestinal cells following Zn treatment is mediated via a PI3K-CDX2 pathway. • Zinc stimulated CDX2 phosphorylation and hephaestin expression in Caco-2 cells. • SiRNA silencing of CDX2 abrogated the zinc induced hephaestin expression. • Antagonist of PI3K abrogated the zinc induced CDX2 phosphorylation as well as hephaestin expression. • Zinc induced hephaestin expression is mediated via a Pi3K -CDX2 dependent mechanism. [ABSTRACT FROM AUTHOR]

Hephaestin (HEPH) belongs to a group of exocytoplasmic ferroxidases which contribute to cellular iron homeostasis by favouring its export. Down-regulation of HEPH expression, possibly by stimulating cell proliferation due to an increase in iron availability, has shown to correlate with poor survival in breast cancer. The lung is particularly sensitive to iron-induced oxidative stress, given the high oxygen tension present, however, HEPH distribution in lung cancer and its influence on prognosis have not been investigated yet. In this study we explored the prognostic value of HEPH and its expression pattern in the most prevalent histotypes of lung cancers, namely lung adenocarcinoma and lung squamous cell carcinoma. In silico analyses, based on UALCAN, Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan–Meier plotter bioinformatics, revealed a significant correlation between higher levels of HEPH expression and favorable prognosis, in both cancer histotypes. Moreover, TIMER web platform showed a statistically significant association between HEPH expression and cell elements belonging to the tumor microenvironment identified as endothelial cells and a subpopulation of cancer-associated fibroblasts, further confirmed by double immunohistochemical labeling with cell type specific markers. Taken together, these data shed a light on the complex mechanisms of local iron handling lung cancer can exploit to support tumorigenesis.

Hephaestin (HEPH) belongs to a group of exocytoplasmic ferroxidases which contribute to cellular iron homeostasis by favouring its export. Down-regulation of HEPH expression, possibly by stimulating cell proliferation due to an increase in iron availability, has shown to correlate with poor survival in breast cancer. The lung is particularly sensitive to iron-induced oxidative stress, given the high oxygen tension present, however, HEPH distribution in lung cancer and its influence on prognosis have not been investigated yet. In this study we explored the prognostic value of HEPH and its expression pattern in the most prevalent histotypes of lung cancers, namely lung adenocarcinoma and lung squamous cell carcinoma. In silico analyses, based on UALCAN, Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan–Meier plotter bioinformatics, revealed a significant correlation between higher levels of HEPH expression and favorable prognosis, in both cancer histotypes. Moreover, TIMER web platform showed a statistically significant association between HEPH expression and cell elements belonging to the tumor microenvironment identified as endothelial cells and a subpopulation of cancer-associated fibroblasts, further confirmed by double immunohistochemical labeling with cell type specific markers. Taken together, these data shed a light on the complex mechanisms of local iron handling lung cancer can exploit to support tumorigenesis. [ABSTRACT FROM AUTHOR]

Hephaestin (Hp) is a trans-membrane protein, which plays a critical role in intestinal iron absorption. Hp was originally identified as the gene responsible for the phenotype of sex-linked anaemia in the sla mouse. The mutation in the sla protein causes accumulation of dietary iron in duodenal cells, causing severe microcytic hypochromic anaemia. Although mucosal uptake of dietary iron is normal, export from the duodenum is inhibited. Hp is homologous to ceruloplasmin (Cp), a member of the family of multi copper ferroxidases (MCFs) and possesses ferroxidase activity that facilitates iron release from the duodenum and load onto the serum iron transport protein transferrin. In the present study, attempts were made to produce biologically active recombinant mouse hephaestin as a secretory form tagged with green fluorescent protein (GFP), Hpsec-GFP. Plasmid expressing Hpsec-GFP was constructed and transfected into COS and CHO cells. The GFP aided the monitoring expression in real time to select the best conditions to maximise expression and provided a tag for purifying and analysing Hpsec-GFP. The protein had detectable oxidase activity as shown by in-gel and solution-based assays. The methods described here can provide the basis for further work to probe the interaction of hephaestin with other proteins using complementary fluorescent tags on target proteins that would facilitate the fluorescence resonance energy transfer measurements, for example with transferrin or colocalisation studies, and help to discover more about hephaestin works at the molecular level. [ABSTRACT FROM AUTHOR]

Iron is one of the most important nonorganic substances that make life possible. Iron plays major roles in oxygen transport (eg, hemoglobin; ∼67% of total body iron [TBI]), short-term oxygen storage (eg, myoglobin; ∼3.5% of TBI), and energy generation (eg, cytochromes; ∼3% of TBI).1 Iron also serves vital roles in various nonheme-containing enzymes (∼2% of TBI). Figure 1 lists heme-containing and nonheme iron-containing proteins. TBI is controlled by the rate of iron absorption; there are no physiologic mechanisms to excrete excess iron. Iron deficiency has many adverse consequences, including anemia, and in children, behavioral and learning disorders.2-4 Iron excess is toxic to the body, harming the heart, liver, skin, pancreatic islet beta cells, bones, joints, and pituitary gland. Maintaining proper iron balance is essential for maintaining homeostasis and health. TBI in adults normally ranges between 3.5 and 5.0 g.5 A total of 75% of TBI is functional, and 25% is stored within cells as ferritin or hemosiderin. Ferritin contains 24 subunits of light chains (L chains; 19.7 kDa) and heavy chains (H chains; 21.1 kDa). The L chains are encoded on chromosome 19q13.33 and are 175 amino acids long. The H chains are encoded on chromosome 11q1 and are 183 amino acids long. Each ferritin molecule can contain as many as approximately 4500 ferric ions. Because the major role of iron is in hemoglobin synthesis, this review will focus on iron, iron transport, and hematopoiesis. [ABSTRACT FROM AUTHOR]

Iron accumulation has been implicated in degenerative retinal diseases. It can catalyze the production of damaging reactive oxygen species. Previous work has demonstrated iron accumulation in multiple retinal diseases, including age-related macular degeneration and diabetic retinopathy. In mice, systemic knockout of the ferroxidases ceruloplasmin (Cp) and hephaestin (Heph), which oxidize iron, results in retinal iron accumulation and iron-induced degeneration. To determine the role of Heph in the retina, we generated a neural retina-specific Heph knockout on a background of systemic Cp knockout. This resulted in elevated neural retina iron. Conversely, retinal ganglion cells had elevated transferrin receptor and decreased ferritin, suggesting diminished iron levels. The retinal degeneration observed in systemic Cp −/− , Heph −/− mice did not occur. These findings indicate that Heph has a local role in regulating neural retina iron homeostasis, but also suggest that preserved Heph function in either the RPE or systemically mitigates the degeneration phenotype observed in the systemic Cp −/− , Heph −/− mice. • Neural retina-specific deletion of hephaestin results in neural retina iron accumulation. • Retinal ganglion cells have elevated transferrin receptor. • Retinal degeneration does not occur in this mouse line. [ABSTRACT FROM AUTHOR]

Iron is essential for life, but excess iron can be toxic. As a potent free radical creator, iron generates hydroxyl radicals leading to significant oxidative stress. Since iron is not excreted from the body, it accumulates with age in tissues, including the retina, predisposing to age-related oxidative insult. Both hereditary and acquired retinal diseases are associated with increased iron levels. For example, retinal degenerations have been found in hereditary iron overload disorders, like aceruloplasminemia, Friedreich's ataxia, and pantothenate kinase-associated neurodegeneration. Similarly, mice with targeted mutation of the iron exporter ceruloplasmin and its homolog hephaestin showed age-related retinal iron accumulation and retinal degeneration with features resembling human age-related macular degeneration (AMD). Post mortem AMD eyes have increased levels of iron in retina compared to age-matched healthy donors. Iron accumulation in AMD is likely to result, in part, from inflammation, hypoxia, and oxidative stress, all of which can cause iron dysregulation. Fortunately, it has been demonstrated by in vitro and in vivo studies that iron in the retinal pigment epithelium (RPE) and retina is chelatable. Iron chelation protects photoreceptors and retinal pigment epithelial cells (RPE) in a variety of mouse models. This has therapeutic potential for diminishing iron-induced oxidative damage to prevent or treat AMD. [ABSTRACT FROM AUTHOR]

Iron is an essential nutrient, but, because it is toxic when present in excess, its levels in the body are tightly controlled. This regulation is affected by controlling the release of iron into the plasma., Most iron enters the plasma from macrophages, which recycle iron from senescent erythrocytes, but dietary iron absorption and the release of hepatocyte storage iron are other major sources., Cellular iron export is mediated by the membrane iron transporter ferroportin 1, in conjunction with an iron oxidase. Hephaestin provides this oxidase activity in the intestine, whereas ceruloplasmin is the oxidase used by most other tissues., The liver-derived peptide hepcidin binds to ferroportin 1 and removes it from the cell surface, thus reducing iron donation to the plasma. The levels of hepcidin, in turn, reflect body iron requirements., At the cellular level, ferroportin 1 can also be regulated independently of hepcidin by hypoxia-inducible factors and the iron regulatory proteins., The hepcidin-ferroportin axis plays a critical role in regulating body iron homeostasis. [ABSTRACT FROM AUTHOR]

Abstract: While intestinal cellular iron entry in vertebrates employs multiple routes including heme and non-heme routes, iron egress from these cells is exclusively channeled through the only known transporter, ferroportin. Reduced intestinal iron export in sex-linked anemia mice implicates hephaestin, a ferroxidase, in this process. Polarized cells are exposed to two distinct environments. Enterocytes contact the gut lumen via the apical surface of the cell, and through the basolateral surface, to the body. Previous studies indicate both local and systemic control of iron uptake. We hypothesized that differences in iron availability at the apical and/or basolateral surface may modulate iron uptake via cellular localization of hephaestin. We therefore characterized the localization of hephaestin in two models of polarized epithelial cell lines, MDCK and Caco2, with varying iron availability at the apical and basolateral surfaces. Our results indicate that hephaestin is expressed in a supra-nuclear compartment in non-polarized cells regardless of the iron status of the cells and in iron deficient and polarized cells. In polarized cells, we found that both apical (as FeSO4) and basolateral iron (as the ratio of apo-transferrin to holo-transferrin) affect mobilization of hephaestin from the supra-nuclear compartment. We find that the presence of apical iron is essential for relocalization of hephaestin to a cellular compartment in close proximity but not overlapping with the basolateral surface. Surface biotinylation studies indicate that hephaestin in the peri-basolateral location is accessible to the extra-cellular environment. These results support the hypothesis that hephaestin is involved in iron mobilization of iron from the intestine to circulation. [Copyright &y& Elsevier]

The process of placental iron transfer is an important physiological process during pregnancy. However, the molecular mechanism of placental iron transport has not been completely elucidated until now. Ferroportin 1 (FPN1) and hephaestin (Heph) have been identified as the important molecules involved in duodenal iron export. However, whether they participate in the placental iron efflux has been undefined until now. In this study, the BeWo cells were treated with desferrioxamine and Holo-transferrin human in different concentrations and harvested at 48 and 72 h. The mRNA expression of FPN1 and Heph was detected with quantitative real-time polymerase chain reaction, and the protein expression was detected with western blots. The results showed an up-regulated FPN1 expression with desferrioxamine treatment and down-regulated expression with Holo-transferrin human supplementation. However, the change of FPN1 expression at protein level was limited. Heph expression enhanced when cells were treated with desferrioxamine although the quantity of Heph expression was low. Heph expression showed no significant change with Holo-transferrin human supplementation. It indicates that FPN1 may participate in placental iron transport, and placental FPN1 expression is obviously not dependent on the iron regular element/iron regular protein regulation. An alternatively spliced FPN1 isoform that lacks an iron regular element may be the predominant expression in BeWo cells. It also demonstrates that Heph is active in placenta but may not play a key role in placental iron transport because it is not the main part of placental copper oxidase. Copyright © 2011 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background: Tanshinone IIA is a lipid-soluble compound extracted from Chinese herb Danshen which was commonly used in the treatment of cerebrovascular diseases. Brain iron homeostasis is very essential for normal physiological functions of neurons, and brain iron accumulation contributes to many neurological disorders. The present study was aimed to determine whether Tanshinone IIA protects against cerebral ischemic injury via maintaining brain iron homeostasis. Methods: Wistar Rats were orally administered with Tanshinone IIA (4, 20 and 100 mg/kg/d) for one week, and then subjected to cerebral ischemia by middle cerebral artery occlusion (MCAO) for 2 h. In vitro, cultured neurons were pretreated with Tanshinone IIA (2, 10 and 50 uM) for one week, and then exposed to hypoxia for 24 h. Brain iron content was determined by Flame Atomic Absorption Spectrophotometer and intracellular free iron level was measured by laser scanning Confocal microscopy. Expression levels of iron transport proteins including DMT1, TfR, Fpn1 and Heph were assessed by Western blot technique. Results: Tanshinone IIA pretreatment resulted in a significant reduction of cerebral infarct volume in MCAO rats. Compared with control rats, cerebral ischemia considerably augmented total iron content in the brain of MCAO rats, which was also effectively restricted by Tanshinone IIA pretreatment. MCAO rats exhibited the increased expression of iron influx proteins DMT1 and TfR, and the decreased expression of iron efflux proteins Fpn1 and Heph compared with control rats, which was responsible for elevated iron content in the ischemic brain. Tanshinone IIA pretreatment prevented the dysregulation of these four iron transport proteins and maintained brain iron homeostasis. In vitro studies also confirmed that Tanshinone IIA alleviated the hypoxia-induced decline of cell viability and the overload of intracellular free iron level in neurons through downregulating the expression of DMT1 and TfR, and upregulating the expression of Fpn1 and Heph. Conclusion: Tanshinone IIA protected brain tissues against ischemic and hypoxic damage in vivo and in vitro by mediating brain iron homeostasis which was associated with the downregulation of DMT1 and TfR, and the upregulation of Fpn1 and Heph. These results provide new insights into molecular mechanisms of ischemia-induced brain iron abnormalities and suggest maintaining brain iron homeostasis may be a novel therapeutic strategy for ischemic cerebrovascular diseases. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Interactions between the essential dietary metals, iron and copper, have been known for many years. This review highlights recent advances in iron-copper interactions with a focus on tissues and cell types important for regulating whole-body iron and copper homeostasis. Cells that mediate dietary assimilation (enterocytes) and storage and distribution (hepatocytes) of iron and copper are considered, along with the principal users (erythroid cells) and recyclers of red cell iron (reticuloendothelial macrophages). Interactions between iron and copper in the brain are also discussed. Many unanswered questions regarding the role of these metals and their interactions in health and disease emerge from this synopsis, highlighting extensive future research opportunities. [ABSTRACT FROM AUTHOR]

Abstract: Iron-induced oxidative stress is thought to play a crucial role in the pathogenesis of Parkinson''s disease (PD). Based on our previous in vivo experiments showing that down-regulation of the iron transporters ferroportin 1 (FP1) and hephaestin (HP) might account for the nigral iron accumulation in 6-hydroxydopamine (6-OHDA)-lesioned animal models, in this study we investigated whether FP1 and HP were involved in cellular iron accumulation and the underlying mechanisms in a cell model of PD. The findings showed that 6-OHDA induced FP1 and HP down-regulation, followed by decreased iron efflux and iron accumulation in primary ventral mesencephalic neurons and MES23.5 dopaminergic cells. Silencing of FP1 but not HP led to increased iron levels and aggravated reactive oxygen species generation in MES23.5 cells. Under iron-overloaded conditions, FP1 showed dose-dependent up-regulation, whereas HP showed no response, indicating their down-regulation was not due to the increased intracellular iron content. In 6-OHDA-treated cells, both iron-regulatory protein (IRP) 1 and IRP2 were up-regulated, and silencing of IRPs in MES23.5 cells dramatically blocked 6-OHDA-induced FP1 down-regulation and reversed HP down-regulation. These results suggest that FP1 but not HP contributes to 6-OHDA-induced intracellular iron accumulation, and down-regulation of FP1 and HP by 6-OHDA is IRPs-dependent. [Copyright &y& Elsevier]

Abstract: Aceruloplasminemia is an autosomal recessive disorder caused by mutations in the ceruloplasmin (CP) gene. It is characterized by iron accumulation in the brain and in visceral organs. However, little is known about the mechanism of iron transport in these regions. Adult CP null (CP−/−) mice show increased iron deposition in several regions of brain, such as the cerebellum and brainstem. In this study, we investigated the expression of the ceruloplasmin homolog hephaestin (Heph) in the brain of CP−/− mice as a function of age. In the cerebral cortex and caudate putamen of 80-week-old CP−/− mice, the expression of Heph increased significantly whilst iron levels remain normal [Patel BN, Dunn RJ, Jeong SY, Zhu Q, Julien JP, David S. Ceruloplasmin regulates iron levels in the CNS and prevents free radical injury. J Neurosci 2002;22(15):6578–6], indicating that Heph might compensate for the loss of CP. In contrast, the substantia nigra and cerebellum of 80-week-old CP−/− mice accumulate iron but do not express high levels or significant decrease of Heph, suggesting that Heph does not replace CP in these regions. These data suggest that Heph may compensate for the loss of CP in a region-specific manner. [Copyright &y& Elsevier]

Elevated iron levels in the substantia nigra (SN) participate in neuronal death in Parkinson's disease (PD). While the mechanisms underlying the increased iron are still unknown, some iron transport proteins may be involved. The nigral iron accumulation could be a result of either increased import or decreased export. The mechanisms of iron import have received considerable attention, but little is known about iron export mechanisms. Ferroportin1 (FP1) and hephaestin (HP), two newly discovered iron export proteins, cooperate in the iron export in the gut. Here, we investigated their expression in the SN of rats lesioned by 6-hydroxydopamine (6-OHDA). Using immunofluorescence, we showed that FP1 and HP were both expressed on astrocytes, microglia, oligodendrocytes and neurons in the SN. By immunohistochemistry, we showed that 1 day after 6-OHDA lesion, the expression of the two proteins decreased compared with the control. When rats began showing rotation behaviour induced by apomorphine, usually 6 weeks after 6-OHDA lesion, they are considered PD models. In these PD models, a further decrease in the two proteins was observed. Reverse transcriptase-polymerase chain reaction showed that the mRNA levels of FP1 and HP decreased 1 day after 6-OHDA lesion compared with the control, and further decrease was also observed in the PD model rats. These results show for the first time that FP1 and HP co-localize in the rat brain, and suggest that decreased expression of these transporters in the SN can account for the increased iron levels. [ABSTRACT FROM AUTHOR]