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Amereh M, Seyfoori A, Shojaei S, Lane S, Zhao T, Shokrollahi Barough M, Lum JJ, Walter P, Akbari M. Tumoroid Model Reveals Synergistic Impairment of Metabolism by Iron Chelators and Temozolomide in Chemo-Resistant Patient-derived Glioblastoma Cells. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2025; 12:e2412505. [PMID: 40285641 PMCID: PMC12120723 DOI: 10.1002/advs.202412505] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/07/2024] [Revised: 03/18/2025] [Indexed: 04/29/2025]
Abstract
Chemoresistance poses a significant clinical challenge in managing glioblastoma (GBM), limiting the long-term success of traditional treatments. Here, a 3D tumoroid model is used to investigate the metabolic sensitivity of temozolomide (TMZ)-resistant GBM cells to iron chelation by deferoxamine (DFO) and deferiprone (DFP). This work shows that TMZ-resistant GBM cells acquire stem-like characteristics, higher intracellular iron levels, higher expression of aconitase, and elevated reliance on oxidative phosphorylation and proteins associated with iron metabolism. Using a microphysiological model of GBM-on-a-chip consisting of extracellular matrix (ECM)-incorporated tumoroids, this work demonstrates that the combination of iron chelators with TMZ induces a synergistic effect on an in vitro tumoroid model of newly diagnosed and recurrent chemo-resistant patient-derived GBM and reduced their size and invasion. Investigating downstream metabolic variations reveal reduced intracellular iron, increased reactive oxygen species (ROS), upregulated hypoxia-inducible factor-1α, reduced viability, increased autophagy, upregulated ribonucleotide reductase (RRM2), arrested proliferation, and induced cell death in normoxic TMZ-resistant cells. Hypoxic cells, while showing similar results, display reduced responses to iron deficiency, less blebbing, and an induced autophagic flux, suggesting an adaptive mechanism associated with hypoxia. These findings show that co-treatment with iron chelators and TMZ induces a synergistic effect, making this combination a promising GBM therapy.
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Affiliation(s)
- Meitham Amereh
- Laboratory for Innovations in Micro Engineering (LiME)Department of Mechanical EngineeringUniversity of VictoriaVictoriaBCV8P 5C2Canada
| | - Amir Seyfoori
- Laboratory for Innovations in Micro Engineering (LiME)Department of Mechanical EngineeringUniversity of VictoriaVictoriaBCV8P 5C2Canada
| | - Shahla Shojaei
- Department of Human Anatomy and Cell ScienceMax Rady College of MedicineRady Faculty of Health SciencesUniversity of ManitobaWinnipegMBR3T 2N2Canada
| | - Sarah Lane
- Department of BiologyUniversity of VictoriaBCCanada
| | - Tian Zhao
- Trev and Joyce Deeley Research CentreBC CancerVictoriaBCV8R 6V5Canada
| | - Mahdieh Shokrollahi Barough
- Laboratory for Innovations in Micro Engineering (LiME)Department of Mechanical EngineeringUniversity of VictoriaVictoriaBCV8P 5C2Canada
| | - Julian J. Lum
- Trev and Joyce Deeley Research CentreBC CancerVictoriaBCV8R 6V5Canada
- Department of Biochemistry and MicrobiologyUniversity of VictoriaVictoriaBCV8W 2Y2Canada
| | | | - Mohsen Akbari
- Laboratory for Innovations in Micro Engineering (LiME)Department of Mechanical EngineeringUniversity of VictoriaVictoriaBCV8P 5C2Canada
- Terasaki Institute for Biomedical InnovationsLos AngelesCA91367USA
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Zhao N, Li S, Wu H, Wei D, Pu N, Wang K, Liu Y, Tao Y, Song Z. Ferroptosis: An Energetic Villain of Age-Related Macular Degeneration. Biomedicines 2025; 13:986. [PMID: 40299661 PMCID: PMC12024642 DOI: 10.3390/biomedicines13040986] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2025] [Revised: 04/01/2025] [Accepted: 04/14/2025] [Indexed: 05/01/2025] Open
Abstract
Iron homeostasis plays an important role in maintaining cellular homeostasis; however, excessive iron can promote the production of reactive oxygen species (ROS). Ferroptosis is iron-dependent programmed cell death that is characterized by excessive iron accumulation, elevated lipid peroxides, and the overproduction of ROS. The maintenance of iron homeostasis is contingent upon the activity of the transferrin receptor (TfR), ferritin (Ft), and ferroportin (FPn). In the retina, iron accumulation and lipid peroxidation can contribute to the development of age-related macular degeneration (AMD). This phenomenon can be explained by the occurrence of the Fenton reaction, in which the interaction between divalent iron and hydrogen peroxide leads to the generation of highly reactive hydroxyl radicals. The hydroxyl radicals exhibit a propensity to attack proteins, lipids, nucleic acids, and carbohydrates, thereby instigating oxidative damage and promoting lipid peroxidation. Ultimately, these processes culminate in cell death and retinal degeneration. In this context, a comprehensive understanding of the exact mechanisms underlying ferroptosis may hold significant importance for developing therapeutic interventions. This review summarizes recent findings on iron metabolism, cellular ferroptosis, and lipid metabolism in the aging retina. We also introduce developments in the therapeutic strategies using iron chelating agents. Further refinements of these knowledges would deepen our comprehension of the pathophysiology of AMD and advance the clinical management of degenerative retinopathy. A comprehensive search strategy was employed to identify relevant studies on the role of ferroptosis in AMD. We performed systematic searches of the PubMed and Web of Science electronic databases from inception to the current date. The keywords used in the search included "ferroptosis", "AMD", "age-related macular degeneration", "iron metabolism", "oxidative stress", and "ferroptosis pathways". Peer-reviewed articles, including original research, reviews, meta-analyses, and clinical studies, were included in this paper, with a focus on the molecular mechanisms of ferroptosis in AMDs. Studies not directly related to ferroptosis, iron metabolism, or oxidative stress in the context of AMD were excluded. Furthermore, articles that lacked sufficient data or were not peer-reviewed (e.g., conference abstracts, editorials, or opinion pieces) were not considered.
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Affiliation(s)
- Na Zhao
- Henan Eye Institute, Henan Eye Hospital, People’s Hospital of Zhengzhou University, Henan University School of Medicine, Henan Provincial People’s Hospital, Zhengzhou 450003, China; (N.Z.); (K.W.); (Y.L.)
- College of Medicine, Zhengzhou University, Zhengzhou 450001, China; (S.L.); (H.W.); (D.W.); (N.P.)
| | - Siyu Li
- College of Medicine, Zhengzhou University, Zhengzhou 450001, China; (S.L.); (H.W.); (D.W.); (N.P.)
| | - Hao Wu
- College of Medicine, Zhengzhou University, Zhengzhou 450001, China; (S.L.); (H.W.); (D.W.); (N.P.)
| | - Dong Wei
- College of Medicine, Zhengzhou University, Zhengzhou 450001, China; (S.L.); (H.W.); (D.W.); (N.P.)
| | - Ning Pu
- College of Medicine, Zhengzhou University, Zhengzhou 450001, China; (S.L.); (H.W.); (D.W.); (N.P.)
| | - Kexin Wang
- Henan Eye Institute, Henan Eye Hospital, People’s Hospital of Zhengzhou University, Henan University School of Medicine, Henan Provincial People’s Hospital, Zhengzhou 450003, China; (N.Z.); (K.W.); (Y.L.)
| | - Yashuang Liu
- Henan Eye Institute, Henan Eye Hospital, People’s Hospital of Zhengzhou University, Henan University School of Medicine, Henan Provincial People’s Hospital, Zhengzhou 450003, China; (N.Z.); (K.W.); (Y.L.)
| | - Ye Tao
- Henan Eye Institute, Henan Eye Hospital, People’s Hospital of Zhengzhou University, Henan University School of Medicine, Henan Provincial People’s Hospital, Zhengzhou 450003, China; (N.Z.); (K.W.); (Y.L.)
| | - Zongming Song
- Henan Eye Institute, Henan Eye Hospital, People’s Hospital of Zhengzhou University, Henan University School of Medicine, Henan Provincial People’s Hospital, Zhengzhou 450003, China; (N.Z.); (K.W.); (Y.L.)
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Ahmadi Badi S, Bereimipour A, Rohani P, Khatami S, Siadat SD. Interplay between gut microbiota and the master iron regulator, hepcidin, in the pathogenesis of liver fibrosis. Pathog Dis 2024; 82:ftae005. [PMID: 38555503 PMCID: PMC10990161 DOI: 10.1093/femspd/ftae005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2023] [Revised: 02/12/2024] [Accepted: 03/28/2024] [Indexed: 04/02/2024] Open
Abstract
INTRODUCTION There is a proven role for hepcidin and the composition of gut microbiota and its derivatives in the pathophysiology of liver fibrosis. AREA COVERED This review focuses on the literature search regarding the effect of hepcidin and gut microbiota on regulating liver physiology. We presented the regulating mechanisms of hepcidin expression and discussed the possible interaction between gut microbiota and hepcidin regulation. Furthermore, we investigated the importance of the hepcidin gene in biological processes and bacterial interactions using bioinformatics analysis. EXPERT OPINION One of the main features of liver fibrosis is iron accumulation in hepatic cells, including hepatocytes. This accumulation can induce an oxidative stress response, inflammation, and activation of hepatic stellate cells. Hepcidin is a crucial regulator of iron by targeting ferroportin expressed on hepatocytes, macrophages, and enterocytes. Various stimuli, such as iron load and inflammatory signals, control hepcidin regulation. Furthermore, a bidirectional relationship exists between iron and the composition and metabolic activity of gut microbiota. We explored the potential of gut microbiota to influence hepcidin expression and potentially manage liver fibrosis, as the regulation of iron metabolism plays a crucial role in this context.
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Affiliation(s)
- Sara Ahmadi Badi
- Biochemistry Department, Pasteur Institute of Iran, Tehran, 1963737611, Iran
- Pediatric Gastroenterology and Hepatology Research Center, Pediatrics Center of Excellence, Children's Medical Center, Tehran University of Medical Science, Tehran, 1416753955, Iran
| | - Ahmad Bereimipour
- Department of Biological Sciences and BioDiscovery Institute, University of North Texas, Denton, TX 76203, USA
| | - Pejman Rohani
- Pediatric Gastroenterology and Hepatology Research Center, Pediatrics Center of Excellence, Children's Medical Center, Tehran University of Medical Science, Tehran, 1416753955, Iran
| | - Shohreh Khatami
- Biochemistry Department, Pasteur Institute of Iran, Tehran, 1963737611, Iran
| | - Seyed Davar Siadat
- Microbiology Research Center, Pasteur Institute of Iran, Tehran, 1963737611, Iran
- Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran,1963737611, Iran
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Abstract
Iron accumulation in the CNS occurs in many neurological disorders. It can contribute to neuropathology as iron is a redox-active metal that can generate free radicals. The reasons for the iron buildup in these conditions are varied and depend on which aspects of iron influx, efflux, or sequestration that help maintain iron homeostasis are dysregulated. Iron was shown recently to induce cell death and damage via lipid peroxidation under conditions in which there is deficient glutathione-dependent antioxidant defense. This form of cell death is called ferroptosis. Iron chelation has had limited success in the treatment of neurological disease. There is therefore much interest in ferroptosis as it potentially offers new drugs that could be more effective in reducing iron-mediated lipid peroxidation within the lipid-rich environment of the CNS. In this review, we focus on the molecular mechanisms that induce ferroptosis. We also address how iron enters and leaves the CNS, as well as the evidence for ferroptosis in several neurological disorders. Finally, we highlight biomarkers of ferroptosis and potential therapeutic strategies.
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Affiliation(s)
- Samuel David
- Centre for Research in Neuroscience, and BRaIN Program, Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada
| | - Fari Ryan
- Centre for Research in Neuroscience, and BRaIN Program, Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada
| | - Priya Jhelum
- Centre for Research in Neuroscience, and BRaIN Program, Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada
| | - Antje Kroner
- Department of Neurosurgery, Medical College of Wisconsin, Milwaukee, WI, USA
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Leischner C, Marongiu L, Piotrowsky A, Niessner H, Venturelli S, Burkard M, Renner O. Relevant Membrane Transport Proteins as Possible Gatekeepers for Effective Pharmacological Ascorbate Treatment in Cancer. Antioxidants (Basel) 2023; 12:antiox12040916. [PMID: 37107291 PMCID: PMC10135768 DOI: 10.3390/antiox12040916] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Revised: 03/23/2023] [Accepted: 04/06/2023] [Indexed: 04/29/2023] Open
Abstract
Despite the increasing number of newly diagnosed malignancies worldwide, therapeutic options for some tumor diseases are unfortunately still limited. Interestingly, preclinical but also some clinical data suggest that the administration of pharmacological ascorbate seems to respond well, especially in some aggressively growing tumor entities. The membrane transport and channel proteins are highly relevant for the use of pharmacological ascorbate in cancer therapy and are involved in the transfer of active substances such as ascorbate, hydrogen peroxide, and iron that predominantly must enter malignant cells to induce antiproliferative effects and especially ferroptosis. In this review, the relevant conveying proteins from cellular surfaces are presented as an integral part of the efficacy of pharmacological ascorbate, considering the already known genetic and functional features in tumor tissues. Accordingly, candidates for diagnostic markers and therapeutic targets are mentioned.
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Affiliation(s)
- Christian Leischner
- Institute of Nutritional Sciences, Department of Nutritional Biochemistry, University of Hohenheim, Garbenstraße 30, 70599 Stuttgart, Germany
| | - Luigi Marongiu
- Institute of Nutritional Sciences, Department of Nutritional Biochemistry, University of Hohenheim, Garbenstraße 30, 70599 Stuttgart, Germany
- Department of Internal Medicine VIII, University Hospital Tuebingen, Otfried-Mueller-Straße 10, 72076 Tuebingen, Germany
| | - Alban Piotrowsky
- Institute of Nutritional Sciences, Department of Nutritional Biochemistry, University of Hohenheim, Garbenstraße 30, 70599 Stuttgart, Germany
| | - Heike Niessner
- Department of Dermatology, Division of Dermatooncology, University of Tuebingen, Liebermeisterstraße 25, 72076 Tuebingen, Germany
- Cluster of Excellence iFIT (EXC 2180) "Image Guided and Functionally Instructed Tumor Therapies", 72076 Tuebingen, Germany
| | - Sascha Venturelli
- Institute of Nutritional Sciences, Department of Nutritional Biochemistry, University of Hohenheim, Garbenstraße 30, 70599 Stuttgart, Germany
- Institute of Physiology, Department of Vegetative and Clinical Physiology, University of Tuebingen, Wilhelmstraße 56, 72074 Tuebingen, Germany
| | - Markus Burkard
- Institute of Nutritional Sciences, Department of Nutritional Biochemistry, University of Hohenheim, Garbenstraße 30, 70599 Stuttgart, Germany
| | - Olga Renner
- Institute of Nutritional Sciences, Department of Nutritional Biochemistry, University of Hohenheim, Garbenstraße 30, 70599 Stuttgart, Germany
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Zhang J, Song Y, Li Y, Lin HB, Fang X. Iron homeostasis in the heart: Molecular mechanisms and pharmacological implications. J Mol Cell Cardiol 2023; 174:15-24. [PMID: 36375319 DOI: 10.1016/j.yjmcc.2022.11.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/20/2022] [Revised: 11/01/2022] [Accepted: 11/02/2022] [Indexed: 11/13/2022]
Abstract
Iron is necessary for the life of practically all living things, yet it may also harm people toxically. Accordingly, humans and other mammals have evolved an effective and tightly regulatory system to maintain iron homeostasis in healthy tissues, including the heart. Iron deficiency is common in patients with heart failure, and is associated with worse prognosis in this population; while the prevalence of iron overload-related cardiovascular disorders is also increasing. Therefore, enhancing the therapy of patients with cardiovascular disorders requires a thorough understanding of iron homeostasis. Here, we give readers an overview of the fundamental mechanisms governing systemic iron homeostasis as well as the most recent knowledge about the intake, storage, use, and export of iron from the heart. Genetic mouse models used for investigation of iron metabolism in various in vivo scenarios are summarized and highlighted. We also go through different clinical conditions and therapeutic approaches that target cardiac iron dyshomeostasis. Finally, we conclude the review by outlining the present knowledge gaps and important open questions in this field in order to guide future research on cardiac iron metabolism.
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Affiliation(s)
- Jiawei Zhang
- Department of Nutrition and Toxicology, School of Public Health, Hangzhou Normal University, Hangzhou, China
| | - Yijing Song
- Department of Nutrition and Toxicology, School of Public Health, Hangzhou Normal University, Hangzhou, China
| | - You Li
- Department of Nutrition and Toxicology, School of Public Health, Hangzhou Normal University, Hangzhou, China
| | - Han-Bin Lin
- Zhongshan Institute for Drug Discovery, SIMM, CAS, Zhongshan, China; Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
| | - Xuexian Fang
- Department of Nutrition and Toxicology, School of Public Health, Hangzhou Normal University, Hangzhou, China; Key Laboratory of Elemene Class Anti-cancer Chinese Medicines of Zhejiang Province, Hangzhou Normal University, Hangzhou, China.
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Zhang X, Liu J, Wang H. The cGAS-STING-autophagy pathway: Novel perspectives in neurotoxicity induced by manganese exposure. ENVIRONMENTAL POLLUTION (BARKING, ESSEX : 1987) 2022; 315:120412. [PMID: 36240967 DOI: 10.1016/j.envpol.2022.120412] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/26/2022] [Revised: 08/28/2022] [Accepted: 10/07/2022] [Indexed: 06/16/2023]
Abstract
Chronic high-level heavy metal exposure increases the risk of developing different neurodegenerative diseases. Chronic excessive manganese (Mn) exposure is known to lead to neurodegenerative diseases. In addition, some evidence suggests that autophagy dysfunction plays an important role in the pathogenesis of various neurodegenerative diseases. Over the past decade, the DNA-sensing receptor cyclic GMP-AMP synthase (cGAS) and its downstream signal-efficient interferon gene stimulator (STING), as well as the molecular composition and regulatory mechanisms of this pathway have been well understood. The cGAS-STING pathway has emerged as a crucial mechanism to induce effective innate immune responses by inducing type I interferons in mammalian cells. Moreover, recent studies have found that Mn2+ is the second activator of the cGAS-STING pathway besides dsDNA, and inducing autophagy is a primitive function for the activation of the cGAS-STING pathway. However, overactivation of the immune response can lead to tissue damage. This review discusses the mechanism of neurotoxicity induced by Mn exposure from the cGAS-STING-autophagy pathway. Future work exploiting the cGAS-STING-autophagy pathway may provide a novel perspective for manganese neurotoxicity.
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Affiliation(s)
- Xin Zhang
- Department of Toxicology, School of Public Health, Lanzhou University, Lanzhou, Gansu, China
| | - Jingjing Liu
- Department of Toxicology, School of Public Health, Lanzhou University, Lanzhou, Gansu, China
| | - Hui Wang
- Department of Toxicology, School of Public Health, Lanzhou University, Lanzhou, Gansu, China.
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Duca L, Granata F, Di Pierro E, Brancaleoni V, Graziadei G, Nava I. Associated Effect of SLC40A1 and TMPRSS6 Polymorphisms on Iron Overload. Metabolites 2022; 12:metabo12100919. [PMID: 36295822 PMCID: PMC9612384 DOI: 10.3390/metabo12100919] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2022] [Revised: 09/15/2022] [Accepted: 09/27/2022] [Indexed: 11/16/2022] Open
Abstract
Mutations in the ferroportin (FPN) gene SLC40A1 alter iron recycling and cause disturbances in iron homeostasis. The variants of TMPRSS6 contribute to the development of iron deficiencies. In this study, we determined the role of FPN and TMPRSS6 gene polymorphisms in the modulation of iron homeostasis based on biochemical parameters. PCR analysis and sequencing were performed to determine the single nucleotide polymorphisms (SNPs) SLC40A1 c.44−24G>C (rs1439816), SLC40A1 c.663T>C (rs2304704), and TMPRSS6 c.2207T>C (rs855791). Hemoglobin concentration and iron status were determined by standard procedures. We studied 79 iron-loaded individuals for SLC40A1 polymorphisms. Interestingly, 35/79 individuals with SLC40A1 SNPs also carried a TMPRSS6 c.2207T>C polymorphism. The biochemical values of the iron overloaded individuals were compared to those of the individuals carrying TMPRSS6 SNPs and the healthy individuals (wild-type group). The ferritin concentration, transferrin saturation % (TS%), and hemoglobin concentration were significantly higher in the participants with FPN SNPs than in the other three groups. The ferritin concentration and TS% were higher in participants with both SLC40A1 and TMPRSS6 SNPs than in the TMPRSS6 and wild-type groups, while hemoglobin concentration was significantly higher than that in the TMPRSS6 SNP group only. The participants with TMPRSS6 SNPs had significantly lower ferritin concentration, TS%, and hemoglobin concentration than all the other groups. SLC40A1 and TMPRSS6 SNPs might act in the opposite direction, preventing the development of severe iron overload, and the modulation of the iron status by TMPRSS6 SNPs might provide protection.
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Iron overload impairs renal function and is associated with vascular calcification in rat aorta. Biometals 2022; 35:1325-1339. [PMID: 36178540 PMCID: PMC9674728 DOI: 10.1007/s10534-022-00449-7] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2022] [Accepted: 09/16/2022] [Indexed: 12/14/2022]
Abstract
Vascular calcification (VC) has been associated with a risk of cardiovascular diseases. Iron may play a critical role in progressive VC. Therefore, we investigated the effects of iron overload on the aorta of rats. A rat model of iron overload was established by intraperitoneal injection of Iron-Dextran. The levels of iron, calcium, and ALP activity were detected. Von Kossa staining and Perl's staining were conducted. The expression of iron metabolism-related and calcification related factors were examined in the aortic tissue of rats. The results showed serum and aortic tissue iron were increased induced by iron overload and excessive iron induced hepatic and renal damage. In iron overload rats, the expression of divalent metal transporter 1 (DMT1) and hepcidin were higher, but ferroportin1 (FPN1) was lower. Von Kossa staining demonstrated calcium deposition in the aorta of iron overload rats. The calcium content and ALP activity in serum and aortic tissue were increased and iron level in aortic tissue highly correlated with calcium content and ALP activity. The expressions of the osteogenic markers were increased while a decrease of Alpha-smooth muscle actin (α-SMA) in the aortic tissue of iron overload rats. IL-24 was increased during the calcification process induced by iron. Overall, we demonstrated excessive iron accumulation in the aortic tissue and induced organs damage. The iron metabolism-related factors were significantly changed during iron overload. Moreover, we found that iron overload leads to calcium deposition in aorta, playing a key role in the pathological process of VC by mediating osteoblast differentiation factors.
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Redox-Active Metal Ions and Amyloid-Degrading Enzymes in Alzheimer's Disease. Int J Mol Sci 2021; 22:ijms22147697. [PMID: 34299316 PMCID: PMC8307724 DOI: 10.3390/ijms22147697] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2021] [Revised: 07/11/2021] [Accepted: 07/16/2021] [Indexed: 12/11/2022] Open
Abstract
Redox-active metal ions, Cu(I/II) and Fe(II/III), are essential biological molecules for the normal functioning of the brain, including oxidative metabolism, synaptic plasticity, myelination, and generation of neurotransmitters. Dyshomeostasis of these redox-active metal ions in the brain could cause Alzheimer’s disease (AD). Thus, regulating the levels of Cu(I/II) and Fe(II/III) is necessary for normal brain function. To control the amounts of metal ions in the brain and understand the involvement of Cu(I/II) and Fe(II/III) in the pathogenesis of AD, many chemical agents have been developed. In addition, since toxic aggregates of amyloid-β (Aβ) have been proposed as one of the major causes of the disease, the mechanism of clearing Aβ is also required to be investigated to reveal the etiology of AD clearly. Multiple metalloenzymes (e.g., neprilysin, insulin-degrading enzyme, and ADAM10) have been reported to have an important role in the degradation of Aβ in the brain. These amyloid degrading enzymes (ADE) could interact with redox-active metal ions and affect the pathogenesis of AD. In this review, we introduce and summarize the roles, distributions, and transportations of Cu(I/II) and Fe(II/III), along with previously invented chelators, and the structures and functions of ADE in the brain, as well as their interrelationships.
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Zhao T, Guo X, Sun Y. Iron Accumulation and Lipid Peroxidation in the Aging Retina: Implication of Ferroptosis in Age-Related Macular Degeneration. Aging Dis 2021; 12:529-551. [PMID: 33815881 PMCID: PMC7990372 DOI: 10.14336/ad.2020.0912] [Citation(s) in RCA: 108] [Impact Index Per Article: 27.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2020] [Accepted: 09/12/2020] [Indexed: 01/19/2023] Open
Abstract
Iron is an essential component in many biological processes in the human body. It is critical for the visual phototransduction cascade in the retina. However, excess iron can be toxic. Iron accumulation and reduced efficiency of intracellular antioxidative defense systems predispose the aging retina to oxidative stress-induced cell death. Age-related macular degeneration (AMD) is characterized by retinal iron accumulation and lipid peroxidation. The mechanisms underlying AMD include oxidative stress-mediated death of retinal pigment epithelium (RPE) cells and subsequent death of retinal photoreceptors. Understanding the mechanism of the disruption of iron and redox homeostasis in the aging retina and AMD is crucial to decipher these mechanisms of cell death and AMD pathogenesis. The mechanisms of retinal cell death in AMD are an area of active investigation; previous studies have proposed several types of cell death as major mechanisms. Ferroptosis, a newly discovered programmed cell death pathway, has been associated with the pathogenesis of several neurodegenerative diseases. Ferroptosis is initiated by lipid peroxidation and is characterized by iron-dependent accumulation. In this review, we provide an overview of the mechanisms of iron accumulation and lipid peroxidation in the aging retina and AMD, with an emphasis on ferroptosis.
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Affiliation(s)
- Tantai Zhao
- 1Department of Ophthalmology, the Second Xiangya Hospital, Central South University, Changsha, Hunan, China.,2Hunan Clinical Research Center of Ophthalmic Disease, Changsha, Hunan, China
| | - Xiaojian Guo
- 1Department of Ophthalmology, the Second Xiangya Hospital, Central South University, Changsha, Hunan, China.,2Hunan Clinical Research Center of Ophthalmic Disease, Changsha, Hunan, China
| | - Yun Sun
- 1Department of Ophthalmology, the Second Xiangya Hospital, Central South University, Changsha, Hunan, China.,2Hunan Clinical Research Center of Ophthalmic Disease, Changsha, Hunan, China
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Evidence for dimerization of ferroportin in a human hepatic cell line using proximity ligation assays. Biosci Rep 2021; 40:222672. [PMID: 32301493 PMCID: PMC7201565 DOI: 10.1042/bsr20191499] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2019] [Revised: 04/08/2020] [Accepted: 04/16/2020] [Indexed: 12/30/2022] Open
Abstract
Mutations in the only known iron exporter ferroportin (FPN) in humans are associated with the autosomal dominantly inherited iron overload disorder ferroportin disease or type IV hereditary hemochromatosis (HH). While our knowledge of the central role of FPN in iron homeostasis has grown in the last 20 years, there exist some questions surrounding the structure and membrane topology of FPN with conflicting data on whether this receptor acts as a monomer or a multimer. To investigate and determine if FPN dimerization occurs in cells, we used novel tools including a variety of different FPN constructs expressing different tagged versions of the protein, a novel antibody that only detects cell surface FPN and proximity ligation assays. The results of the present study suggest that both the carboxy- and amino-termini of the FPN protein are intracellular. We also show that exogenously transfected FPN forms dimers; these dimers can be formed between the wild-type and mutant FPN proteins. This is the first study to examine the intracellular dimerization of FPN protein. Using proximity ligation assays, we show intracellular localization of FPN dimers and the interaction between FPN and hepcidin proteins as well. These results have important implications in the field of iron metabolism and add to our knowledge about FPN membrane topology and physiology of iron transport. This will be of importance in understanding the clinical implications of FPN mutations and of interest to future research aimed at targeting FPN expression to modulate iron homeostasis.
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Deshpande CN, Azucenas CR, Qiao B, Nomura N, Xin V, Font J, Iwata S, Ganz T, Nemeth E, Mackenzie B, Jormakka M. Isolation and thermal stabilization of mouse ferroportin. FEBS Open Bio 2021; 11:26-34. [PMID: 33190422 PMCID: PMC7780100 DOI: 10.1002/2211-5463.13039] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2020] [Revised: 10/30/2020] [Accepted: 11/12/2020] [Indexed: 12/03/2022] Open
Abstract
Ferroportin (Fpn) is an essential mammalian iron transporter that is negatively regulated by the hormone hepcidin. Our current molecular understanding of Fpn-mediated iron efflux and regulation is limited due to a lack of biochemical, biophysical and high-resolution structural studies. A critical step towards understanding the transport mechanism of Fpn is to obtain sufficient quantities of pure and stable protein for downstream studies. As such, we detail here an expression and purification protocol for mouse Fpn yielding milligram quantities of pure protein. We have generated deletion constructs exhibiting enhanced thermal stability and which retained iron-transport activity and hepcidin responsiveness, providing a platform for further biophysical studies of Fpn.
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Affiliation(s)
| | - Corbin R. Azucenas
- Department of Pharmacology & Systems PhysiologyUniversity of Cincinnati College of MedicineOH
- Medical Sciences Baccalaureate ProgramUniversity of Cincinnati College of MedicineOHUSA
| | - Bo Qiao
- Department of MedicineDavid Geffen School of MedicineUniversity of CaliforniaLos AngelesCAUSA
| | - Norimichi Nomura
- Department of Cell BiologyGraduate School of MedicineKyoto UniversityJapan
| | - Vicky Xin
- Structural Biology ProgramCentenary InstituteSydneyNSWAustralia
| | - Josep Font
- Structural Biology ProgramCentenary InstituteSydneyNSWAustralia
- Present address:
Transporter Biology GroupDiscipline of PharmacologySydney Medical SchoolUniversity of SydneyNSW2006Australia
| | - So Iwata
- Department of Cell BiologyGraduate School of MedicineKyoto UniversityJapan
| | - Tomas Ganz
- Department of MedicineDavid Geffen School of MedicineUniversity of CaliforniaLos AngelesCAUSA
- Department of PathologyDavid Geffen School of MedicineUniversity of CaliforniaLos AngelesCAUSA
| | - Elizabeta Nemeth
- Department of MedicineDavid Geffen School of MedicineUniversity of CaliforniaLos AngelesCAUSA
| | - Bryan Mackenzie
- Medical Sciences Baccalaureate ProgramUniversity of Cincinnati College of MedicineOHUSA
- Department of MedicineDavid Geffen School of MedicineUniversity of CaliforniaLos AngelesCAUSA
| | - Mika Jormakka
- Structural Biology ProgramCentenary InstituteSydneyNSWAustralia
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14
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Rishi G, Subramaniam VN. Biology of the iron efflux transporter, ferroportin. ADVANCES IN PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY 2020; 123:1-16. [PMID: 33485480 DOI: 10.1016/bs.apcsb.2020.10.005] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Iron, the most common metal in the earth, is also an essential component for almost all living organisms. While these organisms require iron for many biological processes, too much or too little iron itself poses many issues; this is most easily recognized in human beings. The control of body iron levels is thus an important metabolic process which is regulated essentially by controlling the expression, activity and levels of the iron transporter ferroportin. Ferroportin is the only known iron exporter. The function and activity of ferroportin is influenced by its interaction with the iron-regulatory peptide hepcidin, which itself is regulated by many factors. Here we review the current state of understanding of the mechanisms that regulate ferroportin and its function.
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Affiliation(s)
- Gautam Rishi
- Hepatogenomics Research Group, School of Biomedical Sciences, Faculty of Health, Queensland University of Technology (QUT), Brisbane, QLD, Australia
| | - V Nathan Subramaniam
- Hepatogenomics Research Group, School of Biomedical Sciences, Faculty of Health, Queensland University of Technology (QUT), Brisbane, QLD, Australia
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15
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The role of cellular iron deficiency in controlling iron export. Biochim Biophys Acta Gen Subj 2020; 1865:129829. [PMID: 33340587 DOI: 10.1016/j.bbagen.2020.129829] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2020] [Revised: 11/25/2020] [Accepted: 12/14/2020] [Indexed: 12/12/2022]
Abstract
BACKGROUND Iron export via the transport protein ferroportin (Fpn) plays a critical role in the regulation of dietary iron absorption and iron recycling in macrophages. Fpn plasma membrane expression is controlled by the hepatic iron-regulated hormone hepcidin in response to high iron availability and inflammation. Hepcidin binds to the central cavity of the Fpn transporter to block iron export either directly or by inducing Fpn internalization and lysosomal degradation. Here, we investigated whether iron deficiency affects Fpn protein turnover. METHODS We ectopically expressed Fpn in HeLa cells and used cycloheximide chase experiments to study basal and hepcidin-induced Fpn degradation under extracellular and intracellular iron deficiency. CONCLUSIONS/GENERAL SIGNIFICANCE We show that iron deficiency does not affect basal Fpn turnover but causes a significant delay in hepcidin-induced degradation when cytosolic iron levels are low. These data have important mechanistic implications supporting the hypothesis that iron export is required for efficient targeting of Fpn by hepcidin. Additionally, we show that Fpn degradation is not involved in protecting cells from intracellular iron deficiency.
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16
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Structural basis of ion transport and inhibition in ferroportin. Nat Commun 2020; 11:5686. [PMID: 33173040 PMCID: PMC7655804 DOI: 10.1038/s41467-020-19458-6] [Citation(s) in RCA: 48] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2020] [Accepted: 10/15/2020] [Indexed: 01/01/2023] Open
Abstract
Ferroportin is an iron exporter essential for releasing cellular iron into circulation. Ferroportin is inhibited by a peptide hormone, hepcidin. In humans, mutations in ferroportin lead to ferroportin diseases that are often associated with accumulation of iron in macrophages and symptoms of iron deficiency anemia. Here we present the structures of the ferroportin from the primate Philippine tarsier (TsFpn) in the presence and absence of hepcidin solved by cryo-electron microscopy. TsFpn is composed of two domains resembling a clamshell and the structure defines two metal ion binding sites, one in each domain. Both structures are in an outward-facing conformation, and hepcidin binds between the two domains and reaches one of the ion binding sites. Functional studies show that TsFpn is an electroneutral H+/Fe2+ antiporter so that transport of each Fe2+ is coupled to transport of two H+ in the opposite direction. Perturbing either of the ion binding sites compromises the coupled transport of H+ and Fe2+. These results establish the structural basis of metal ion binding, transport and inhibition in ferroportin and provide a blueprint for targeting ferroportin in pharmacological intervention of ferroportin diseases. Ferroportin is an iron exporter essential for releasing cellular iron into circulation and is inhibited by a peptide hormone, hepcidin. Here authors present cryo-EM structures of the ferroportin from the primate Philippine tarsier (TsFpn) with and without hepcidin and show that TsFpn is an electroneutral H+ /Fe2+ antiporter.
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17
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Tsuji Y. Transmembrane protein western blotting: Impact of sample preparation on detection of SLC11A2 (DMT1) and SLC40A1 (ferroportin). PLoS One 2020; 15:e0235563. [PMID: 32645092 PMCID: PMC7347119 DOI: 10.1371/journal.pone.0235563] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2020] [Accepted: 06/18/2020] [Indexed: 12/24/2022] Open
Abstract
Western blotting has been widely used for investigation of protein expression, posttranslational modifications, and interactions. Because western blotting usually involves heat-denaturation of samples prior to gel loading, clarification of detailed procedures for sample preparation have been omitted or neglected in many publications. We show here the case that even excellent primary antibodies failed to detect a specific protein of interest due to a routine heating practice of protein samples. We performed western blotting for transmembrane iron transporter proteins; SLC11A2 (divalent metal transporter 1, DMT1), SLC40A1 (ferroportin 1, Fpn1), and transferrin receptor-1 (TfR1), along with cytoplasmic iron storage protein ferritin H. Our results in 12 human culture cell lysates indicated that only unheated samples prior to gel loading gave rise to clear resolution of DMT1 protein, while heated samples (95°C, 5min) caused the loss of resolution due to DMT1 protein aggregates. Unheated samples also resulted in better resolution for Fpn1 and TfR1 western blots. Conversely, only heated samples allowed to detect ferritin H, otherwise ferritin polymers failed to get into the gel. Neither different lysis/sample loading buffers nor sonication improved the resolution of DMT1 and Fpn1 western blots. Thus, heating samples most critically affected the outcome of western blotting, suggesting the similar cases for thousands of other transmembrane and heat-sensitive proteins.
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Affiliation(s)
- Yoshiaki Tsuji
- Department of Biological Sciences, Toxicology Program, North Carolina State University, Raleigh, NC, United States of America
- * E-mail:
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18
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Piperno A, Pelucchi S, Mariani R. Inherited iron overload disorders. Transl Gastroenterol Hepatol 2020; 5:25. [PMID: 32258529 DOI: 10.21037/tgh.2019.11.15] [Citation(s) in RCA: 79] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/30/2019] [Accepted: 11/12/2019] [Indexed: 12/21/2022] Open
Abstract
Hereditary iron overload includes several disorders characterized by iron accumulation in tissues, organs, or even single cells or subcellular compartments. They are determined by mutations in genes directly involved in hepcidin regulation, cellular iron uptake, management and export, iron transport and storage. Systemic forms are characterized by increased serum ferritin with or without high transferrin saturation, and with or without functional iron deficient anemia. Hemochromatosis includes five different genetic forms all characterized by high transferrin saturation and serum ferritin, but with different penetrance and expression. Mutations in HFE, HFE2, HAMP and TFR2 lead to inadequate or severely reduced hepcidin synthesis that, in turn, induces increased intestinal iron absorption and macrophage iron release leading to tissue iron overload. The severity of hepcidin down-regulation defines the severity of iron overload and clinical complications. Hemochromatosis type 4 is caused by dominant gain-of-function mutations of ferroportin preventing hepcidin-ferroportin binding and leading to hepcidin resistance. Ferroportin disease is due to loss-of-function mutation of SLC40A1 that impairs the iron export efficiency of ferroportin, causes iron retention in reticuloendothelial cell and hyperferritinemia with normal transferrin saturation. Aceruloplasminemia is caused by defective iron release from storage and lead to mild microcytic anemia, low serum iron, and iron retention in several organs including the brain, causing severe neurological manifestations. Atransferrinemia and DMT1 deficiency are characterized by iron deficient erythropoiesis, severe microcytic anemia with high transferrin saturation and parenchymal iron overload due to secondary hepcidin suppression. Diagnosis of the different forms of hereditary iron overload disorders involves a sequential strategy that combines clinical, imaging, biochemical, and genetic data. Management of iron overload relies on two main therapies: blood removal and iron chelators. Specific therapeutic options are indicated in patients with atransferrinemia, DMT1 deficiency and aceruloplasminemia.
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Affiliation(s)
- Alberto Piperno
- Department of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy.,Centre for Rare Diseases, Disorder of Iron Metabolism, ASST-Monza, S. Gerardo Hospital, Monza, Italy
| | - Sara Pelucchi
- Department of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy
| | - Raffaella Mariani
- Centre for Rare Diseases, Disorder of Iron Metabolism, ASST-Monza, S. Gerardo Hospital, Monza, Italy
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19
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Vlasveld LT, Janssen R, Bardou-Jacquet E, Venselaar H, Hamdi-Roze H, Drakesmith H, Swinkels DW. Twenty Years of Ferroportin Disease: A Review or An Update of Published Clinical, Biochemical, Molecular, and Functional Features. Pharmaceuticals (Basel) 2019; 12:ph12030132. [PMID: 31505869 PMCID: PMC6789780 DOI: 10.3390/ph12030132] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2019] [Revised: 08/14/2019] [Accepted: 08/20/2019] [Indexed: 12/14/2022] Open
Abstract
Iron overloading disorders linked to mutations in ferroportin have diverse phenotypes in vivo, and the effects of mutations on ferroportin in vitro range from loss of function (LOF) to gain of function (GOF) with hepcidin resistance. We reviewed 359 patients with 60 ferroportin variants. Overall, macrophage iron overload and low/normal transferrin saturation (TSAT) segregated with mutations that caused LOF, while GOF mutations were linked to high TSAT and parenchymal iron accumulation. However, the pathogenicity of individual variants is difficult to establish due to the lack of sufficiently reported data, large inter-assay variability of functional studies, and the uncertainty associated with the performance of available in silico prediction models. Since the phenotypes of hepcidin-resistant GOF variants are indistinguishable from the other types of hereditary hemochromatosis (HH), these variants may be categorized as ferroportin-associated HH, while the entity ferroportin disease may be confined to patients with LOF variants. To further improve the management of ferroportin disease, we advocate for a global registry, with standardized clinical analysis and validation of the functional tests preferably performed in human-derived enterocytic and macrophagic cell lines. Moreover, studies are warranted to unravel the definite structure of ferroportin and the indispensable residues that are essential for functionality.
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Affiliation(s)
- L Tom Vlasveld
- Department of Internal Medicine, Haaglanden MC-Bronovo, 2597AX The Hague, The Netherlands
| | - Roel Janssen
- Department of Laboratory Medicine, Translational Metabolic Laboratory, Radboud University Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
| | - Edouard Bardou-Jacquet
- Liver Diseases Department, French Reference Centre for Rare Iron Overload Diseases of Genetic Origin, University Hospital Pontchaillou, 35033 Rennes, France
| | - Hanka Venselaar
- Centre for Molecular and Biomolecular Informatics, Radboud Institute for Molecular Life Sciences, Radboud, University Medical Center, P.O. Box 9191, 6500 HB Nijmegen, The Netherlands
| | - Houda Hamdi-Roze
- Molecular Genetics Department, French Reference Centre for Rare Iron Overload Diseases of Genetic Origin, University Hospital Pontchaillou, 35033 Rennes, France
| | - Hal Drakesmith
- MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX39DS, UK
| | - Dorine W Swinkels
- Department of Laboratory Medicine, Translational Metabolic Laboratory, Radboud University Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
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20
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New Insights into the Hepcidin-Ferroportin Axis and Iron Homeostasis in iPSC-Derived Cardiomyocytes from Friedreich's Ataxia Patient. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2019; 2019:7623023. [PMID: 31049138 PMCID: PMC6458886 DOI: 10.1155/2019/7623023] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/11/2018] [Accepted: 12/04/2018] [Indexed: 12/18/2022]
Abstract
Iron homeostasis in the cardiac tissue as well as the involvement of the hepcidin-ferroportin (HAMP-FPN) axis in this process and in cardiac functionality are not fully understood. Imbalance of iron homeostasis occurs in several cardiac diseases, including iron-overload cardiomyopathies such as Friedreich's ataxia (FRDA, OMIM no. 229300), a hereditary neurodegenerative disorder. Exploiting the induced pluripotent stem cells (iPSCs) technology and the iPSC capacity to differentiate into specific cell types, we derived cardiomyocytes of a FRDA patient and of a healthy control subject in order to study the cardiac iron homeostasis and the HAMP-FPN axis. Both CTR and FRDA iPSCs-derived cardiomyocytes express cardiac differentiation markers; in addition, FRDA cardiomyocytes maintain the FRDA-like phenotype. We found that FRDA cardiomyocytes show an increase in the protein expression of HAMP and FPN. Moreover, immunofluorescence analysis revealed for the first time an unexpected nuclear localization of FPN in both CTR and FRDA cardiomyocytes. However, the amount of the nuclear FPN was less in FRDA cardiomyocytes than in controls. These and other data suggest that iron handling and the HAMP-FPN axis regulation in FRDA cardiac cells are hampered and that FPN may have new, still not fully understood, functions. These findings underline the complexity of the cardiac iron homeostasis.
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21
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Choi EK, Nguyen TT, Iwase S, Seo YA. Ferroportin disease mutations influence manganese accumulation and cytotoxicity. FASEB J 2019; 33:2228-2240. [PMID: 30247984 PMCID: PMC6338638 DOI: 10.1096/fj.201800831r] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2018] [Accepted: 08/27/2018] [Indexed: 12/12/2022]
Abstract
Hemochromatosis is a frequent genetic disorder, characterized by the accumulation of excess iron across tissues. Mutations in the FPN1 gene, encoding a cell surface iron exporter [ferroportin (Fpn)], are responsible for hemochromatosis type 4, also known as ferroportin disease. Recently, Fpn has been implicated in the regulation of manganese (Mn), another essential nutrient required for numerous cellular enzymes. However, the roles of Fpn in Mn regulation remain ill-defined, and the impact of disease mutations on cellular Mn levels is unknown. Here, we provide evidence that Fpn can export Mn from cells into extracellular space. Fpn seems to play protective roles in Mn-induced cellular toxicity and oxidative stress. Finally, disease mutations interfere with the role of Fpn in controlling Mn levels as well as the stability of Fpn. These results define the function of Fpn as an exporter of both iron and Mn and highlight the potential involvement of Mn dysregulation in ferroportin disease.-Choi, E.-K., Nguyen, T.-T., Iwase, S., Seo, Y. A. Ferroportin disease mutations influence manganese accumulation and cytotoxicity.
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Affiliation(s)
- Eun-Kyung Choi
- Department of Nutritional Sciences, University of Michigan School of Public Health, Ann Arbor, Michigan, USA; and
| | - Trang-Tiffany Nguyen
- Department of Nutritional Sciences, University of Michigan School of Public Health, Ann Arbor, Michigan, USA; and
| | - Shigeki Iwase
- Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, USA
| | - Young Ah Seo
- Department of Nutritional Sciences, University of Michigan School of Public Health, Ann Arbor, Michigan, USA; and
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22
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Dlouhy AC, Bailey DK, Steimle BL, Parker HV, Kosman DJ. Fluorescence resonance energy transfer links membrane ferroportin, hephaestin but not ferroportin, amyloid precursor protein complex with iron efflux. J Biol Chem 2019; 294:4202-4214. [PMID: 30647129 DOI: 10.1074/jbc.ra118.005142] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2018] [Revised: 01/08/2019] [Indexed: 11/06/2022] Open
Abstract
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 Fe3+ 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 59Fe 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.
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Affiliation(s)
- Adrienne C Dlouhy
- From the Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York 14203
| | - Danielle K Bailey
- From the Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York 14203
| | - Brittany L Steimle
- From the Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York 14203
| | - Haley V Parker
- From the Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York 14203
| | - Daniel J Kosman
- From the Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York 14203
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23
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Zhou Y, Yang Y, Liu Y, Chang H, Liu K, Zhang X, Chang Y. Irp2 Knockout Causes Osteoporosis by Inhibition of Bone Remodeling. Calcif Tissue Int 2019; 104:70-78. [PMID: 30191282 DOI: 10.1007/s00223-018-0469-2] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/04/2018] [Accepted: 08/22/2018] [Indexed: 02/07/2023]
Abstract
It has been found that iron disorder may lead to osteoporosis. However, the mechanism has been little explored. In the present study, we try to investigate the effects of iron disorder on bone metabolism using Irp2 knockout (Irp2-/-) mice. Female Irp2-/- mice were used in this study. Bone mineral density (BMD) was measured by Micro-CT. Serum markers for bone turnover were measured by enzyme-linked immunosorbent assay. Content of iron was measured in bone and liver tissue, and Vitamin D 25-hydroxylase (CYP2R1) content was measured in liver tissue. Relative gene expression involved in iron export and uptake, and some genes involved in activities of osteoblast and osteoclast were all measured by real-time PCR and western blot. Compared to wild-type mice, Irp2-/- mice exhibited reduced BMD, bone iron deficiency, and hepatic iron overload. Serum levels of 25(OH)D3 and markers for bone formation such as bone alkaline phosphatase (Balp), bone-gla-protein (BGP), and type I collagen alpha1 chain (Col I α1) were decreased, while markers for bone resorption including cathepsin K (Ctsk) and tartrate-resistant acid phosphatase (Trap) were all significantly increased. Hepatic CYP2R1 level was decreased in Irp2-/- mice compared with wild-type control mice. Compared to wild-type C57BL6 control mice, the expression of genes involved in osteoblast activity such as Balp, BGP, and Col I α1 were all significantly decreased in bone tissue, while genes for osteoclast activity such as Ctsk and Trap were all markedly increased in Irp2-/- mice at mRNA level. Genes involved in iron storage, uptake, and exporting were also measured in bone tissue. Posttranscriptionally decreased ferritin (FTL), ferroportin 1 (FPN1), and increased transferrin receptor 1 (TfR1) gene expressions have been unexpectedly found in bone tissue of Irp2-/- mice. Irp2-/- mice exhibit reduced bone iron content and osteoporosis. Decreased circulating 25(OH)D3 levels promoted activity of osteoclast, while impaired activity of osteoblast may contribute to pathogenesis of osteoporosis. And, reduced bone iron content may not be totally caused by TfR1-dependent pathways.
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Affiliation(s)
- Yaru Zhou
- Department of Endocrinology, Third Hospital of Hebei Medical University, Shijiazhuang, China.
| | - Yu Yang
- Department of Endocrinology, Third Hospital of Hebei Medical University, Shijiazhuang, China
| | - Yan Liu
- Department of Endocrinology, Third Hospital of Hebei Medical University, Shijiazhuang, China
| | - Hengrui Chang
- Department of Orthopaedic Surgery, Third Hospital of Hebei Medical University, Shijiazhuang, China
| | - Kuanzhi Liu
- Department of Endocrinology, Third Hospital of Hebei Medical University, Shijiazhuang, China
| | - Xiaojuan Zhang
- Department of Endocrinology, Third Hospital of Hebei Medical University, Shijiazhuang, China
| | - Yanzhong Chang
- College of Life Science, Hebei Normal University, Shijiazhuang, 050051, China.
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24
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Abstract
The liver orchestrates systemic iron balance by producing and secreting hepcidin. Known as the iron hormone, hepcidin induces degradation of the iron exporter ferroportin to control iron entry into the bloodstream from dietary sources, iron recycling macrophages, and body stores. Under physiologic conditions, hepcidin production is reduced by iron deficiency and erythropoietic drive to increase the iron supply when needed to support red blood cell production and other essential functions. Conversely, hepcidin production is induced by iron loading and inflammation to prevent the toxicity of iron excess and limit its availability to pathogens. The inability to appropriately regulate hepcidin production in response to these physiologic cues underlies genetic disorders of iron overload and deficiency, including hereditary hemochromatosis and iron-refractory iron deficiency anemia. Moreover, excess hepcidin suppression in the setting of ineffective erythropoiesis contributes to iron-loading anemias such as β-thalassemia, whereas excess hepcidin induction contributes to iron-restricted erythropoiesis and anemia in chronic inflammatory diseases. These diseases have provided key insights into understanding the mechanisms by which the liver senses plasma and tissue iron levels, the iron demand of erythrocyte precursors, and the presence of potential pathogens and, importantly, how these various signals are integrated to appropriately regulate hepcidin production. This review will focus on recent insights into how the liver senses body iron levels and coordinates this with other signals to regulate hepcidin production and systemic iron homeostasis.
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25
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Doguer C, Ha JH, Collins JF. Intersection of Iron and Copper Metabolism in the Mammalian Intestine and Liver. Compr Physiol 2018; 8:1433-1461. [PMID: 30215866 DOI: 10.1002/cphy.c170045] [Citation(s) in RCA: 111] [Impact Index Per Article: 15.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Iron and copper have similar physiochemical properties; thus, physiologically relevant interactions seem likely. Indeed, points of intersection between these two essential trace minerals have been recognized for many decades, but mechanistic details have been lacking. Investigations in recent years have revealed that copper may positively influence iron homeostasis, and also that iron may antagonize copper metabolism. For example, when body iron stores are low, copper is apparently redistributed to tissues important for regulating iron balance, including enterocytes of upper small bowel, the liver, and blood. Copper in enterocytes may positively influence iron transport, and hepatic copper may enhance biosynthesis of a circulating ferroxidase, ceruloplasmin, which potentiates iron release from stores. Moreover, many intestinal genes related to iron absorption are transactivated by a hypoxia-inducible transcription factor, hypoxia-inducible factor-2α (HIF2α), during iron deficiency. Interestingly, copper influences the DNA-binding activity of the HIF factors, thus further exemplifying how copper may modulate intestinal iron homeostasis. Copper may also alter the activity of the iron-regulatory hormone hepcidin. Furthermore, copper depletion has been noted in iron-loading disorders, such as hereditary hemochromatosis. Copper depletion may also be caused by high-dose iron supplementation, raising concerns particularly in pregnancy when iron supplementation is widely recommended. This review will cover the basic physiology of intestinal iron and copper absorption as well as the metabolism of these minerals in the liver. Also considered in detail will be current experimental work in this field, with a focus on molecular aspects of intestinal and hepatic iron-copper interplay and how this relates to various disease states. © 2018 American Physiological Society. Compr Physiol 8:1433-1461, 2018.
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Affiliation(s)
- Caglar Doguer
- Food Science and Human Nutrition Department, University of Florida, Florida, Gainesville, USA.,Nutrition and Dietetics Department, Namık Kemal University, Tekirdag, Turkey
| | - Jung-Heun Ha
- Food Science and Human Nutrition Department, University of Florida, Florida, Gainesville, USA.,Department of Food and Nutrition, Chosun University Note: Caglar Doguer and Jung-Heun Ha have contributed equally to this work., Gwangju, Korea
| | - James F Collins
- Food Science and Human Nutrition Department, University of Florida, Florida, Gainesville, USA
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26
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Expression of iron-regulators in the bone tissue of rats with and without iron overload. Biometals 2018; 31:749-757. [PMID: 30027360 DOI: 10.1007/s10534-018-0133-3] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2018] [Accepted: 05/21/2018] [Indexed: 12/14/2022]
Abstract
Recently, more and more studies indicate that iron overload would cause osteopenia or osteoporosis. However, the molecular mechanism of it remains unclear. Moreover, very little is known about the iron metabolism in bone tissue at present. Therefore, the mRNA expression of iron-regulators, transferrin receptor1 (Tfr1), divalent metal transporter1 (Dmt1 + IRE and Dmt1 - IRE), ferritin (FtH and FtL), and ferroportin1 (Ireg1), and the localization of ferroportin1 protein were examined in the bone tissue of rats. In addition, the mRNA expression of each gene was compared between groups of rats with and without iron overload. The results showed that ferroportin1 protein was localized in the cytoplasm of osteoblast, osteocyte, chondrocyte and osteoclast of rats' femur. The six iron-regulatory genes, Tfr1, ferritin (FtH and FtL), (Dmt1 + IRE and Dmt1 - IRE) and ferroportin1 (Ireg1), were found in femurs of rats. In addition, significantly up-regulated expression of FtH and FtL mRNA, and markedly down-regulated expression of Tfr1, Dmt1 + IRE and Ireg1 mRNA, were observed in the iron overload group compared with the control group. The result indicates that ferroportin1 protein is localized in the cytoplasm of bone cells of rats. Tfr1, Dmt1, ferritin and ferroportin1 exist in bone tissue of rats, and they may be involved in the pathological process of iron overload-induced bone lesion.
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Collins JF, Flores SR, Wang X, Anderson GJ. Mechanisms and Regulation of Intestinal Iron Transport. PHYSIOLOGY OF THE GASTROINTESTINAL TRACT 2018:1451-1483. [DOI: 10.1016/b978-0-12-809954-4.00060-8] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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Structure-function analysis of ferroportin defines the binding site and an alternative mechanism of action of hepcidin. Blood 2017; 131:899-910. [PMID: 29237594 DOI: 10.1182/blood-2017-05-786590] [Citation(s) in RCA: 237] [Impact Index Per Article: 29.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2017] [Accepted: 11/30/2017] [Indexed: 02/07/2023] Open
Abstract
Nonclassical ferroportin disease (FD) is a form of hereditary hemochromatosis caused by mutations in the iron transporter ferroportin (Fpn), resulting in parenchymal iron overload. Fpn is regulated by the hormone hepcidin, which induces Fpn endocytosis and cellular iron retention. We characterized 11 clinically relevant and 5 nonclinical Fpn mutations using stably transfected, inducible isogenic cell lines. All clinical mutants were functionally resistant to hepcidin as a consequence of either impaired hepcidin binding or impaired hepcidin-dependent ubiquitination despite intact hepcidin binding. Mapping the residues onto 2 computational models of the human Fpn structure indicated that (1) mutations that caused ubiquitination-resistance were positioned at helix-helix interfaces, likely preventing the hepcidin-induced conformational change, (2) hepcidin binding occurred within the central cavity of Fpn, (3) hepcidin interacted with up to 4 helices, and (4) hepcidin binding should occlude Fpn and interfere with iron export independently of endocytosis. We experimentally confirmed hepcidin-mediated occlusion of Fpn in the absence of endocytosis in multiple cellular systems: HEK293 cells expressing an endocytosis-defective Fpn mutant (K8R), Xenopus oocytes expressing wild-type or K8R Fpn, and mature human red blood cells. We conclude that nonclassical FD is caused by Fpn mutations that decrease hepcidin binding or hinder conformational changes required for ubiquitination and endocytosis of Fpn. The newly documented ability of hepcidin and its agonists to occlude iron transport may facilitate the development of broadly effective treatments for hereditary iron overload disorders.
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Ross SL, Biswas K, Rottman J, Allen JR, Long J, Miranda LP, Winters A, Arvedson TL. Identification of Antibody and Small Molecule Antagonists of Ferroportin-Hepcidin Interaction. Front Pharmacol 2017; 8:838. [PMID: 29209212 PMCID: PMC5702341 DOI: 10.3389/fphar.2017.00838] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2017] [Accepted: 11/03/2017] [Indexed: 12/21/2022] Open
Abstract
The iron exporter ferroportin and its ligand, the hormone hepcidin, control fluxes of stored and recycled iron for use in a variety of essential biochemical processes. Inflammatory disorders and malignancies are often associated with high hepcidin levels, leading to ferroportin down-regulation, iron sequestration in tissue macrophages and subsequent anemia. The objective of this research was to develop reagents to characterize the expression of ferroportin, the interaction between ferroportin and hepcidin, as well as to identify novel ferroportin antagonists capable of maintaining iron export in the presence of hepcidin. Development of investigative tools that enabled cell-based screening assays is described in detail, including specific and sensitive monoclonal antibodies that detect endogenously-expressed human and mouse ferroportin and fluorescently-labeled chemically-synthesized human hepcidin. Large and small molecule antagonists inhibiting hepcidin-mediated ferroportin internalization were identified, and unique insights into the requirements for interaction between these two key iron homeostasis molecules are provided.
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Affiliation(s)
- Sandra L Ross
- Department of Oncology Research, Amgen Inc., Thousand Oaks, CA, United States
| | - Kaustav Biswas
- Department of Hybrid Modality Engineering, Amgen Inc., Thousand Oaks, CA, United States
| | - James Rottman
- Department of Comparative Biology and Safety Sciences, Amgen Inc., Thousand Oaks, CA, United States
| | - Jennifer R Allen
- Department of Medicinal Chemistry, Amgen Inc., Thousand Oaks, CA, United States
| | - Jason Long
- Department of Hybrid Modality Engineering, Amgen Inc., Thousand Oaks, CA, United States
| | - Les P Miranda
- Department of Hybrid Modality Engineering, Amgen Inc., Thousand Oaks, CA, United States
| | - Aaron Winters
- Department of Therapeutic Discovery, Amgen Inc., Thousand Oaks, CA, United States
| | - Tara L Arvedson
- Department of Oncology Research, Amgen Inc., Thousand Oaks, CA, United States
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Pietrangelo A. Ferroportin disease: pathogenesis, diagnosis and treatment. Haematologica 2017; 102:1972-1984. [PMID: 29101207 PMCID: PMC5709096 DOI: 10.3324/haematol.2017.170720] [Citation(s) in RCA: 101] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2017] [Accepted: 09/25/2017] [Indexed: 12/14/2022] Open
Abstract
Ferroportin Disease (FD) is an autosomal dominant hereditary iron loading disorder associated with heterozygote mutations of the ferroportin-1 (FPN) gene. It represents one of the commonest causes of genetic hyperferritinemia, regardless of ethnicity. FPN1 transfers iron from the intestine, macrophages and placenta into the bloodstream. In FD, loss-of-function mutations of FPN1 limit but do not impair iron export in enterocytes, but they do severely affect iron transfer in macrophages. This leads to progressive and preferential iron trapping in tissue macrophages, reduced iron release to serum transferrin (i.e. inappropriately low transferrin saturation) and a tendency towards anemia at menarche or after intense bloodletting. The hallmark of FD is marked iron accumulation in hepatic Kupffer cells. Numerous FD-associated mutations have been reported worldwide, with a few occurring in different populations and some more commonly reported (e.g. Val192del, A77D, and G80S). FPN1 polymorphisms also represent the gene variants most commonly responsible for hyperferritinemia in Africans. Differential diagnosis includes mainly hereditary hemochromatosis, the syndrome commonly due to either HFE or TfR2, HJV, HAMP, and, in rare instances, FPN1 itself. Here, unlike FD, hyperferritinemia associates with high transferrin saturation, iron-spared macrophages, and progressive parenchymal cell iron load. Abdominal magnetic resonance imaging (MRI), the key non-invasive diagnostic tool for the diagnosis of FD, shows the characteristic iron loading SSL triad (spleen, spine and liver). A non-aggressive phlebotomy regimen is recommended, with careful monitoring of transferrin saturation and hemoglobin due to the risk of anemia. Family screening is mandatory since siblings and offspring have a 50% chance of carrying the pathogenic mutation.
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Affiliation(s)
- Antonello Pietrangelo
- Center for Hemochromatosis, Department of Internal Medicine II, University of Modena and Reggio Emilia Policlinico, Modena, Italy
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Hamp1 but not Hamp2 regulates ferroportin in fish with two functionally distinct hepcidin types. Sci Rep 2017; 7:14793. [PMID: 29093559 PMCID: PMC5665920 DOI: 10.1038/s41598-017-14933-5] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2017] [Accepted: 10/18/2017] [Indexed: 01/01/2023] Open
Abstract
Hepcidin is a small cysteine rich peptide that regulates the sole known cellular iron exporter, ferroportin, effectively controlling iron metabolism. Contrary to humans, where a single hepcidin exists, many fish have two functionally distinct hepcidin types, despite having a single ferroportin gene. This raises the question of whether ferroportin is similarly regulated by the iron regulator Hamp1 and the antimicrobial Hamp2. In sea bass (Dicentrarchus labrax), iron overload prompted a downregulation of ferroportin, associated with an upregulation of hamp1, whereas an opposite response was observed during anemia, with no changes in hamp2 in either situation. During infection, ferroportin expression decreased, indicating iron withholding to avoid microbial proliferation. In vivo administration of Hamp1 but not Hamp2 synthetic peptides caused significant reduction in ferroportin expression, indicating that in teleost fish with two hepcidin types, ferroportin activity is mediated through the iron-regulator Hamp1, and not through the dedicated antimicrobial Hamp2. Additionally, in vitro treatment of mouse macrophages with fish Hamp1 but not Hamp2 caused a decrease in ferroportin levels. These results raise questions on the evolution of hepcidin and ferroportin functional partnership and open new possibilities for the pharmaceutical use of selected fish Hamp2 hepcidins during infections, with no impact on iron homeostasis.
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Wallace DF, McDonald CJ, Ostini L, Iser D, Tuckfield A, Subramaniam VN. The dynamics of hepcidin-ferroportin internalization and consequences of a novel ferroportin disease mutation. Am J Hematol 2017; 92:1052-1061. [PMID: 28681497 DOI: 10.1002/ajh.24844] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2017] [Revised: 06/29/2017] [Accepted: 06/30/2017] [Indexed: 12/31/2022]
Abstract
The hepcidin-ferroportin axis underlies the pathophysiology of many iron-associated disorders and is a key target for the development of therapeutics for treating iron-associated disorders. The aims of this study were to investigate the dynamics of hepcidin-mediated ferroportin internalization and the consequences of a novel disease-causing mutation on ferroportin function. Specific reagents for ferroportin are limited; we developed and characterized antibodies against the largest extracellular loop of ferroportin and developed a novel cell-based assay for studying hepcidin-ferroportin function. We show that hepcidin-mediated ferroportin internalization is a rapid process and could be induced using low concentrations of hepcidin. Targeted next-generation sequencing utilizing an iron metabolism gene panel developed in our group identified a novel ferroportin p.D84E variant in a patient with iron overload. Wild-type and mutant ferroportin constructs were generated, transfected into HEK293 cells and analysed using an all-in-one flow-cytometry-based assay to study the effects on hepcidin-mediated internalization and iron transport. Consistent with the classical phenotype of ferroportin disease, the p.D84E mutation results in an inability to transport iron and hepcidin insensitivity. These results validate a recently proposed 3D-structural model of ferroportin and highlight the significance of this variant in the structure and function of ferroportin. Our novel ferroportin antibody and assay will be valuable tools for investigating the regulation of hepcidin/ferroportin function and the development of novel approaches for the therapeutic modulation of iron homeostasis.
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Affiliation(s)
- Daniel F. Wallace
- Institute of Health and Biomedical Innovation and School of Biomedical Sciences. Queensland University of Technology (QUT); Brisbane Queensland Australia
- Membrane Transport Laboratory; QIMR Berghofer Medical Research Institute; Brisbane Queensland Australia
| | - Cameron J. McDonald
- Membrane Transport Laboratory; QIMR Berghofer Medical Research Institute; Brisbane Queensland Australia
| | - Lesa Ostini
- Membrane Transport Laboratory; QIMR Berghofer Medical Research Institute; Brisbane Queensland Australia
| | - David Iser
- Department of Gastroenterology; St Vincent's Hospital; Fitzroy Victoria Australia
| | | | - V. Nathan Subramaniam
- Institute of Health and Biomedical Innovation and School of Biomedical Sciences. Queensland University of Technology (QUT); Brisbane Queensland Australia
- Membrane Transport Laboratory; QIMR Berghofer Medical Research Institute; Brisbane Queensland Australia
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Sabelli M, Montosi G, Garuti C, Caleffi A, Oliveto S, Biffo S, Pietrangelo A. Human macrophage ferroportin biology and the basis for the ferroportin disease. Hepatology 2017; 65:1512-1525. [PMID: 28027576 PMCID: PMC5413859 DOI: 10.1002/hep.29007] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/10/2016] [Accepted: 12/15/2016] [Indexed: 01/01/2023]
Abstract
Ferroportin (FPN1) is the sole iron exporter in mammals, but its cell-specific function and regulation are still elusive. This study examined FPN1 expression in human macrophages, the cells that are primarily responsible on a daily basis for plasma iron turnover and are central in the pathogenesis of ferroportin disease (FD), the disease attributed to lack-of-function FPN1 mutations. We characterized FPN1 protein expression and traffic by confocal microscopy, western blotting, gel filtration, and immunoprecipitation studies in macrophages from control blood donors (donor) and patients with either FPN1 p.A77D, p.G80S, and p.Val162del lack-of-function or p.A69T gain-of-function mutations. We found that in normal macrophages, FPN1 cycles in the early endocytic compartment does not multimerize and is promptly degraded by hepcidin (Hepc), its physiological inhibitor, within 3-6 hours. In FD macrophages, endogenous FPN1 showed a similar localization, except for greater accumulation in lysosomes. However, in contrast with previous studies using overexpressed mutant protein in cell lines, FPN1 could still reach the cell surface and be normally internalized and degraded upon exposure to Hepc. However, when FD macrophages were exposed to large amounts of heme iron, in contrast to donor and p.A69T macrophages, FPN1 could no longer reach the cell surface, leading to intracellular iron retention. CONCLUSION FPN1 cycles as a monomer within the endocytic/plasma membrane compartment and responds to its physiological inhibitor, Hepc, in both control and FD cells. However, in FD, FPN1 fails to reach the cell surface when cells undergo high iron turnover. Our findings provide a basis for the FD characterized by a preserved iron transfer in the enterocytes (i.e., cells with low iron turnover) and iron retention in cells exposed to high iron flux, such as liver and spleen macrophages. (Hepatology 2017;65:1512-1525).
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Affiliation(s)
- Manuela Sabelli
- Division of Internal Medicine 2 and Center for HemochromatosisUniversity Hospital of ModenaModenaItaly
| | - Giuliana Montosi
- Division of Internal Medicine 2 and Center for HemochromatosisUniversity Hospital of ModenaModenaItaly
| | - Cinzia Garuti
- Division of Internal Medicine 2 and Center for HemochromatosisUniversity Hospital of ModenaModenaItaly
| | - Angela Caleffi
- Division of Internal Medicine 2 and Center for HemochromatosisUniversity Hospital of ModenaModenaItaly
| | | | - Stefano Biffo
- INGM, ‘Romeo ed Enrica Invernizzi’MilanoItaly
- Department of BiosciencesUniversity of MilanMilanItaly
| | - Antonello Pietrangelo
- Division of Internal Medicine 2 and Center for HemochromatosisUniversity Hospital of ModenaModenaItaly
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Zhao MH, Liang S, Guo J, Choi JW, Kim NH, Lu WF, Cui XS. Analysis of Ferrous on Ten-Eleven Translocation Activity and Epigenetic Modifications of Early Mouse Embryos by Fluorescence Microscopy. MICROSCOPY AND MICROANALYSIS : THE OFFICIAL JOURNAL OF MICROSCOPY SOCIETY OF AMERICA, MICROBEAM ANALYSIS SOCIETY, MICROSCOPICAL SOCIETY OF CANADA 2016; 22:342-348. [PMID: 26947808 DOI: 10.1017/s1431927616000040] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/05/2023]
Abstract
Iron is an essential trace element that plays important roles in the cellular function of all organs and systems. However, the function of Fe(II) in mammalian embryo development is unknown. In this study, we investigated the role of Fe(II) during preimplantation embryo development. Depletion of Fe(II) using thiosemicarbazone-24 (TSC24), a specific Fe(II) chelator, rescued quenching of the Fe(II)-sensitive fluorophore phen green-SK. After in vitro fertilization, TSC24 significantly reduced the cleavage rate as well as blastocyst formation. The hatch rate of blastocysts was also reduced with 1 pM TSC24 treatment (20.25±1.86 versus 42.28±12.96%, p<0.05). Blastocysts were cultured in leukemia inhibitory factor-free mouse embryonic stem cell culture medium with or without TSC24, and those with depleted Fe(II) displayed delayed attachment and lost the ability to induce embryoid body formation. To further explore the mechanism of Fe(II) in embryo development, we assessed the expression of 5-hydroxymethylcytosine (5hmC) and OCT4 in the pronuclear and blastocyst stages, respectively. We observed that Fe(II) reduced 5hmC and OCT4 expression, which could be explained by low ten-eleven translocation (TET) enzyme activity induced by TSC24 treatment. These findings demonstrate that Fe(II) is required for mammalian embryo development and that it facilitates the process via regulation of TET activity.
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Affiliation(s)
- Ming-Hui Zhao
- 1Department of Animal Sciences,Chungbuk National University,Cheongju 361-763,Republic of Korea
| | - Shuang Liang
- 1Department of Animal Sciences,Chungbuk National University,Cheongju 361-763,Republic of Korea
| | - Jing Guo
- 1Department of Animal Sciences,Chungbuk National University,Cheongju 361-763,Republic of Korea
| | - Jeong-Woo Choi
- 1Department of Animal Sciences,Chungbuk National University,Cheongju 361-763,Republic of Korea
| | - Nam-Hyung Kim
- 1Department of Animal Sciences,Chungbuk National University,Cheongju 361-763,Republic of Korea
| | - Wen-Fa Lu
- 2College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China
| | - Xiang-Shun Cui
- 1Department of Animal Sciences,Chungbuk National University,Cheongju 361-763,Republic of Korea
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Wallace DF, Subramaniam VN. The global prevalence of HFE and non-HFE hemochromatosis estimated from analysis of next-generation sequencing data. Genet Med 2015; 18:618-26. [PMID: 26633544 DOI: 10.1038/gim.2015.140] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2015] [Accepted: 08/27/2015] [Indexed: 12/31/2022] Open
Abstract
PURPOSE The prevalence of HFE-related hereditary hemochromatosis (HH) among European populations has been well studied. There are no prevalence data for atypical forms of HH caused by mutations in HFE2, HAMP, TFR2, or SLC40A1. The purpose of this study was to estimate the population prevalence of these non-HFE forms of HH. METHODS A list of HH pathogenic variants in publically available next-generation sequence (NGS) databases was compiled and allele frequencies were determined. RESULTS Of 161 variants previously associated with HH, 43 were represented among the NGS data sets; an additional 40 unreported functional variants also were identified. The predicted prevalence of HFE HH and the p.Cys282Tyr mutation closely matched previous estimates from similar populations. Of the non-HFE forms of iron overload, TFR2-, HFE2-, and HAMP-related forms are predicted to be rare, with pathogenic allele frequencies in the range of 0.00007 to 0.0005. Significantly, SLC40A1 variants that have been previously associated with autosomal-dominant ferroportin disease were identified in several populations (pathogenic allele frequency 0.0004), being most prevalent among Africans. CONCLUSION We have, for the first time, estimated the population prevalence of non-HFE HH. This methodology could be applied to estimate the population prevalence of a wide variety of genetic disorders.Genet Med 18 6, 618-626.
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Affiliation(s)
- Daniel F Wallace
- Membrane Transport Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia.,Faculty of Medicine and Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia
| | - V Nathan Subramaniam
- Membrane Transport Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia.,Faculty of Medicine and Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia
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Effects of mouse hepcidin 1 treatment on osteoclast differentiation and intracellular iron concentration. Inflammation 2015; 38:718-27. [PMID: 25059214 DOI: 10.1007/s10753-014-9982-2] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
Hepcidin is a key player in the regulation of mammalian iron homeostasis. Because iron overload may be one of the causes of osteoporosis, hepcidin may have therapeutic potential for osteoporosis patients. However, the effects of hepcidin on bone metabolism are not fully clear. We recently found that hepcidin can increase intracellular iron and calcium levels and promote mineralization in osteoblasts. The present study was designed to evaluate the effects of hepcidin on osteoclasts. Our results showed that mouse hepcidin 1 (MH1) can increase the number of TRAP-positive MNCs concomitant in both bone marrow-derived macrophages (BMMs) and RAW264.7 cells and upregulate mRNA levels of TRAP, cathepsin K, and MMP-9 and increase TRAP-5b protein secretion in RAW264.7 cells. Moreover, MH1 can downregulate the level of FPN1 protein and increase intracellular iron in RAW 264.7 cells. Therefore, we conclude that MH1 can significantly facilitate osteoclast differentiation in vitro. The mechanism behind accelerated differentiation may be associated with increased levels of intracellular iron. These findings may facilitate understanding of the effects of hepcidin on bone metabolism.
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Abstract
Maintaining physiologic iron concentrations in tissues is critical for metabolism and host defense. Iron absorption in the duodenum, recycling of iron from senescent erythrocytes, and iron mobilization from storage in macrophages and hepatocytes constitute the major iron flows into plasma for distribution to tissues, predominantly for erythropoiesis. All iron transfer to plasma occurs through the iron exporter ferroportin. The concentration of functional membrane-associated ferroportin is controlled by its ligand, the iron-regulatory hormone hepcidin, and fine-tuned by regulatory mechanisms serving iron homeostasis, oxygen utilization, host defense, and erythropoiesis. Fundamental questions about the structure and biology of ferroportin remain to be answered.
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Gu Z, Wang H, Xia J, Yang Y, Jin Z, Xu H, Shi J, De Domenico I, Tricot G, Zhan F. Decreased ferroportin promotes myeloma cell growth and osteoclast differentiation. Cancer Res 2015; 75:2211-21. [PMID: 25855377 DOI: 10.1158/0008-5472.can-14-3804] [Citation(s) in RCA: 84] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2014] [Accepted: 03/27/2015] [Indexed: 11/16/2022]
Abstract
Iron homeostasis is disrupted in multiple myeloma, a difficult-to-cure plasma cell malignancy with lytic bone lesions. Here, we systematically analyzed iron gene expression signature and demonstrated that mRNA expression of iron exporter ferroportin (FPN1) is significantly downregulated in myeloma cells and correlates negatively with clinic outcome. Restoring expression of FPN1 reduces intracellular liable iron pool, inhibits STAT3-MCL-1 signaling, and suppresses myeloma cells growth. Furthermore, we demonstrated that mRNA of FPN1 is also downregulated at the initial stages of osteoclast differentiation and suppresses myeloma cell-induced osteoclast differentiation through regulating iron regulator TFRC, NF-κB, and JNK pathways. Altogether, we demonstrated that downregulation of FPN1 plays critical roles in promoting myeloma cell growth and bone resorption in multiple myeloma.
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Affiliation(s)
- Zhimin Gu
- Department of Internal Medicine, Carver College of Medicine, The University of Iowa, Iowa City, Iowa
| | - He Wang
- Department of Internal Medicine, Carver College of Medicine, The University of Iowa, Iowa City, Iowa
| | - Jiliang Xia
- Department of Internal Medicine, Carver College of Medicine, The University of Iowa, Iowa City, Iowa
| | - Ye Yang
- Department of Internal Medicine, Carver College of Medicine, The University of Iowa, Iowa City, Iowa
| | - Zhendong Jin
- Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, The University of Iowa, Iowa City, Iowa
| | - Hongwei Xu
- Department of Internal Medicine, Carver College of Medicine, The University of Iowa, Iowa City, Iowa
| | - Jumei Shi
- Department of Hematology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai, China
| | - Ivana De Domenico
- Department of Internal Medicine, University of Utah, Salt Lake City, Utah
| | - Guido Tricot
- Department of Internal Medicine, Carver College of Medicine, The University of Iowa, Iowa City, Iowa.
| | - Fenghuang Zhan
- Department of Internal Medicine, Carver College of Medicine, The University of Iowa, Iowa City, Iowa.
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Impact of D181V and A69T on the function of ferroportin as an iron export pump and hepcidin receptor. Biochim Biophys Acta Mol Basis Dis 2014; 1842:1406-12. [DOI: 10.1016/j.bbadis.2014.05.011] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2013] [Revised: 05/06/2014] [Accepted: 05/14/2014] [Indexed: 01/03/2023]
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Zhao GY, DI DH, Wang B, Zhang P, Xu YJ. Iron regulates the expression of ferroportin 1 in the cultured hFOB 1.19 osteoblast cell line. Exp Ther Med 2014; 8:826-830. [PMID: 25120608 PMCID: PMC4113530 DOI: 10.3892/etm.2014.1823] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2014] [Accepted: 06/11/2014] [Indexed: 12/16/2022] Open
Abstract
Iron metabolism is tightly regulated in osteoblasts, and ferroportin 1 (FPN1) is the only identified iron exporter in mammals to date. In the present study, the regulation of FNP1 in human osteoblasts was investigated following various iron treatments. The human osteoblast cell line hFOB 1.19 was treated with ferric ammonium citrate (FAC) or desferrioxamine (DFO) of various concentrations. The intracellular iron ion levels were measured using a confocal laser scanning microscope. In addition, the mRNA and protein expression levels of FPN1 were detected by quantitative polymerase chain reaction, western blot analysis and immunofluorescence. The results demonstrated that increasing iron concentrations via FAC treatment increased the expression of FPN1. By contrast, decreasing the iron concentration by DFO treatment decreased FNP1 expression levels. In addition to demonstrating that the FNP1 expression changed according to the iron concentration, the observations indicated that changes in FPN1 expression may contribute to the maintenance of the intracellular iron balance in osteoblasts.
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Affiliation(s)
- Guo-Yang Zhao
- Department of Orthopedics, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212000, P.R. China
| | - Dong-Hua DI
- Department of Orthopedics, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212000, P.R. China
| | - Bo Wang
- Department of Orthopedics, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212000, P.R. China
| | - Peng Zhang
- Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215004, P.R. China
| | - You-Jia Xu
- Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215004, P.R. China
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Musci G, Polticelli F, Bonaccorsi di Patti MC. Ceruloplasmin-ferroportin system of iron traffic in vertebrates. World J Biol Chem 2014; 5:204-215. [PMID: 24921009 PMCID: PMC4050113 DOI: 10.4331/wjbc.v5.i2.204] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/13/2014] [Accepted: 02/19/2014] [Indexed: 02/05/2023] Open
Abstract
Safe trafficking of iron across the cell membrane is a delicate process that requires specific protein carriers. While many proteins involved in iron uptake by cells are known, only one cellular iron export protein has been identified in mammals: ferroportin (SLC40A1). Ceruloplasmin is a multicopper enzyme endowed with ferroxidase activity that is found as a soluble isoform in plasma or as a membrane-associated isoform in specific cell types. According to the currently accepted view, ferrous iron transported out of the cell by ferroportin would be safely oxidized by ceruloplasmin to facilitate loading on transferrin. Therefore, the ceruloplasmin-ferroportin system represents the main pathway for cellular iron egress and it is responsible for physiological regulation of cellular iron levels. The most recent findings regarding the structural and functional features of ceruloplasmin and ferroportin and their relationship will be described in this review.
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Bonaccorsi di Patti MC, Polticelli F, Cece G, Cutone A, Felici F, Persichini T, Musci G. A structural model of human ferroportin and of its iron binding site. FEBS J 2014; 281:2851-60. [PMID: 24767627 DOI: 10.1111/febs.12825] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2013] [Revised: 03/17/2014] [Accepted: 04/24/2014] [Indexed: 11/29/2022]
Abstract
A structural model of human ferroportin has been built using two Escherichia coli proteins belonging to the major facilitator superfamily of transporters. A potential iron binding site was identified in the inward-open conformation of the model, and its relevance was tested through measurement of iron export of HEK293T cells expressing wild-type or mutated ferroportin. Aspartates 39 and 181 were found to be essential for the transport ability of the protein. Noteworthy, the D181V mutation is naturally found in type 4 hemochromatosis with reticuloendothelial system iron retention phenotype. The outward-open conformation of ferroportin was also predicted, and showed that significant conformational changes must occur in the inward- to outward-open transition of ferroportin. In particular, putative iron ligands move several ångströms away from each other, leading to the logical conclusion that the iron binding site is not occupied by the metal in the outward-open conformation of ferroportin.
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Callebaut I, Joubrel R, Pissard S, Kannengiesser C, Gérolami V, Ged C, Cadet E, Cartault F, Ka C, Gourlaouen I, Gourhant L, Oudin C, Goossens M, Grandchamp B, De Verneuil H, Rochette J, Férec C, Le Gac G. Comprehensive functional annotation of 18 missense mutations found in suspected hemochromatosis type 4 patients. Hum Mol Genet 2014; 23:4479-90. [PMID: 24714983 DOI: 10.1093/hmg/ddu160] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Abstract
Hemochromatosis type 4 is a rare form of primary iron overload transmitted as an autosomal dominant trait caused by mutations in the gene encoding the iron transport protein ferroportin 1 (SLC40A1). SLC40A1 mutations fall into two functional categories (loss- versus gain-of-function) underlying two distinct clinical entities (hemochromatosis type 4A versus type 4B). However, the vast majority of SLC40A1 mutations are rare missense variations, with only a few showing strong evidence of causality. The present study reports the results of an integrated approach collecting genetic and phenotypic data from 44 suspected hemochromatosis type 4 patients, with comprehensive structural and functional annotations. Causality was demonstrated for 10 missense variants, showing a clear dichotomy between the two hemochromatosis type 4 subtypes. Two subgroups of loss-of-function mutations were distinguished: one impairing cell-surface expression and one altering only iron egress. Additionally, a new gain-of-function mutation was identified, and the degradation of ferroportin on hepcidin binding was shown to probably depend on the integrity of a large extracellular loop outside of the hepcidin-binding domain. Eight further missense variations, on the other hand, were shown to have no discernible effects at either protein or RNA level; these were found in apparently isolated patients and were associated with a less severe phenotype. The present findings illustrate the importance of combining in silico and biochemical approaches to fully distinguish pathogenic SLC40A1 mutations from benign variants. This has profound implications for patient management.
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Affiliation(s)
- Isabelle Callebaut
- IMPMC, Sorbonne Universités - UMR CNRS 7590, UPMC Univ Paris 06, Muséum d'Histoire Naturelle, IRD UMR 206, Paris, France
| | - Rozenn Joubrel
- Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Inserm U1078, Université de Brest, SFR SnInBioS, CHRU de Brest, Etablissement Français du Sang - Bretagne, Brest, France
| | - Serge Pissard
- Laboratoire de Génétique, UPEC (Université Paris Est Creteil), GHU Henri Mondor, Créteil, France
| | - Caroline Kannengiesser
- Hôpital Bichat, Département de Génétique, Inserm U1149 - Center for Research on Inflammation, Université Paris Diderot, AP-HP, Paris, France
| | | | - Cécile Ged
- Inserm U1035, Biothérapies des Maladies Génétiques et Cancers, Université de Bordeaux, CHU de Bordeaux, Pôle de Biologie et Pathologie, Bordeaux, France
| | - Estelle Cadet
- Laboratoire de Génétique Moléculaire, UPJV EA4666, CHU d'Amiens, Amiens, France
| | | | - Chandran Ka
- Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Inserm U1078, Université de Brest, SFR SnInBioS, CHRU de Brest, Etablissement Français du Sang - Bretagne, Brest, France
| | - Isabelle Gourlaouen
- Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Inserm U1078, Université de Brest, SFR SnInBioS, CHRU de Brest, Etablissement Français du Sang - Bretagne, Brest, France
| | | | - Claire Oudin
- Hôpital Bichat, Département de Génétique, Inserm U1149 - Center for Research on Inflammation, Université Paris Diderot, AP-HP, Paris, France
| | - Michel Goossens
- Laboratoire de Génétique, UPEC (Université Paris Est Creteil), GHU Henri Mondor, Créteil, France
| | - Bernard Grandchamp
- Hôpital Bichat, Département de Génétique, Inserm U1149 - Center for Research on Inflammation, Université Paris Diderot, AP-HP, Paris, France
| | - Hubert De Verneuil
- Inserm U1035, Biothérapies des Maladies Génétiques et Cancers, Université de Bordeaux, CHU de Bordeaux, Pôle de Biologie et Pathologie, Bordeaux, France
| | - Jacques Rochette
- Laboratoire de Génétique Moléculaire, UPJV EA4666, CHU d'Amiens, Amiens, France
| | - Claude Férec
- Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Inserm U1078, Université de Brest, SFR SnInBioS, CHRU de Brest, Etablissement Français du Sang - Bretagne, Brest, France
| | - Gérald Le Gac
- Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Inserm U1078, Université de Brest, SFR SnInBioS, CHRU de Brest, Etablissement Français du Sang - Bretagne, Brest, France CHRU de Brest, Inserm CIC0502, Brest, France
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Practice guidelines for the diagnosis and management of microcytic anemias due to genetic disorders of iron metabolism or heme synthesis. Blood 2014; 123:3873-86; quiz 4005. [PMID: 24665134 DOI: 10.1182/blood-2014-01-548776] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
During recent years, our understanding of the pathogenesis of inherited microcytic anemias has gained from the identification of several genes and proteins involved in systemic and cellular iron metabolism and heme syntheses. Numerous case reports illustrate that the implementation of these novel molecular discoveries in clinical practice has increased our understanding of the presentation, diagnosis, and management of these diseases. Integration of these insights into daily clinical practice will reduce delays in establishing a proper diagnosis, invasive and/or costly diagnostic tests, and unnecessary or even detrimental treatments. To assist the clinician, we developed evidence-based multidisciplinary guidelines on the management of rare microcytic anemias due to genetic disorders of iron metabolism and heme synthesis. These genetic disorders may present at all ages, and therefore these guidelines are relevant for pediatricians as well as clinicians who treat adults. This article summarizes these clinical practice guidelines and includes background on pathogenesis, conclusions, and recommendations and a diagnostic flowchart to facilitate using these guidelines in the clinical setting.
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Wang Q, Zhou J, Zhong D, Wang Q, Huang J. Ferroportin in the progression and prognosis of hepatocellular carcinoma. Eur J Med Res 2013; 18:59. [PMID: 24360312 PMCID: PMC3878504 DOI: 10.1186/2047-783x-18-59] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2013] [Accepted: 11/25/2013] [Indexed: 01/04/2023] Open
Abstract
BACKGROUND Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor in men and the seventh in women and understanding the molecular mechanisms of HCC and establishing more effective therapies are critical and urgent issues. Our objective was to study the expression of ferroportin in hepatocellular carcinoma (HCC) tissue samples and the relationship between ferroportin expression and HCC characteristics. METHODS Sixty HCC tissues and their corresponding para-cancer liver tissues (PCLT) were obtained from sixty HCC patients who had undergone hepatectomy in the Second Xiangya Hospital of Central South University. Ten normal liver tissue samples were also obtained as a control. Immunohistochemistry (IHC) was performed to analyze the ferroportin expression in HCC, and the relationship between ferroportin expression and HCC clinical pathological characteristics also was analyzed. For the evaluation of IHC results, the comprehensive scoring criteria were met according to the staining intensity and the number of positive staining cells. Western blotting was performed to detect the expression level of ferroportin in HCC cell lines. RESULTS Ferroportin expression in HCC tissue was significantly lower compared to PCLT and normal liver tissue (P <0.05). Moreover, ferroportin expression was related to liver cancer cell de-differentiation, the severity degree in TNM staging, Edmondson-Steiner grading, intrahepatic metastasis and portal vein invasion. In addition, high expression of ferroportin was observed in normal human liver cell lines L02 and HL7702, whereas weak positive expression and even negative expression of ferroportin were observed in HCC cell lines FOCUS, MHCC-97H, HepG2 and SMMC-7721. Furthermore, among the four kinds of HCC cell lines, the expression level of ferroportin was the lowest in MHCC-97H cells. CONCLUSIONS Ferroportin expression level declines along with the progression of liver cancer, suggesting that the reduction of ferroportin may serve as an important marker for poor HCC prognosis and as a new therapeutic target.
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Affiliation(s)
| | - Jun Zhou
- Department of Minimal Invasive surgery, The Second Xiangya Hospital of Central South University, 139 Renmin Middle Road, Changsha 410011, Hunan Province, China.
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Abstract
The iron hormone hepcidin and its receptor and cellular iron exporter ferroportin control the major fluxes of iron into blood plasma: intestinal iron absorption, the delivery of recycled iron from macrophages, and the release of stored iron from hepatocytes. Because iron losses are comparatively very small, iron absorption and its regulation by hepcidin and ferroportin determine total body iron content. Hepcidin is in turn feedback-regulated by plasma iron concentration and iron stores, and negatively regulated by the activity of erythrocyte precursors, the dominant consumers of iron. Hepcidin and ferroportin also play a role in host defense and inflammation, and hepcidin synthesis is induced by inflammatory signals including interleukin-6 and activin B. This review summarizes and discusses recent progress in molecular characterization of systemic iron homeostasis and its disorders, and identifies areas for further investigation.
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Alexander SPH, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Spedding M, Peters JA, Harmar AJ. The Concise Guide to PHARMACOLOGY 2013/14: transporters. Br J Pharmacol 2013; 170:1706-96. [PMID: 24528242 PMCID: PMC3892292 DOI: 10.1111/bph.12450] [Citation(s) in RCA: 120] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. Transporters are one of the seven major pharmacological targets into which the Guide is divided, with the others being G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear hormone receptors and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors and Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and the Guide to Receptors and Channels, providing a permanent, citable, point-in-time record that will survive database updates.
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Affiliation(s)
- Stephen PH Alexander
- School of Life Sciences, University of Nottingham Medical SchoolNottingham, NG7 2UH, UK
| | - Helen E Benson
- The University/BHF Centre for Cardiovascular Science, University of EdinburghEdinburgh, EH16 4TJ, UK
| | - Elena Faccenda
- The University/BHF Centre for Cardiovascular Science, University of EdinburghEdinburgh, EH16 4TJ, UK
| | - Adam J Pawson
- The University/BHF Centre for Cardiovascular Science, University of EdinburghEdinburgh, EH16 4TJ, UK
| | - Joanna L Sharman
- The University/BHF Centre for Cardiovascular Science, University of EdinburghEdinburgh, EH16 4TJ, UK
| | | | - John A Peters
- Neuroscience Division, Medical Education Institute, Ninewells Hospital and Medical School, University of DundeeDundee, DD1 9SY, UK
| | - Anthony J Harmar
- The University/BHF Centre for Cardiovascular Science, University of EdinburghEdinburgh, EH16 4TJ, UK
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Détivaud L, Island ML, Jouanolle AM, Ropert M, Bardou-Jacquet E, Le Lan C, Mosser A, Leroyer P, Deugnier Y, David V, Brissot P, Loréal O. Ferroportin diseases: functional studies, a link between genetic and clinical phenotype. Hum Mutat 2013; 34:1529-36. [PMID: 23943237 DOI: 10.1002/humu.22396] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2013] [Accepted: 08/05/2013] [Indexed: 12/21/2022]
Abstract
Ferroportin (FPN) mediates iron export from cells and this function is modulated by serum hepcidin. Mutations in the FPN gene (SLC40A1) lead to autosomal dominant iron overload diseases related either to loss or to gain of function, and usually characterized by normal or low transferrin saturation versus elevated transferrin saturation, respectively. However, for the same mutation, the phenotypic expression may vary from one patient to another. Using in vitro overexpression of wild-type or mutant FPN proteins, we characterized the functional impact of five recently identified FPN gene mutations regarding FPN localization, cell iron status, and hepcidin sensitivity. Our aim was to integrate functional results and biological findings in probands and relatives. We show that while the p.Arg371Gln (R371Q) mutation had no impact on studied parameters, the p.Trp158Leu (W158L), p.Arg88Gly (R88G), and p.Asn185Asp (N185D) mutations caused an iron export defect and were classified as loss-of-function mutations. The p.Gly204Ser (G204S) mutation induced a gain of FPN function. Functional studies are useful to determine whether or not a FPN gene mutation found in an iron overloaded patient is deleterious and to characterize its biological impact, especially when family studies are not fully informative and/or additional confounding factors may affect bio-clinical expression.
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Affiliation(s)
- Lénaïck Détivaud
- CHU Rennes, French Reference Centre for Rare Iron Overload Diseases of Genetic Origin, Rennes, France; CHU Rennes, Department of Molecular Biology, Rennes, France; CHU Rennes, Department of Liver Diseases, Rennes, France; INSERM UMR 991, Equipe Fer et Foie, Rennes, France
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Le Gac G, Ka C, Joubrel R, Gourlaouen I, Lehn P, Mornon JP, Férec C, Callebaut I. Structure-function analysis of the human ferroportin iron exporter (SLC40A1): effect of hemochromatosis type 4 disease mutations and identification of critical residues. Hum Mutat 2013; 34:1371-80. [PMID: 23784628 DOI: 10.1002/humu.22369] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2013] [Accepted: 06/06/2013] [Indexed: 11/06/2022]
Abstract
Ferroportin (SLC40A1) is the only known iron exporter in mammals and is considered a key coordinator of the iron balance between intracellular and systemic iron homeostasis. However, the structural organization of ferroportin in the lipid bilayer remains controversial and very little is known about the mechanism underlying iron egress. In the present study, we have developed an approach based on comparative modeling, which has led to the construction of a model of the three-dimensional (3D) structure of ferroportin by homology to the crystal structure of a Major Facilitator Superfamily member (EmrD). This model predicts atomic details for the organization of ferroportin transmembrane helices and is in agreement with our current understanding of the ferroportin function and its interaction with hepcidin. Using in vitro experiments, we demonstrate that this model can be used to identify novel critical amino acids. In particular, we show that the tryptophan residue 42 (p.Trp42), which is localized within the extracellular end of the ferroportin pore, is likely involved in both the iron export function and in the mechanism of inhibition by hepcidin. Thus, our 3D model provides a new perspective for understanding the molecular basis of ferroportin functions and dysfunctions.
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Affiliation(s)
- Gérald Le Gac
- Inserm UMR1078, Université de Brest, SFR SnInBioS, Centre Hospitalier Régional Universitaire - Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Etablissement Français du Sang - Bretagne, Brest, France
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Montalbetti N, Simonin A, Kovacs G, Hediger MA. Mammalian iron transporters: families SLC11 and SLC40. Mol Aspects Med 2013; 34:270-87. [PMID: 23506870 DOI: 10.1016/j.mam.2013.01.002] [Citation(s) in RCA: 99] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2012] [Accepted: 12/14/2012] [Indexed: 01/13/2023]
Abstract
This review is focused on the mammalian SLC11 and SLC40 families and their roles in iron homeostasis. The SLC11 family is composed of two members, SLC11A1 and SLC11A2. SLC11A1 is expressed in the lysosomal compartment of macrophages and in the tertiary granules of neutrophils, playing a key role in innate resistance against infection by intracellular microbes. SLC11A2 is a key player in iron metabolism and is ubiquitously expressed, most notably in the proximal duodenum, immature erythroid cells, brain, placenta and kidney. Intestinal iron absorption is mediated by SLC11A2 at the apical membrane of enterocytes, followed by basolateral exit via SLC40A1. To meet the daily requirement for iron, approximately 80% of the iron comes from the breakdown of hemoglobin following macrophage phagocytosis of senescent erythrocytes (iron recycling). Both SLC11A1 and SLC11A2 play an important role in macrophage iron recycling. SLC11A2 also transports iron into the cytosol across the membrane of endocytotic vesicles of the transferrin receptor-cycle. SLC40A1 is the sole member of the SLC40 family and is involved in the only cellular iron efflux mechanism described. SLC40A1 is highly expressed in several tissues and cells that play a critical role in body iron homeostasis. The signaling pathways that regulate SLC11A2 and SLC40A1 expression at transcriptional, post-transcriptional and post-translational levels are discussed. The roles of SLC11A2 and/or SLC40A1 in iron-associated disorders such as hemochromatosis, neurodegenerative diseases, and breast cancer are also summarized.
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Affiliation(s)
- Nicolas Montalbetti
- Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland.
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