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Man RC, Idrus RBH, Ibrahim WIW, Saim AB, Lokanathan Y. Secretome Analysis of Human Nasal Fibroblast Identifies Proteins That Promote Wound Healing. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2024; 1450:59-76. [PMID: 37247133 DOI: 10.1007/5584_2023_777] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/30/2023]
Abstract
Conditioned medium from cultured fibroblast cells is recognized to promote wound healing and growth through the secretion of enzymes, extracellular matrix proteins, and various growth factors and cytokines. The objective of this study was to profile the secreted proteins present in nasal fibroblast conditioned medium (NFCM). Nasal fibroblasts isolated from human nasal turbinates were cultured for 72 h in Defined Keratinocytes Serum Free Medium (DKSFM) or serum-free F12: Dulbecco's Modified Eagle's Medium (DMEM) to collect conditioned medium, denoted as NFCM_DKSFM and NFCM_FD, respectively. SDS-PAGE was performed to detect the presence of protein bands, followed by MALDI-TOF and mass spectrometry analysis. SignalP, SecretomeP, and TMHMM were used to identify the secreted proteins in conditioned media. PANTHER Classification System was performed to categorize the protein according to protein class, whereas STRING 10 was carried out to evaluate the predicted proteins interactions. SDS-PAGE results showed the presence of various protein with molecular weight ranging from ~10 kDa to ~260 kDa. Four protein bands were identified using MALDI-TOF. The analyses identified 104, 83, and 7 secreted proteins in NFCM_FD, NFCM_DKSFM, and DKSFM, respectively. Four protein classes involved in wound healing were identified, namely calcium-binding proteins, cell adhesion molecules, extracellular matrix proteins, and signaling molecules. STRING10 protein prediction successfully identified various pathways regulated by secretory proteins in NFCM. In conclusion, this study successfully profiled the secreted proteins of nasal fibroblasts and these proteins are predicted to play important roles in RECs wound healing through various pathways.
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Affiliation(s)
- Rohaina Che Man
- Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
| | - Ruszymah Binti Hj Idrus
- Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
- Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
| | - Wan Izlina Wan Ibrahim
- Medical Biotechnology Laboratory, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
| | - Aminuddin Bin Saim
- Ear, Nose & Throat Consultant Clinic, Ampang Puteri Specialist Hospital, Selangor, Malaysia
| | - Yogeswaran Lokanathan
- Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.
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Al-Hassi HO, Ng O, Evstatiev R, Mangalika M, Worton N, Jambrich M, Khare V, Phipps O, Keeler B, Gasche C, Acheson AG, Brookes MJ. Intravenous iron is non-inferior to oral iron regarding cell growth and iron metabolism in colorectal cancer associated with iron-deficiency anaemia. Sci Rep 2021; 11:13699. [PMID: 34211054 PMCID: PMC8249613 DOI: 10.1038/s41598-021-93155-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2020] [Accepted: 06/10/2021] [Indexed: 01/25/2023] Open
Abstract
Oral iron promotes intestinal tumourigenesis in animal models. In humans, expression of iron transport proteins are altered in colorectal cancer. This study examined whether the route of iron therapy alters iron transport and tumour growth. Colorectal adenocarcinoma patients with pre-operative iron deficiency anaemia received oral ferrous sulphate (n = 15), or intravenous ferric carboxymaltose (n = 15). Paired (normal and tumour tissues) samples were compared for expression of iron loading, iron transporters, proliferation, apoptosis and Wnt signalling using immunohistochemistry and RT-PCR. Iron loading was increased in tumour and distributed to the stroma in intravenous treatment and to the epithelium in oral treatment. Protein and mRNA expression of proliferation and iron transporters were increased in tumours compared to normal tissues but there were no significant differences between the treatment groups. However, intravenous iron treatment reduced ferritin mRNA levels in tumours and replenished body iron stores. Iron distribution to non-epithelial cells in intravenous iron suggests that iron is less bioavailable to tumour cells. Therefore, intravenous iron may be a better option in the treatment of colorectal cancer patients with iron deficiency anaemia due to its efficiency in replenishing iron levels while its effect on proliferation and iron metabolism is similar to that of oral iron treatment.
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Affiliation(s)
- Hafid O Al-Hassi
- Research Institute in Healthcare Science, Faculty of Science and Engineering, University of Wolverhampton, Wolverhampton, UK
| | - Oliver Ng
- NIHR Nottingham Biomedical Research Centre and the University of Nottingham, Nottingham, UK
| | - Rayko Evstatiev
- Department of Internal Medicine III, Division of Gastroenterology and Hepatology, Medical University of Vienna, Vienna, Austria
| | | | | | - Manuela Jambrich
- Department of Internal Medicine III, Division of Gastroenterology and Hepatology, Medical University of Vienna, Vienna, Austria
| | - Vineeta Khare
- Department of Internal Medicine III, Division of Gastroenterology and Hepatology, Medical University of Vienna, Vienna, Austria
| | - Oliver Phipps
- Research Institute in Healthcare Science, Faculty of Science and Engineering, University of Wolverhampton, Wolverhampton, UK
| | - Barrie Keeler
- NIHR Nottingham Biomedical Research Centre and the University of Nottingham, Nottingham, UK
| | - Christoph Gasche
- Department of Internal Medicine III, Division of Gastroenterology and Hepatology, Medical University of Vienna, Vienna, Austria
| | - Austin G Acheson
- NIHR Nottingham Biomedical Research Centre and the University of Nottingham, Nottingham, UK
| | - Matthew J Brookes
- Research Institute in Healthcare Science, Faculty of Science and Engineering, University of Wolverhampton, Wolverhampton, UK. .,The Royal Wolverhampton NHS Trust, Wolverhampton, UK.
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Abstract
Meiosis is a highly conserved process, and is responsible for the production of haploid gametes and generation of genetic diversity. We previously identified the transferrin receptor (TFRC) in the proteome profile of mice neonatal testes, indicating the involvement of the TFRC in meiosis. However, the exact molecular role of the TFRC in meiosis remains unclear. In this study, we aimed to determine the function of the TFRC in neonatal testicular development by TFRC knockdown using the testis culture platform. Our results showed high TFRC expression in 2-week testes, corresponding to the first meiotic division. Targeting TFRC using morpholino oligonucleotides resulted in clear spermatocyte apoptosis. In addition, we used the chromosomal spread technique to show that a deficiency of TFRC caused the accumulation of leptotene and zygotene spermatocytes, and a decrease of pachytene spermatocytes, indicating early meiotic arrest. Moreover, the chromosomes of TFRC-deficient pachytene spermatocytes displayed sustained γH2AX association, as well as SYCP1/SYCP3 dissociation beyond the sex body. Therefore, our results demonstrated that the TFRC is essential for the progression of spermatocyte meiosis, particularly for DNA double-stranded break repair and chromosomal synapsis.
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Mendes BB, Gómez-Florit M, Osório H, Vilaça A, Domingues RMA, Reis RL, Gomes ME. Cellulose nanocrystals of variable sulfation degrees can sequester specific platelet lysate-derived biomolecules to modulate stem cell response. Chem Commun (Camb) 2020; 56:6882-6885. [DOI: 10.1039/d0cc01850c] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Cellulose nanocrystals can bind different patterns of platelet lysate-derived protein in a surface sulfation dependent manner. The potential to direct stem cell fate by solid-phase presentation of defined protein coronas is demonstrated.
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Affiliation(s)
- Bárbara B. Mendes
- 3B's Research Group
- I3Bs – Research Institute on Biomaterials
- Biodegradables and Biomimetics
- University of Minho
- Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine
| | - Manuel Gómez-Florit
- 3B's Research Group
- I3Bs – Research Institute on Biomaterials
- Biodegradables and Biomimetics
- University of Minho
- Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine
| | - Hugo Osório
- Instituto de Investigação e Inovação em Saúde (I3S)
- Universidade do Porto
- Porto
- Portugal
| | - Adriana Vilaça
- 3B's Research Group
- I3Bs – Research Institute on Biomaterials
- Biodegradables and Biomimetics
- University of Minho
- Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine
| | - Rui M. A. Domingues
- 3B's Research Group
- I3Bs – Research Institute on Biomaterials
- Biodegradables and Biomimetics
- University of Minho
- Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine
| | - Rui L. Reis
- 3B's Research Group
- I3Bs – Research Institute on Biomaterials
- Biodegradables and Biomimetics
- University of Minho
- Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine
| | - Manuela E. Gomes
- 3B's Research Group
- I3Bs – Research Institute on Biomaterials
- Biodegradables and Biomimetics
- University of Minho
- Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine
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Lewandowska H, Stępkowski TM, Męczyńska-Wielgosz S, Sikorska K, Sadło J, Dudek J, Kruszewski M. LDL dinitrosyl iron complex acts as an iron donor in mouse macrophages. J Inorg Biochem 2018; 188:29-37. [PMID: 30119015 DOI: 10.1016/j.jinorgbio.2018.08.004] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2018] [Revised: 07/22/2018] [Accepted: 08/02/2018] [Indexed: 12/27/2022]
Abstract
[Fe(NO)2] - modified nanoparticles of low-density protein (DNICLDL) can serve as conveyors of iron in the form of stable complexes with ApoB100 protein. As reported recently, in human hepatoma cells DNICLDL significantly increased the total iron content, while showing low toxicity. In the present work, we focused on the effects of internalization of DNIC-modified lipoproteins in macrophages, with special regards to cytotoxicity. DNICLDL was administered to a model macrophage cell line, RAW 264.7. Administration of DNICLDL considerably increased total iron content. High increase of iron was accompanied by moderate toxicity. As shown by in vitro plasmid nicking assay, chelation of iron in the form of DNIC strongly reduced the iron-related reactive oxygen species (ROS) -induced DNA damage. In addition, DNICLDL, plausibly due to its NO-donating activity, did not induce inducible nitric oxide synthase (iNOS) expression, as opposed to other forms of low-density protein (LDL).
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Affiliation(s)
- Hanna Lewandowska
- Institute of Nuclear Chemistry and Technology, 16 Dorodna Str., 03-195 Warsaw, Poland.
| | - Tomasz M Stępkowski
- Laboratory of Mitochondrial Biogenesis, Centre of New Technologies UW, Banacha 2c, 02-097 Warsaw, Poland
| | | | - Katarzyna Sikorska
- Institute of Nuclear Chemistry and Technology, 16 Dorodna Str., 03-195 Warsaw, Poland
| | - Jarosław Sadło
- Institute of Nuclear Chemistry and Technology, 16 Dorodna Str., 03-195 Warsaw, Poland
| | - Jakub Dudek
- Institute of Nuclear Chemistry and Technology, 16 Dorodna Str., 03-195 Warsaw, Poland
| | - Marcin Kruszewski
- Institute of Nuclear Chemistry and Technology, 16 Dorodna Str., 03-195 Warsaw, Poland; Faculty of Medicine, University of Information Technology and Management in Rzeszów, ul. Sucharskiego 2, 35-225 Rzeszów, Poland; Department of Molecular Biology and Translational Research, Institute of Rural Health, Jaczewskiego 2, 20-090 Lublin, Poland
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Shang N, Wu J. Egg White Ovotransferrin Shows Osteogenic Activity in Osteoblast Cells. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2018; 66:2775-2782. [PMID: 29502401 DOI: 10.1021/acs.jafc.8b00069] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/08/2023]
Abstract
Ovotransferrin, the major protein in egg white, is a member of transferrin family. The objective of this study was to study the effects of ovotransferrin on cell proliferation, differentiation, mineralization and osteoclastogenesis of bone osteoblast cells. Effect of ovotransferrin (concentrations ranging from 1 to 1000 μg/mL) on the proliferation, differentiation, and mineralization of mouse osteoblast cells MC3T3-E1 was determined by 5-bromo-2-deoxyuridine (BrdU) incorporation assay, Western blot, immunofluorescence, and Alizarin-S red staining, respectively. Our results showed that ovotransferrin stimulated cell proliferation (enhanced BrdU incorporation), differentiation (enhanced expression of alkaline phosphatase and type-I collagen), and mineralization (increased calcium deposits) in a dose-dependent manner. Furthermore, ovotransferrin could increase the expression of osteoprotegerin (OPG) while decreasing the expression of receptor activator of nuclear factor kappa-B ligand (RANKL), suggesting its role in inhibition of bone resorption. This study demonstrated for the first time that ovotransferrin might promote bone formation while preventing bone resorption, which might open up a new application of egg white protein ovotransferrin as a functional ingredient in bone health management.
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Affiliation(s)
- Nan Shang
- Department of Agricultural, Food and Nutritional Science , University of Alberta , Edmonton , Alberta , Canada T6G 2P5
| | - Jianping Wu
- Department of Agricultural, Food and Nutritional Science , University of Alberta , Edmonton , Alberta , Canada T6G 2P5
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Ahmed U, Oates PS. Dietary fat level affects tissue iron levels but not the iron regulatory gene HAMP in rats. Nutr Res 2012; 33:126-35. [PMID: 23399663 DOI: 10.1016/j.nutres.2012.11.012] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2012] [Revised: 10/26/2012] [Accepted: 11/14/2012] [Indexed: 02/06/2023]
Abstract
Because dietary fats affect the regulation and use of body iron, we hypothesized that iron regulatory and transport genes may be affected by dietary fat. A model of early-stage I to II, nonalcoholic fatty liver was used in which rats were fed standard (35% energy from fat) or high-fat (71% energy from fat) liquid diets with normal iron content (STD/HF groups). In addition, intraperitoneal injections of iron dextran were given to iron-loaded (STD+/HF+ groups) and iron-deficient diets to STD-/HF- groups. Plasma osmolality, hemoglobin level, and mean corpuscular hemoglobin concentration were increased in all STD diet groups compared with all HF diet groups. Plasma iron and transferrin saturation were affected by an interaction between dietary fat and iron. They were high in the STD group (normal iron) compared with their respective HF group. Similarly, this group also showed a 4-fold increase in the messenger RNA expression of the hepatic hemochromatosis gene. Spleen iron was high in the iron-loaded STD+ group compared with all other groups. Hepatic iron and messenger RNA expression of peroxisome proliferator-activated receptor-γ, CCAAT/enhancer binding protein α, interleukin-6, and iron transport genes (transferrin receptor 2, divalent metal transporter 1 iron-responsive element, and divalent metal transporter 1 non-iron-responsive element) were increased, whereas tumor necrosis factor α was decreased in the HF diet groups. The expression of iron regulatory gene HAMP was not increased in the HF diet groups. Iron regulatory and transport genes involved in cellular and systemic iron homeostasis may be affected by the macronutrient composition of the diet.
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Affiliation(s)
- Umbreen Ahmed
- Department of Physiology, National University of Sciences and Technology, Rawalpindi, Pakistan
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8
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Bae S, Kang D, Hong J, Chung B, Choi J, Jhun H, Hong K, Kim E, Jo S, Lee S, Kim SH, Kim S. Characterizing antiviral mechanism of interleukin-32 and a circulating soluble isoform in viral infection. Cytokine 2012; 58:79-86. [PMID: 22277801 DOI: 10.1016/j.cyto.2011.12.024] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2011] [Revised: 12/23/2011] [Accepted: 12/31/2011] [Indexed: 11/24/2022]
Abstract
Interleukin-32 (IL-32) is an inflammatory cytokine, and its activity is associated with various auto-inflammatory disorders as well as infectious pathogens such as Mycobacterium tuberculosis, and viral infections. However, the precise antiviral mechanism of IL-32 remains unclear. We assessed the IL-32 level in the sera of H1N1 influenza A patients and IL-32 level was significantly elevated. Next we examined the antiviral activity of recombinant IL-32γ (rIL-32γ) with WISH cells infected by vesicular stomatitis virus (VSV) but no antiviral activity was observed. Therefore we investigated the supernatant of rIL-32-treated THP-1 cells since this cell line effectively responded to rIL-32γ. The supernatant of rIL-32-treated THP-1 cell possessed an antiviral effect and in addition, an agonistic monoclonal antibody further enhanced a specific antiviral activity of rIL-32γ. The fractionation and mass spectrometer analysis of the THP-1 cell supernatant revealed that the antiviral activity of rIL-32γ is via a THP-1 cell-produced factor, transferrin, rather than the direct effects of rIL-32γ on epithelial cells. We also characterized a secreted soluble IL-32γ protein in serum of IL-32γ transgenic mouse (TG), but not in that of IL-32α TG. The present results suggest that IL-32γ expression and its genetic variation in individual could be an important aspect of viral infections.
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Affiliation(s)
- Suyoung Bae
- Laboratory of Cytokine Immunology, Department of Biomedical Science and Technology, Konkuk University, Seoul 143-701, Republic of Korea
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Tsuchiya H, Ashla AA, Hoshikawa Y, Matsumi Y, Kanki K, Enjoji M, Momosaki S, Nakamuta M, Taketomi A, Maehara Y, Shomori K, Kurimasa A, Hisatome I, Ito H, Shiota G. Iron state in association with retinoid metabolism in non-alcoholic fatty liver disease. Hepatol Res 2010; 40:1227-38. [PMID: 20880062 DOI: 10.1111/j.1872-034x.2010.00719.x] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
AIM We have recently reported that hyperdynamic state of retinoid metabolism, which may lead to the shortage of retinoid, is observed in patients with non-alcoholic fatty liver disease (NAFLD). Hepatic iron overload, which causes production of reactive oxygen species (ROS), is also frequently seen in NAFLD patients. The aim of the study is to examine iron state and retinoid metabolic state simultaneously, and to clarify the relationship between two disorders. METHODS Thirty-six persons, comprising 17 patients with simple steatosis (SS), 11 with NASH, and 8 normal controls (N), were examined on hepatic expression of iron metabolism-related genes including hemojuvelin (HJV), hepcidin (HEPC), transferrin receptor 1 and 2 (TfR1, TfR2), ferroportin (FPN), neogenin (NEO) and ferritin heavy chain (FtH) and hepatic iron contents in addition to expression 51 genes which is involved in retinoid metabolism and antioxidative action. RESULTS In patients with NAFLD, expression of HJV, TfR2, FPN, TfR1, FtH, SOD and catalase was increased, compared with that in N. In addition, hepatic iron content, which was increased in NASH, was correlated with expression level of TfR2. Expression of cellular retinoid binding protein (CRBP1), alcohol dehydrogenase 1 (ADH1) and cytochrome P450 26A1(CYP26A1) was significantly correlated with that of HJV, TfR2 and FPN, respectively. CONCLUSION The results of the present study suggest that the reasons responsible for iron accumulation in NASH in the present study may partly be due to enhanced expression of TfRs, especially TfR2, and hyperdynamic state of retinoid metabolism is closely related to iron metabolism in the disease.
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Affiliation(s)
- Hiroyuki Tsuchiya
- Division of Molecular and Genetic Medicine Division of Regenerative Therapeutics, Department of Genetic Medicine and, Regenerative Therapeutics, Graduate School of Medicine Division of Organ Pathology, Department of Microbiology and Pathology, Tottori University, Japan
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Herbison CE, Thorstensen K, Chua ACG, Graham RM, Leedman P, Olynyk JK, Trinder D. The role of transferrin receptor 1 and 2 in transferrin-bound iron uptake in human hepatoma cells. Am J Physiol Cell Physiol 2009; 297:C1567-75. [PMID: 19828835 DOI: 10.1152/ajpcell.00649.2008] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Transferrin receptor (TFR) 1 and 2 are expressed in the liver; TFR1 levels are regulated by cellular iron levels while TFR2 levels are regulated by transferrin saturation. The aims of this study were to 1) determine the relative importance of TFR1 and TFR2 in transferrin-bound iron (TBI) uptake by HuH7 human hepatoma cells and 2) characterize the role of metal-transferrin complexes in the regulation of these receptors. TFR expression was altered by 1) incubation with metal-transferrin (Tf) complexes, 2) TFR1 and TFR2 small interfering RNA knockdown, and 3) transfection with a human TFR2 plasmid. TBI uptake was measured using (59)Fe-(125)I-labeled Tf and mRNA and protein expression by real-time PCR and Western blot analysis, respectively. Fe(2)Tf, Co(2)Tf, and Mn(2)Tf increased TFR2 protein expression, indicating that the upregulation was not specifically regulated by iron-transferrin but also other metal-transferrins. In addition, Co(2)Tf and Mn(2)Tf upregulated TFR1, reduced ferritin, and increased hypoxia-inducible factor-1alpha protein expression, suggesting that TFR1 upregulation was due to a combination of iron deficiency and chemical hypoxia. TBI uptake correlated with changes in TFR1 but not TFR2 expression. TFR1 knockdown reduced iron uptake by 80% while TFR2 knockdown did not affect uptake. At 5 microM transferrin, iron uptake was not affected by combined TFR1 and TFR2 knockdown. Transfection with a hTFR2 plasmid increased TFR2 protein expression, causing a 15-20% increase in iron uptake and ferritin levels. This shows for the first time that TFR-mediated TBI uptake is mediated primarily via TFR1 but not TFR2 and that a high-capacity TFR-independent pathway exists in hepatoma cells.
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Anderson GJ, Vulpe CD. Mammalian iron transport. Cell Mol Life Sci 2009; 66:3241-61. [PMID: 19484405 PMCID: PMC11115736 DOI: 10.1007/s00018-009-0051-1] [Citation(s) in RCA: 225] [Impact Index Per Article: 14.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2009] [Revised: 04/21/2009] [Accepted: 05/12/2009] [Indexed: 02/07/2023]
Abstract
Iron is essential for basic cellular processes but is toxic when present in excess. Consequently, iron transport into and out of cells is tightly regulated. Most iron is delivered to cells bound to plasma transferrin via a process that involves transferrin receptor 1, divalent metal-ion transporter 1 and several other proteins. Non-transferrin-bound iron can also be taken up efficiently by cells, although the mechanism is poorly understood. Cells can divest themselves of iron via the iron export protein ferroportin in conjunction with an iron oxidase. The linking of an oxidoreductase to a membrane permease is a common theme in membrane iron transport. At the systemic level, iron transport is regulated by the liver-derived peptide hepcidin which acts on ferroportin to control iron release to the plasma.
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Affiliation(s)
- Gregory Jon Anderson
- Iron Metabolism Laboratory, Queensland Institute of Medical Research, PO Royal Brisbane Hospital, QLD, Australia.
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12
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Proteomic analysis of HCV cirrhosis and HCV-induced HCC: identifying biomarkers for monitoring HCV-cirrhotic patients awaiting liver transplantation. Transplantation 2009; 87:143-52. [PMID: 19136905 DOI: 10.1097/tp.0b013e318191c68d] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
BACKGROUND Progression from chronic hepatitis C virus (HCV) infection to cirrhosis and hepatocellular carcinoma (HCC) results in protein changes in the peripheral blood. We evaluated global protein expression in plasma samples of HCV-cirrhotic and HCV-cirrhotic-HCC patients. PATIENTS AND METHODS Plasma samples from 25 HCV-cirrhotic-HCC and 10 HCV-cirrhotic patients were quantitatively evaluated for protein expression. Tryptic peptides were analyzed using Thermo linear ion-trap mass spectrometer (LTQ) coupled with a Surveyor HPLC system (Thermo). SEQUEST and X!Tandem database search algorithms were used for peptide sequence identification. Protein relative quantification was performed using the area under the curve from the select ion chromatogram. A significant fold change between groups was based on controlling the false discovery rate (FDR) at less than 5%. RESULTS We identified and quantified 2320 proteins from the analysis of the different protein pattern between HCV-cirrhosis and HCV-HCC samples. Gene ontology terms classified the more important biologic process related to these proteins as signal transduction, regulation of transcription DNA-dependent, protein amino acid phosphorylation, cell adhesion, transport, and immune response. Seven proteins showed significant expression changes with a FDR less than 5% between cirrhosis and tumor groups. Moreover, 18 proteins showed significant expression changes (FDR <5%) when plasma samples from HCV-cirrhosis were compared with early HCV-HCC. CONCLUSIONS Differential protein expression was observed between samples from HCV patients with cirrhosis with and without HCC. Also, differences were observed between early and advanced HCV-HCC samples. This study provides important information for discovery of potential biomarkers for early HCC diagnosis in HCV cirrhotic patients.
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13
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Tsuchiya H, Akechi Y, Ikeda R, Nishio R, Sakabe T, Terabayashi K, Matsumi Y, Ashla AA, Hoshikawa Y, Kurimasa A, Suzuki T, Ishibashi N, Yanagida S, Shiota G. Suppressive effects of retinoids on iron-induced oxidative stress in the liver. Gastroenterology 2009; 136:341-350.e8. [PMID: 18952085 DOI: 10.1053/j.gastro.2008.09.027] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/22/2008] [Revised: 08/26/2008] [Accepted: 09/18/2008] [Indexed: 02/06/2023]
Abstract
BACKGROUND & AIMS We previously reported that impaired retinoid signaling in the liver causes steatohepatitis and hepatocellular carcinoma. Recently, oxidative stress induced by hepatic iron overload has emerged as an important factor for the progression of liver disease in patients with chronic hepatitis C, alcoholic liver disease, and nonalcoholic steatohepatitis. In this study, the relationship between retinoid signaling and iron metabolism in the liver was investigated. METHODS The effect of retinoids on the iron metabolism was examined in HuH7 cells treated with all-trans retinoic acid and acyclic retinoid NIK-333. In in vivo experiments, we used the mice expressing the dominant negative form of retinoic acid receptor alpha gene under the control of albumin enhancer/promoter (RAR-E Tg) and iron-overloaded wild mice fed with retinoid-deficient and retinoid-excess diets. RESULTS Hepatic iron accumulation and increased expression of hemojuvelin were observed in RAR-E Tg mouse liver. Retinoid treatment significantly suppressed expression of hemojuvelin and mildly suppressed expression of transferrin receptor type 2 and hepcidin, accompanied by decreased hepatic iron content and iron-induced oxidative stress in vitro and in vivo. Overexpression of hemojuvelin in HuH7 hepatoma cells led to a significant increase in cellular iron content. CONCLUSIONS Our results suggest that retinoids are involved in hepatic iron metabolism through transcriptional regulation of hemojuvelin. This study demonstrated a novel functional role of retinoids in preventing iron-induced oxidative stress in the liver.
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Affiliation(s)
- Hiroyuki Tsuchiya
- Division of Molecular and Genetic Medicine, Department of Genetic Medicine and Regenerative Therapeutics, Graduate School of Medicine, Tottori University, Yonago, Japan.
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Youn P, Kim S, Ahn JH, Kim Y, Park JD, Ryu DY. Regulation of iron metabolism-related genes in diethylnitrosamine-induced mouse liver tumors. Toxicol Lett 2008; 184:151-8. [PMID: 19061943 DOI: 10.1016/j.toxlet.2008.11.002] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2008] [Revised: 11/05/2008] [Accepted: 11/07/2008] [Indexed: 01/12/2023]
Abstract
BACKGROUND It has been suggested that the altered iron metabolism in liver tumors, characterized by the iron-deficient phenotype, is of importance for tumor growth. AIM This study was performed to elucidate the mechanisms underlying iron deficiency in liver tumors by examining how the liver tumor development affects the expression of iron metabolism-related genes. METHODS Iron metabolism reference values were analyzed in the sera of diethylnitrosamine-induced hepatocellular adenoma-bearing mice. Expression of iron metabolism-related genes was analyzed in adenomas and surrounding non-tumor tissues, and a subgroup of adenoma-bearing mice loaded with iron 72h before sacrifice. RESULTS Iron content of the adenoma tissues was 2.0-2.5-fold lower compared to surrounding and age-matched control tissues. There was no significant difference in serum iron levels between the adenoma-bearing and control mice, while the adenoma-bearing mice exhibited a 2.4-fold lower level of serum transferrin saturation. Expression of iron metabolism-related genes was dysregulated in the adenomas. Iron loading affected protein expression similarly in the adenomas and surrounding tissues suggesting that iron-responsive regulation of the proteins was not impaired. However, the mRNA expression for ceruloplasmin and divalent metal transporter 1 (DMT1) IRE(+) in the adenomas was altered independently of iron status, and the dysregulation may contribute to diminished iron content. CONCLUSION These findings suggest that diethylnitrosamine-induced liver adenoma-bearing mice have abnormal iron metabolism and that dysregulation of iron metabolism-related genes contributes to iron deficiency in the adenomas.
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Affiliation(s)
- Pilju Youn
- College of Veterinary Medicine, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
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Chua ACG, Herbison CE, Drake SF, Graham RM, Olynyk JK, Trinder D. The role of Hfe in transferrin-bound iron uptake by hepatocytes. Hepatology 2008; 47:1737-44. [PMID: 18393371 DOI: 10.1002/hep.22180] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
UNLABELLED HFE-related hereditary hemochromatosis results in hepatic iron overload. Hepatocytes acquire transferrin-bound iron via transferrin receptor (Tfr) 1 and Tfr1-independent pathways (possibly Tfr2-mediated). In this study, the role of Hfe in the regulation of hepatic transferrin-bound iron uptake by these pathways was investigated using Hfe knockout mice. Iron and transferrin uptake by hepatocytes from Hfe knockout, non-iron-loaded and iron-loaded wild-type mice were measured after incubation with 50 nM (125)I-Tf-(59)Fe (Tfr1 pathway) and 5 microM (125)I-Tf-(59)Fe (Tfr1-independent or putative Tfr2 pathway). Tfr1 and Tfr2 messenger RNA (mRNA) and protein expression were measured by real-time polymerase chain reaction and western blotting, respectively. Tfr1-mediated iron and transferrin uptake by Hfe knockout hepatocytes were increased by 40% to 70% compared with iron-loaded wild-type hepatocytes with similar iron levels and Tfr1 expression. Iron and transferrin uptake by the Tfr1-independent pathway was approximately 100-fold greater than by the Tfr1 pathway and was not affected by the absence of Hfe. Diferric transferrin increased hepatocyte Tfr2 protein expression, resulting in a small increase in transferrin but not iron uptake by the Tfr1-independent pathway. CONCLUSION Tfr1-mediated iron uptake is regulated by Hfe in hepatocytes. The Tfr1-independent pathway exhibited a much greater capacity for iron uptake than the Tfr1 pathway but it was not regulated by Hfe. Diferric transferrin up-regulated hepatocyte Tfr2 protein expression but not iron uptake, suggesting that Tfr2 may have a limited role in the Tfr1-independent pathway.
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Affiliation(s)
- Anita C G Chua
- School of Medicine and Pharmacology, The University of Western Australia, Fremantle Hospital, Western Australia, Australia
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Chua ACG, Graham RM, Trinder D, Olynyk JK. The regulation of cellular iron metabolism. Crit Rev Clin Lab Sci 2008; 44:413-59. [PMID: 17943492 DOI: 10.1080/10408360701428257] [Citation(s) in RCA: 109] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
While iron is an essential trace element required by nearly all living organisms, deficiencies or excesses can lead to pathological conditions such as iron deficiency anemia or hemochromatosis, respectively. A decade has passed since the discovery of the hemochromatosis gene, HFE, and our understanding of hereditary hemochromatosis (HH) and iron metabolism in health and a variety of diseases has progressed considerably. Although HFE-related hemochromatosis is the most widespread, other forms of HH have subsequently been identified. These forms are not attributed to mutations in the HFE gene but rather to mutations in genes involved in the transport, storage, and regulation of iron. This review is an overview of cellular iron metabolism and regulation, describing the function of key proteins involved in these processes, with particular emphasis on the liver's role in iron homeostasis, as it is the main target of iron deposition in pathological iron overload. Current knowledge on their roles in maintaining iron homeostasis and how their dysregulation leads to the pathogenesis of HH are discussed.
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Affiliation(s)
- Anita C G Chua
- School of Medicine and Pharmacology, University of Western Australia, Fremantle, Western Australia, Australia
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Abstract
The liver plays a central role in iron metabolism. It is the major storage site for iron and also expresses a complex range of molecules which are involved in iron transport and regulation of iron homeostasis. An increasing number of genes associated with hepatic iron transport or regulation have been identified. These include transferrin receptors (TFR1 and 2), a ferrireductase (STEAP3), the transporters divalent metal transporter-1 (DMT1) and ferroportin (FPN) as well as the haemochromatosis protein, HFE and haemojuvelin (HJV), which are signalling molecules. Many of these genes also participate in iron regulatory pathways which focus on the hepatic peptide hepcidin. However, we are still only beginning to understand the complex interactions between liver iron transport and iron homeostasis. This review outlines our current knowledge of molecules of iron metabolism and their roles in iron transport and regulation of iron homeostasis.
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Affiliation(s)
- Ross-M Graham
- School of Medicine and Pharmacology, Fremantle Hospital, University of Western Australia, PO Box 480, Fremantle 6959, Western Australia, Australia
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Drake SF, Morgan EH, Herbison CE, Delima R, Graham RM, Chua ACG, Leedman PJ, Fleming RE, Bacon BR, Olynyk JK, Trinder D. Iron absorption and hepatic iron uptake are increased in a transferrin receptor 2 (Y245X) mutant mouse model of hemochromatosis type 3. Am J Physiol Gastrointest Liver Physiol 2007; 292:G323-8. [PMID: 16935854 DOI: 10.1152/ajpgi.00278.2006] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Hereditary hemochromatosis type 3 is an iron (Fe)-overload disorder caused by mutations in transferrin receptor 2 (TfR2). TfR2 is expressed highly in the liver and regulates Fe metabolism. The aim of this study was to investigate duodenal Fe absorption and hepatic Fe uptake in a TfR2 (Y245X) mutant mouse model of hereditary hemochromatosis type 3. Duodenal Fe absorption and hepatic Fe uptake were measured in vivo by 59Fe-labeled ascorbate in TfR2 mutant mice, wild-type mice, and Fe-loaded wild-type mice (2% dietary carbonyl Fe). Gene expression was measured by real-time RT-PCR. Liver nonheme Fe concentration increased progressively with age in TfR2 mutant mice compared with wild-type mice. Fe absorption (both duodenal Fe uptake and transfer) was increased in TfR2 mutant mice compared with wild-type mice. Likewise, expression of genes participating in duodenal Fe uptake (Dcytb, DMT1) and transfer (ferroportin) were increased in TfR2 mutant mice. Nearly all of the absorbed Fe was taken up rapidly by the liver. Despite hepatic Fe loading, hepcidin expression was decreased in TfR2 mutant mice compared with wild-type mice. Even when compared with Fe-loaded wild-type mice, TfR2 mutant mice had increased Fe absorption, increased duodenal Fe transport gene expression, increased liver Fe uptake, and decreased liver hepcidin expression. In conclusion, despite systemic Fe loading, Fe absorption and liver Fe uptake were increased in TfR2 mutant mice in association with decreased expression of hepcidin. These findings support a model in which TfR2 is a sensor of Fe status and regulates duodenal Fe absorption and liver Fe uptake.
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Affiliation(s)
- S F Drake
- School of Medicine and Pharmacology, Fremantle Hospital, University of Western Australia, PO Box 480, Fremantle, 6959, WA, Australia
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West AR, Oates PS. Decreased sucrase and lactase activity in iron deficiency is accompanied by reduced gene expression and upregulation of the transcriptional repressor PDX-1. Am J Physiol Gastrointest Liver Physiol 2005; 289:G1108-14. [PMID: 16081762 DOI: 10.1152/ajpgi.00195.2005] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Disaccharidases are important digestive enzymes whose activities can be reduced by iron deficiency. We hypothesise that this is due to reduced gene expression, either by impairment to enterocyte differentiation or by iron-sensitive mechanisms that regulate mRNA levels in enterocytes. Iron-deficient Wistar rats were generated by dietary means. The enzyme activities and kinetics of sucrase and lactase were tested as well as the activity of intestinal alkaline phosphatase (IAP)-II because it is unrelated to carbohydrate digestion. mRNA levels of beta-actin, sucrase, lactase, and the associated transcription factors pancreatic duodenal homeobox (PDX)-1, caudal-related homeobox (CDX)-2, GATA-binding protein (GATA)-4, and hepatocyte nuclear factor (HNF)-1 were measured by real-time PCR. Spatial patterns of protein and gene expression were assessed by immunofluorescence and in situ hybridization, respectively. It was found that iron-deficient rats had significantly lower sucrase (19.5% lower) and lactase (56.8% lower) but not IAP-II activity than control rats. Kinetic properties of both enzymes remained unchanged from controls, suggesting a decrease in the quantity of enzyme present. Sucrase and lactase mRNA levels were reduced by 44.5% and 67.9%, respectively, by iron deficiency, suggesting that enzyme activity is controlled primarily by gene expression. Iron deficiency did not affect the pattern of protein and gene expression along the crypt to villus axis. Expression of PDX-1, a repressor of sucrase and lactase promoters, was 4.5-fold higher in iron deficiency, whereas CDX-2, GATA-4, and HNF-1 levels were not significantly different. These data suggest that decreases in sucrase and lactase activities result from a reduction in gene expression, following from increased levels of the transcriptional repressor PDX-1.
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Affiliation(s)
- Adrian R West
- Physiology, School of Biomedical and Chemical Sciences, The University of Western Australia, Crawley, Western Australia, Australia
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Robb AD, Ericsson M, Wessling-Resnick M. Transferrin receptor 2 mediates a biphasic pattern of transferrin uptake associated with ligand delivery to multivesicular bodies. Am J Physiol Cell Physiol 2004; 287:C1769-75. [PMID: 15317665 DOI: 10.1152/ajpcell.00337.2004] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
The physiological role of transferrin (Tf) receptor 2 (TfR2), a homolog of the well-characterized TfR1, is unclear. Mutations in TfR2 result in hemochromatosis, indicating that this receptor has a unique role in iron metabolism. We report that HepG2 cells, which endogenously express TfR2, display a biphasic pattern of Tf uptake when presented with ligand concentrations up to 2 muM. The apparently nonsaturating pathway of Tf endocytosis resembles TfR1-independent Tf uptake, a process previously characterized in some liver cell types. Exogenous expression of TfR2 but not TfR1 induces a similar biphasic pattern of Tf uptake in HeLa cells, supporting a role for TfR2 in this process. Immunoelectron microscopy reveals that while Tf, TfR1, and TfR2 are localized in the plasma membrane and tubulovesicular endosomes, TfR2 expression is associated with the additional appearance of Tf in multivesicular bodies. These combined results imply that unlike TfR1, which recycles apo-Tf back to the cell surface after the release of iron, TfR2 promotes the intracellular deposition of ligand. Tf delivered by TfR2 does not appear to be degraded, which suggests that its delivery to this organelle may be functionally relevant to the storage of iron in overloaded states.
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Affiliation(s)
- Aeisha D Robb
- Department of Genetics and Complex Diseases, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115, USA
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Chua ACG, Olynyk JK, Leedman PJ, Trinder D. Nontransferrin-bound iron uptake by hepatocytes is increased in the Hfe knockout mouse model of hereditary hemochromatosis. Blood 2004; 104:1519-25. [PMID: 15155457 DOI: 10.1182/blood-2003-11-3872] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Hereditary hemochromatosis (HH) is an iron-overload disorder caused by a C282Y mutation in the HFE gene. In HH, plasma nontransferrin-bound iron (NTBI) levels are increased and NTBI is bound mainly by citrate. The aim of this study was to examine the importance of NTBI in the pathogenesis of hepatic iron loading in Hfe knockout mice. Plasma NTBI levels were increased 2.5-fold in Hfe knockout mice compared with control mice. Total ferric citrate uptake by hepatocytes isolated from Hfe knockout mice (34.1 +/- 2.8 pmol Fe/mg protein/min) increased by 2-fold compared with control mice (17.8 +/- 2.7 pmol Fe/mg protein/min; P <.001; mean +/- SEM; n = 7). Ferrous ion chelators, bathophenanthroline disulfonate, and 2',2-bipyridine inhibited ferric citrate uptake by hepatocytes from both mouse types. Divalent metal ions inhibited ferric citrate uptake by hepatocytes, as did diferric transferrin. Divalent metal transporter 1 (DMT1) mRNA and protein expression was increased approximately 2-fold by hepatocytes from Hfe knockout mice. We conclude that NTBI uptake by hepatocytes from Hfe knockout mice contributed to hepatic iron loading. Ferric ion was reduced to ferrous ion and taken up by hepatocytes by a pathway shared with diferric transferrin. Inhibition of uptake by divalent metals and up-regulation of DMT1 expression suggested that NTBI uptake was mediated by DMT1.
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Affiliation(s)
- Anita C G Chua
- School of Medicine and Pharmacology, The University of Western Australia, Fremantle Hospital, PO Box 480, Fremantle 6959, WA, Australia
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