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Horeau M, Delalande M, Ropert M, Leroyer P, Martin B, Orfila L, Loréal O, Derbré F. Sex similarities and divergences in systemic and muscle iron metabolism adaptations to extreme physical inactivity in rats. J Cachexia Sarcopenia Muscle 2024; 15:1989-1998. [PMID: 39049183 PMCID: PMC11446688 DOI: 10.1002/jcsm.13547] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/19/2023] [Revised: 05/15/2024] [Accepted: 06/26/2024] [Indexed: 07/27/2024] Open
Abstract
BACKGROUND Previous data in humans suggest that extreme physical inactivity (EPI) affects iron metabolism differently between sexes. Our objective was to deepen the underlying mechanisms by studying rats of both sexes exposed to hindlimb unloading (HU), the reference experimental model mimicking EPI. METHODS Eight-week-old male and female Wistar rats were assigned to control (CTL) or hindlimb unloading (HU) conditions (n = 12/group). After 7 days of HU, serum, liver, spleen, and soleus muscle were removed. Iron parameters were measured in serum samples, and ICP-MS was used to quantify iron in tissues. Iron metabolism genes and proteins were analysed by RT-qPCR and Western blot. RESULTS Compared with control males, control females exhibited higher iron concentrations in serum (+43.3%, p < 0.001), liver (LIC; +198%, P < 0.001), spleen (SIC; +76.1%, P < 0.001), and transferrin saturation (TS) in serum (+53.3%, P < 0.001), contrasting with previous observations in humans. HU rat males, but not females, exhibited an increase of LIC (+54% P < 0.001) and SIC (+30.1%, P = 0.023), along with a rise of H-ferritin protein levels (+60.9% and +134%, respectively, in liver and spleen; P < 0.05) and a decrease of TFRC protein levels (-36%; -50%, respectively, P < 0.05). HU males also exhibited an increase of splenic HO-1 and NRF2 mRNA levels, (p < 0.001), as well as HU females (P < 0.001). Concomitantly to muscle atrophy observed in HU animals, the iron concentration increased in soleus in females (+26.7, P = 0.004) while only a trend is observed in males (+17.5%, P = 0.088). In addition, the H-ferritin and myoglobin protein levels in soleus were increased in males (+748%, P < 0.001, +22%, P = 0.011, respectively) and in females (+369%, P < 0.001, +21.9%, P = 0.007, respectively), whereas TFRC and ferroportin (FPN) protein levels were reduced in males (-68.9%, P < 0.001, -76.8%, P < 0.001, respectively) and females (-75.9%, P < 0.001, -62.9%, P < 0.001, respectively). Interestingly, in both sexes, heme exporter FLVCR1 mRNA increased in soleus, while protein levels decreased (-39.9% for males P = 0.010 and -49.1% for females P < 0.001). CONCLUSIONS Taken together, these data support that, in rats (1) extreme physical inactivity differently impacts the distribution of iron in both sexes, (2) splenic erythrophagocytosis could play a role in this iron misdistribution. The higher iron concentrations in atrophied soleus from both sexes are associated with a decoupling between the increase in iron storage proteins (i.e., ferritin and myoglobin) and the decrease in levels of iron export proteins (i.e., FPN and FLVCR1), thus supporting an iron sequestration in skeletal muscle under extreme physical inactivity.
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Affiliation(s)
- Mathieu Horeau
- Laboratory “Movement Sport and Health Sciences” EA7470University of Rennes/ENSRennesFrance
- INSERM, University of Rennes, INRAE, UMR 1317Nutrition Metabolisms and Cancer (NuMeCan) InstituteRennesFrance
| | - Melissa Delalande
- Laboratory “Movement Sport and Health Sciences” EA7470University of Rennes/ENSRennesFrance
| | - Martine Ropert
- INSERM, University of Rennes, INRAE, UMR 1317Nutrition Metabolisms and Cancer (NuMeCan) InstituteRennesFrance
- Elemental Analysis and Metabolism of Metals (AEM2) PlatformUniv Rennes CHU PontchaillouRennesFrance
| | - Patricia Leroyer
- INSERM, University of Rennes, INRAE, UMR 1317Nutrition Metabolisms and Cancer (NuMeCan) InstituteRennesFrance
| | - Brice Martin
- Laboratory “Movement Sport and Health Sciences” EA7470University of Rennes/ENSRennesFrance
| | - Luz Orfila
- Laboratory “Movement Sport and Health Sciences” EA7470University of Rennes/ENSRennesFrance
| | - Olivier Loréal
- INSERM, University of Rennes, INRAE, UMR 1317Nutrition Metabolisms and Cancer (NuMeCan) InstituteRennesFrance
- Elemental Analysis and Metabolism of Metals (AEM2) PlatformUniv Rennes CHU PontchaillouRennesFrance
| | - Frédéric Derbré
- Laboratory “Movement Sport and Health Sciences” EA7470University of Rennes/ENSRennesFrance
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Baschant U, Fuqua BK, Ledesma-Colunga M, Vulpe CD, McLachlan S, Hofbauer LC, Lusis AJ, Rauner M. Effects of dietary iron deficiency or overload on bone: Dietary details matter. Bone 2024; 184:117092. [PMID: 38575048 DOI: 10.1016/j.bone.2024.117092] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/13/2024] [Revised: 03/28/2024] [Accepted: 03/31/2024] [Indexed: 04/06/2024]
Abstract
PURPOSE Bone is susceptible to fluctuations in iron homeostasis, as both iron deficiency and overload are linked to poor bone strength in humans. In mice, however, inconsistent results have been reported, likely due to different diet setups or genetic backgrounds. Here, we assessed the effect of different high and low iron diets on bone in six inbred mouse strains (C57BL/6J, A/J, BALB/cJ, AKR/J, C3H/HeJ, and DBA/2J). METHODS Mice received a high (20,000 ppm) or low-iron diet (∼10 ppm) after weaning for 6-8 weeks. For C57BL/6J males, we used two dietary setups with similar amounts of iron, yet different nutritional compositions that were either richer ("TUD study") or poorer ("UCLA study") in minerals and vitamins. After sacrifice, liver, blood and bone parameters as well as bone turnover markers in the serum were analyzed. RESULTS Almost all mice on the UCLA study high iron diet had a significant decrease of cortical and trabecular bone mass accompanied by high bone resorption. Iron deficiency did not change bone microarchitecture or turnover in C57BL/6J, A/J, and DBA/2J mice, but increased trabecular bone mass in BALB/cJ, C3H/HeJ and AKR/J mice. In contrast to the UCLA study, male C57BL/6J mice in the TUD study did not display any changes in trabecular bone mass or turnover on high or low iron diet. However, cortical bone parameters were also decreased in TUD mice on the high iron diet. CONCLUSION Thus, these data show that cortical bone is more susceptible to iron overload than trabecular bone and highlight the importance of a nutrient-rich diet to potentially mitigate the negative effects of iron overload on bone.
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Affiliation(s)
- Ulrike Baschant
- Department of Medicine III & Center for Healthy Aging, Technische Universität Dresden, Germany
| | - Brie K Fuqua
- Department of Medicine, Division of Cardiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA
| | - Maria Ledesma-Colunga
- Department of Medicine III & Center for Healthy Aging, Technische Universität Dresden, Germany
| | - Christopher D Vulpe
- Department of Physiological Sciences, University of Florida Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL 32611, USA
| | | | - Lorenz C Hofbauer
- Department of Medicine III & Center for Healthy Aging, Technische Universität Dresden, Germany
| | - Aldons J Lusis
- Department of Medicine, Division of Cardiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA
| | - Martina Rauner
- Department of Medicine III & Center for Healthy Aging, Technische Universität Dresden, Germany.
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Brie B, Sarmento-Cabral A, Pascual F, Cordoba-Chacon J, Kineman RD, Becu-Villalobos D. Modifications of the GH Axis Reveal Unique Sexually Dimorphic Liver Signatures for Lcn13, Asns, Hamp2, Hao2, and Pgc1a. J Endocr Soc 2024; 8:bvae015. [PMID: 38370444 PMCID: PMC10872697 DOI: 10.1210/jendso/bvae015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/23/2023] [Indexed: 02/20/2024] Open
Abstract
Growth hormone (GH) modifies liver gene transcription in a sexually dimorphic manner to meet liver metabolic demands related to sex; thus, GH dysregulation leads to sex-biased hepatic disease. We dissected the steps of the GH regulatory cascade modifying GH-dependent genes involved in metabolism, focusing on the male-predominant genes Lcn13, Asns, and Cyp7b1, and the female-predominant genes Hao2, Pgc1a, Hamp2, Cyp2a4, and Cyp2b9. We explored mRNA expression in 2 settings: (i) intact liver GH receptor (GHR) but altered GH and insulin-like growth factor 1 (IGF1) levels (NeuroDrd2KO, HiGH, aHepIGF1kd, and STAT5bCA mouse lines); and (ii) liver loss of GHR, with or without STAT5b reconstitution (aHepGHRkd, and aHepGHRkd + STAT5bCA). Lcn13 was downregulated in males in most models, while Asns and Cyp7b1 were decreased in males by low GH levels or action, or constant GH levels, but unexpectedly upregulated in both sexes by the loss of liver Igf1 or constitutive Stat5b expression. Hao, Cyp2a4, and Cyp2b9 were generally decreased in female mice with low GH levels or action (NeuroDrd2KO and/or aHepGHRkd mice) and increased in HiGH females, while in contrast, Pgc1a was increased in female NeuroDrd2KO but decreased in STAT5bCA and aHepIGF1kd females. Bioinformatic analysis of RNAseq from aHepGHRkd livers stressed the greater impact of GHR loss on wide gene expression in males and highlighted that GH modifies almost completely different gene signatures in each sex. Concordantly, we show that altering different steps of the GH cascade in the liver modified liver expression of Lcn13, Asns, Cyp7b1, Hao2, Hamp2, Pgc1a, Cyp2a4, and Cyp2b9 in a sex- and gene-specific manner.
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Affiliation(s)
- Belen Brie
- Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), 1428 Ciudad de Buenos Aires, Argentina
| | - Andre Sarmento-Cabral
- Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Florencia Pascual
- Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), 1428 Ciudad de Buenos Aires, Argentina
| | - Jose Cordoba-Chacon
- Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Rhonda Denise Kineman
- Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, University of Illinois at Chicago, Chicago, IL 60612, USA
- Research and Development Division, Jesse Brown Veterans Affairs Medical Center, Chicago, IL 60612, USA
| | - Damasia Becu-Villalobos
- Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), 1428 Ciudad de Buenos Aires, Argentina
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Robin F, Chappard D, Leroyer P, Latour C, Mabilleau G, Monbet V, Cavey T, Horeau M, Derbré F, Roth MP, Ropert M, Guggenbuhl P, Loréal O. Differences in bone microarchitecture between genetic and secondary iron-overload mouse models suggest a role for hepcidin deficiency in iron-related osteoporosis. FASEB J 2023; 37:e23245. [PMID: 37874260 DOI: 10.1096/fj.202301184r] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2023] [Revised: 09/04/2023] [Accepted: 09/25/2023] [Indexed: 10/25/2023]
Abstract
Iron overload is one of the secondary osteoporosis etiologies. Cellular and molecular mechanisms involved in iron-related osteoporosis are not fully understood. AIM The aim of the study was to investigate the respective roles of iron excess and hepcidin, the systemic iron regulator, in the development of iron-related osteoporosis. MATERIAL AND METHODS We used mice models with genetic iron overload (GIO) related to hepcidin deficiency (Hfe-/- and Bmp6-/- ) and secondary iron overload (SIO) exhibiting a hepcidin increase secondary to iron excess. Iron concentration and transferrin saturation levels were evaluated in serum and hepatic, spleen, and bone iron concentrations were assessed by ICP-MS and Perl's staining. Gene expression was evaluated by quantitative RT-PCR. Bone micro-architecture was evaluated by micro-CT. The osteoblastic MC3T3 murine cells that are able to mineralize were exposed to iron and/or hepcidin. RESULTS Despite an increase of bone iron concentration in all overloaded mice models, bone volume/total volume (BV/TV) and trabecular thickness (Tb.Th) only decreased significantly in GIO, at 12 months for Hfe-/- and from 6 months for Bmp6-/- . Alterations in bone microarchitecture in the Bmp6-/- model were positively correlated with hepcidin levels (BV/TV (ρ = +.481, p < .05) and Tb.Th (ρ = +.690, p < .05). Iron deposits were detected in the bone trabeculae of Hfe-/- and Bmp6-/- mice, while iron deposits were mainly visible in bone marrow macrophages in secondary iron overload. In cell cultures, ferric ammonium citrate exposure abolished the mineralization process for concentrations above 5 μM, with a parallel decrease in osteocalcin, collagen 1, and alkaline phosphatase mRNA levels. Hepcidin supplementation of cells had a rescue effect on the collagen 1 and alkaline phosphatase expression level decrease. CONCLUSION Together, these data suggest that iron in excess alone is not sufficient to induce osteoporosis and that low hepcidin levels also contribute to the development of osteoporosis.
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Affiliation(s)
- François Robin
- INSERM, Univ Rennes, INRAE, CHU Rennes, U 1241, Institut NuMeCan (Nutrition Metabolisms and Cancer), Rennes, France
| | - Daniel Chappard
- GEROM, LHEA, IRIS-IBS Biology Institut, Angers cedex, France
| | - Patricia Leroyer
- INSERM, Univ Rennes, INRAE, CHU Rennes, U 1241, Institut NuMeCan (Nutrition Metabolisms and Cancer), Rennes, France
| | - Chloé Latour
- IRSD, Univ Toulouse, INSERM, INRAE, ENVT, UPS, Toulouse, France
| | - Guillaume Mabilleau
- Univ Angers, Nantes Université, Oniris, Inserm, RMeS, REGOS, SFR ICAT, Angers, France
| | | | - Thibault Cavey
- INSERM, Univ Rennes, INRAE, CHU Rennes, U 1241, Institut NuMeCan (Nutrition Metabolisms and Cancer), Rennes, France
| | - Mathieu Horeau
- INSERM, Univ Rennes, INRAE, CHU Rennes, U 1241, Institut NuMeCan (Nutrition Metabolisms and Cancer), Rennes, France
- Laboratory "Movement Sport and Health Sciences" EA7470, University of Rennes/ENS Rennes, Rennes, France
| | - Frédéric Derbré
- Laboratory "Movement Sport and Health Sciences" EA7470, University of Rennes/ENS Rennes, Rennes, France
| | | | - Martine Ropert
- INSERM, Univ Rennes, INRAE, CHU Rennes, U 1241, Institut NuMeCan (Nutrition Metabolisms and Cancer), Rennes, France
- AEM2 Platform, Univ Rennes, University Hospital, Rennes, France
| | - Pascal Guggenbuhl
- INSERM, Univ Rennes, INRAE, CHU Rennes, U 1241, Institut NuMeCan (Nutrition Metabolisms and Cancer), Rennes, France
| | - Olivier Loréal
- INSERM, Univ Rennes, INRAE, CHU Rennes, U 1241, Institut NuMeCan (Nutrition Metabolisms and Cancer), Rennes, France
- AEM2 Platform, Univ Rennes, University Hospital, Rennes, France
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5
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Erythroid overproduction of erythroferrone causes iron overload and developmental abnormalities in mice. Blood 2022; 139:439-451. [PMID: 34614145 PMCID: PMC8777203 DOI: 10.1182/blood.2021014054] [Citation(s) in RCA: 24] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2021] [Accepted: 10/04/2021] [Indexed: 02/08/2023] Open
Abstract
The hormone erythroferrone (ERFE) is produced by erythroid cells in response to hemorrhage, hypoxia, or other erythropoietic stimuli, and it suppresses the hepatic production of the iron-regulatory hormone hepcidin, thereby mobilizing iron for erythropoiesis. Suppression of hepcidin by ERFE is believed to be mediated by interference with paracrine bone morphogenetic protein (BMP) signaling that regulates hepcidin transcription in hepatocytes. In anemias with ineffective erythropoiesis, ERFE is pathologically overproduced, but its contribution to the clinical manifestations of these anemias is not well understood. We generated 3 lines of transgenic mice with graded erythroid overexpression of ERFE and found that they developed dose-dependent iron overload, impaired hepatic BMP signaling, and relative hepcidin deficiency. These findings add to the evidence that ERFE is a mediator of iron overload in conditions in which ERFE is overproduced, including anemias with ineffective erythropoiesis. At the highest levels of ERFE overexpression, the mice manifested decreased perinatal survival, impaired growth, small hypofunctional kidneys, decreased gonadal fat depots, and neurobehavioral abnormalities, all consistent with impaired organ-specific BMP signaling during development. Neutralizing excessive ERFE in congenital anemias with ineffective erythropoiesis may not only prevent iron overload but may have additional benefits for growth and development.
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Development of insulin resistance preceded major changes in iron homeostasis in mice fed a high-fat diet. J Nutr Biochem 2020; 84:108441. [PMID: 32629238 PMCID: PMC7115812 DOI: 10.1016/j.jnutbio.2020.108441] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2019] [Revised: 03/10/2020] [Accepted: 05/22/2020] [Indexed: 02/06/2023]
Abstract
Type 2 diabetes mellitus (T2DM) and insulin resistance (IR) have been associated with dysregulation of iron metabolism. The basis for this association is not completely understood. To attempt to investigate this, we studied temporal associations between onset of insulin resistance (IR) and dysregulated iron homeostasis, in a mouse model of T2DM. Male C57Bl/6 mice (aged 8 weeks) were fed a high-fat diet (HFD; 60% energy from fat) or a control diet (CD; 10% energy from fat) for 4, 8, 12, 16, 20 and 24 weeks. Development of IR was documented, and various metabolic, inflammatory and iron-related parameters were studied in these mice. HFD-feeding induced weight gain, hepato-steatosis and IR in the mice. Onset of IR occurred from 12 weeks onwards. Hepatic iron stores progressively declined from 16 weeks onwards. Accompanying changes included a decrease in hepatic hepcidin (Hamp1) mRNA expression and serum hepcidin levels and an increase in iron content in the epididymal white adipose tissue (eWAT). Iron content in the liver negatively correlated with that in the eWAT. Factors known to regulate hepatic Hamp1 expression (such as serum iron levels, systemic inflammation, and bone marrow-derived erythroid regulators) were not affected by HFD-feeding. In conclusion, the results show that the onset of IR in HFD-fed mice preceded dysregulation of iron homeostasis, evidence of which were found both in the liver and visceral adipose tissue.
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McManus JF, Nguyen NYN, Davey RA, MacLean HE, Pomilio G, McCormack MP, Chiu WS, Wei AH, Zajac JD, Curtis DJ. Androgens stimulate erythropoiesis through the DNA-binding activity of the androgen receptor in non-hematopoietic cells. Eur J Haematol 2020; 105:247-254. [PMID: 32311143 DOI: 10.1111/ejh.13431] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2020] [Revised: 04/10/2020] [Accepted: 04/13/2020] [Indexed: 12/27/2022]
Abstract
BACKGROUND Androgens function through DNA and non-DNA binding-dependent signalling of the androgen receptor (AR). How androgens promote erythropoiesis is not fully understood. DESIGN AND METHODS To identify the androgen signalling pathway, we treated male mice lacking the second zinc finger of the DNA-binding domain of the AR (ARΔZF2 ) with non-aromatizable 5α-dihydrotestosterone (5α-DHT) or aromatizable testosterone. To distinguish direct hematopoietic and non-hematopoietic mechanisms, we performed bone marrow reconstitution experiments. RESULTS In wild-type mice, 5α-DHT had greater erythroid activity than testosterone, which can be aromatized to estradiol. The erythroid response in wild-type mice following 5α-DHT treatment was associated with increased serum erythropoietin (EPO) and its downstream target erythroferrone, and hepcidin suppression. 5α-DHT had no erythroid activity in ARΔZF2 mice, proving the importance of DNA binding by the AR. Paradoxically, testosterone, but not 5α-DHT, suppressed EPO levels in ARΔZF2 mice, suggesting testosterone following aromatization may oppose the erythroid-stimulating effects of androgens. Female wild-type mice reconstituted with ARΔZF2 bone marrow cells remained responsive to 5α-DHT. In contrast, ARΔZF2 mice reconstituted with female wild-type bone marrow cells showed no response to 5α-DHT. CONCLUSION Erythroid promoting effects of androgens are mediated through DNA binding-dependent actions of the AR in non-hematopoietic cells, including stimulating EPO expression.
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Affiliation(s)
- Julie F McManus
- Central Clinical School, Australian Centre for Blood Diseases, Monash University, Melbourne, Vic., Australia.,Human Molecular Pathology, Alfred Pathology Service, Alfred Health, Melbourne, Vic., Australia
| | - Nhu-Y N Nguyen
- Cartherics Pty Ltd, Melbourne, Vic., Australia.,Hudson Institute of Medical Research, Melbourne, Vic., Australia
| | - Rachel A Davey
- Department of Medicine, Austin Health, The University of Melbourne, Melbourne, Vic., Australia
| | - Helen E MacLean
- Department of Medicine, Austin Health, The University of Melbourne, Melbourne, Vic., Australia
| | - Giovanna Pomilio
- Central Clinical School, Australian Centre for Blood Diseases, Monash University, Melbourne, Vic., Australia.,Department of Clinical Haematology, Alfred Health, Melbourne, Vic., Australia
| | - Matthew P McCormack
- Central Clinical School, Australian Centre for Blood Diseases, Monash University, Melbourne, Vic., Australia
| | - Wan Sze Chiu
- Department of Medicine, Austin Health, The University of Melbourne, Melbourne, Vic., Australia
| | - Andrew H Wei
- Central Clinical School, Australian Centre for Blood Diseases, Monash University, Melbourne, Vic., Australia.,Department of Clinical Haematology, Alfred Health, Melbourne, Vic., Australia
| | - Jeffrey D Zajac
- Department of Medicine, Austin Health, The University of Melbourne, Melbourne, Vic., Australia
| | - David J Curtis
- Central Clinical School, Australian Centre for Blood Diseases, Monash University, Melbourne, Vic., Australia.,Department of Clinical Haematology, Alfred Health, Melbourne, Vic., Australia
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Sanyear C, Butthep P, Eamsaard W, Fucharoen S, Svasti S, Masaratana P. Iron homeostasis in a mouse model of thalassemia intermedia is altered between adolescence and adulthood. PeerJ 2020; 8:e8802. [PMID: 32219031 PMCID: PMC7085893 DOI: 10.7717/peerj.8802] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2019] [Accepted: 02/25/2020] [Indexed: 01/03/2023] Open
Abstract
Background Iron overload is one of common complications of β-thalassemia. Systemic iron homeostasis is regulated by iron-regulatory hormone, hepcidin, which inhibits intestinal iron absorption and iron recycling by reticuloendothelial system. In addition, body iron status and requirement can be altered with age. In adolescence, iron requirement is increased due to blood volume expansion and growth spurt. Heterozygous β-globin knockout mice (Hbbth3/+; BKO) is a mouse model of thalassemia widely used to study iron homeostasis under this pathological condition. However, effects of age on iron homeostasis, particularly the expression of genes involved in hemoglobin metabolism as well as erythroid regulators in the spleen, during adolescence have not been explored in this mouse model. Methods Iron parameters as well as the mRNA expression of hepcidin and genes involved in iron transport and metabolism in wildtype (WT) and BKO mice during adolescence (6–7 weeks old) and adulthood (16–20 weeks old) were analyzed and compared by 2-way ANOVA. Results The transition of adolescence to adulthood was associated with reductions in duodenal iron transporter mRNA expression and serum iron levels of both WT and BKO mice. Erythrocyte parameters in BKO mice remained abnormal in both age groups despite persistent induction of genes involved in hemoglobin metabolism in the spleen and progressively increased extramedullary erythropiesis. In BKO mice, adulthood was associated with increased liver hepcidin and ferroportin mRNA expression along with splenic erythroferrone mRNA suppression compared to adolescence. Conclusion Our results demonstrate that iron homeostasis in a mouse model of thalassemia intermedia is altered between adolescence and adulthood. The present study underscores the importance of the age of thalassemic mice in the study of molecular or pathophysiological changes under thalassemic condition.
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Affiliation(s)
- Chanita Sanyear
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Punnee Butthep
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Wiraya Eamsaard
- Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
| | - Suthat Fucharoen
- Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Bangkok, Thailand
| | - Saovaros Svasti
- Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Bangkok, Thailand
| | - Patarabutr Masaratana
- Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
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Kenawi M, Rouger E, Island ML, Leroyer P, Robin F, Rémy S, Tesson L, Anegon I, Nay K, Derbré F, Brissot P, Ropert M, Cavey T, Loréal O. Ceruloplasmin deficiency does not induce macrophagic iron overload: lessons from a new rat model of hereditary aceruloplasminemia. FASEB J 2019; 33:13492-13502. [DOI: 10.1096/fj.201901106r] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Affiliation(s)
- Moussa Kenawi
- INSERM, Univ Rennes, INRA, Platform Analyse Elémentaire et Métabolisme des Métaux, UMR 1241 Institut NuMeCan (Nutrition, Metabolism, and Cancer), Centre Hospitalier Universitaire (CHU) Pontchaillou, Rennes, France
| | - Emmanuel Rouger
- INSERM, Univ Rennes, INRA, Platform Analyse Elémentaire et Métabolisme des Métaux, UMR 1241 Institut NuMeCan (Nutrition, Metabolism, and Cancer), Centre Hospitalier Universitaire (CHU) Pontchaillou, Rennes, France
| | - Marie-Laure Island
- INSERM, Univ Rennes, INRA, Platform Analyse Elémentaire et Métabolisme des Métaux, UMR 1241 Institut NuMeCan (Nutrition, Metabolism, and Cancer), Centre Hospitalier Universitaire (CHU) Pontchaillou, Rennes, France
| | - Patricia Leroyer
- INSERM, Univ Rennes, INRA, Platform Analyse Elémentaire et Métabolisme des Métaux, UMR 1241 Institut NuMeCan (Nutrition, Metabolism, and Cancer), Centre Hospitalier Universitaire (CHU) Pontchaillou, Rennes, France
| | - François Robin
- INSERM, Univ Rennes, INRA, Platform Analyse Elémentaire et Métabolisme des Métaux, UMR 1241 Institut NuMeCan (Nutrition, Metabolism, and Cancer), Centre Hospitalier Universitaire (CHU) Pontchaillou, Rennes, France
| | - Séverine Rémy
- INSERM UMR 1064- Centre de Recherches en Transplantation et Immunologie (CRTI), Transgenic Rats ImmunoPhenomic facility, Nantes, France
| | - Laurent Tesson
- INSERM UMR 1064- Centre de Recherches en Transplantation et Immunologie (CRTI), Transgenic Rats ImmunoPhenomic facility, Nantes, France
| | - Ignacio Anegon
- INSERM UMR 1064- Centre de Recherches en Transplantation et Immunologie (CRTI), Transgenic Rats ImmunoPhenomic facility, Nantes, France
| | - Kévin Nay
- Laboratory Movement, Sport, and Health Sciences (M2S-EA7470), University Rennes 2–Ecole Normale Supérieure (ENS) Rennes, Bruz, France
| | - Frédéric Derbré
- Laboratory Movement, Sport, and Health Sciences (M2S-EA7470), University Rennes 2–Ecole Normale Supérieure (ENS) Rennes, Bruz, France
| | - Pierre Brissot
- INSERM, Univ Rennes, INRA, Platform Analyse Elémentaire et Métabolisme des Métaux, UMR 1241 Institut NuMeCan (Nutrition, Metabolism, and Cancer), Centre Hospitalier Universitaire (CHU) Pontchaillou, Rennes, France
| | - Martine Ropert
- INSERM, Univ Rennes, INRA, Platform Analyse Elémentaire et Métabolisme des Métaux, UMR 1241 Institut NuMeCan (Nutrition, Metabolism, and Cancer), Centre Hospitalier Universitaire (CHU) Pontchaillou, Rennes, France
| | - Thibault Cavey
- INSERM, Univ Rennes, INRA, Platform Analyse Elémentaire et Métabolisme des Métaux, UMR 1241 Institut NuMeCan (Nutrition, Metabolism, and Cancer), Centre Hospitalier Universitaire (CHU) Pontchaillou, Rennes, France
| | - Olivier Loréal
- INSERM, Univ Rennes, INRA, Platform Analyse Elémentaire et Métabolisme des Métaux, UMR 1241 Institut NuMeCan (Nutrition, Metabolism, and Cancer), Centre Hospitalier Universitaire (CHU) Pontchaillou, Rennes, France
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10
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Mallett CL, Hix JML, Kiupel M, Shapiro EM. Effect of mouse strain and diet on feasibility of MRI-based cell tracking in the liver. Magn Reson Med 2019; 83:2276-2283. [PMID: 31765493 DOI: 10.1002/mrm.28081] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2019] [Revised: 10/23/2019] [Accepted: 10/25/2019] [Indexed: 11/11/2022]
Abstract
PURPOSE MRI-based cell tracking identifies the location of magnetically labeled cells with hypointense voxels. Here we demonstrate a strain-dependent effect of liver MRI background on the feasibility of MRI-based cell tracking of transplanted cells in the mouse liver. METHODS FVB mice (GFP-LUC and NOG) and C57BL/6 mice (GFP+ and wild-type) were fed 3 different diets with varying iron content. In vivo T 2 ∗ -weighted images and T 2 ∗ maps of the liver were acquired at different ages. Magnetically labeled cancer cells were injected intrasplenically for hepatic migration; then, mice were imaged by in vivo MRI and bioluminescence imaging. Livers were also imaged ex vivo by magnetic particle imaging. RESULTS R 2 ∗ increased with age in FVBNOG and FVBGFP-LUC mice that were fed diets sufficient in iron. FVBNOG mice developed a mottled appearance in their livers with age that did not occur in FVBGFP-LUC mice. R 2 ∗ was unchanging with age in C57BL/6GFP mice, and the liver remained bright and homogenous. Labeled cells were not detectable by MRI in some livers despite successful engraftment as shown by bioluminescence imaging and magnetic particle imaging. CONCLUSION Strain, diet, and age are important considerations for MRI-based cell tracking in the liver. If a model with excessive liver iron must be used, alternative imaging methods such as magnetic particle imaging can be considered.
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Affiliation(s)
- Christiane L Mallett
- Department of Radiology and Institute for Quantitative Health Science and Engineering, Michigan State University, East Lansing, Michigan
| | - Jeremy M L Hix
- Department of Radiology and Institute for Quantitative Health Science and Engineering, Michigan State University, East Lansing, Michigan
| | - Matti Kiupel
- Veterinary Diagnostic Laboratory, Michigan State University, East Lansing, Michigan
| | - Erik M Shapiro
- Department of Radiology and Institute for Quantitative Health Science and Engineering, Michigan State University, East Lansing, Michigan
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11
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Transferrin receptor 1 controls systemic iron homeostasis by fine-tuning hepcidin expression to hepatocellular iron load. Blood 2018; 133:344-355. [PMID: 30538134 DOI: 10.1182/blood-2018-05-850404] [Citation(s) in RCA: 65] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2018] [Accepted: 12/04/2018] [Indexed: 02/06/2023] Open
Abstract
Transferrin receptor 1 (Tfr1) mediates uptake of circulating transferrin-bound iron to developing erythroid cells and other cell types. Its critical physiological function is highlighted by the embryonic lethal phenotype of Tfr1-knockout (Tfrc-/-) mice and the pathologies of several tissue-specific knockouts. We generated TfrcAlb-Cre mice bearing hepatocyte-specific ablation of Tfr1 to explore implications in hepatocellular and systemic iron homeostasis. TfrcAlb-Cre mice are viable and do not display any apparent liver pathology. Nevertheless, their liver iron content (LIC) is lower compared with that of control Tfrcfl/fl littermates as a result of the reduced capacity of Tfr1-deficient hepatocytes to internalize iron from transferrin. Even though liver Hamp messenger RNA (mRNA) and serum hepcidin levels do not differ between TfrcAlb-Cre and Tfrcfl/fl mice, Hamp/LIC and hepcidin/LIC ratios are significantly higher in the former. Importantly, this is accompanied by modest hypoferremia and microcytosis, and it predisposes TfrcAlb-Cre mice to iron-deficiency anemia. TfrcAlb-Cre mice appropriately regulate Hamp expression following dietary iron manipulations or holo-transferrin injection. Holo-transferrin also triggers proper induction of Hamp mRNA, ferritin, and Tfr2 in primary TfrcAlb-Cre hepatocytes. We further show that these cells can acquire 59Fe from 59Fe-transferrin, presumably via Tfr2. We conclude that Tfr1 is redundant for basal hepatocellular iron supply but essential for fine-tuning hepcidin responses according to the iron load of hepatocytes. Our data are consistent with an inhibitory function of Tfr1 on iron signaling to hepcidin via its interaction with Hfe. Moreover, they highlight hepatocellular Tfr1 as a link between cellular and systemic iron-regulatory pathways.
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12
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Mirciov CSG, Wilkins SJ, Hung GCC, Helman SL, Anderson GJ, Frazer DM. Circulating iron levels influence the regulation of hepcidin following stimulated erythropoiesis. Haematologica 2018; 103:1616-1626. [PMID: 29903760 PMCID: PMC6165793 DOI: 10.3324/haematol.2017.187245] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2017] [Accepted: 06/11/2018] [Indexed: 01/01/2023] Open
Abstract
The stimulation of erythrocyte formation increases the demand for iron by the bone marrow and this in turn may affect the levels of circulating diferric transferrin. As this molecule influences the production of the iron regulatory hormone hepcidin, we hypothesized that erythropoiesis-driven changes in diferric transferrin levels could contribute to the decrease in hepcidin observed following the administration of erythropoietin. To examine this, we treated mice with erythropoietin and examined diferric transferrin at various time points up to 18 hours. We also investigated the effect of altering diferric transferrin levels on erythropoietin-induced inhibition of Hamp1, the gene encoding hepcidin. We detected a decrease in diferric transferrin levels 5 hours after erythropoietin injection and prior to any inhibition of the hepatic Hamp1 message. Diferric transferrin returned to control levels 12 hours after erythropoietin injection and had increased beyond control levels by 18 hours. Increasing diferric transferrin levels via intravenous iron injection prevented the inhibition of Hamp1 expression by erythropoietin without altering hepatic iron concentration or the expression of Erfe, the gene encoding erythroferrone. These results suggest that diferric transferrin likely contributes to the inhibition of hepcidin production in the period shortly after injection of erythropoietin and that, under the conditions examined, increasing diferric transferrin levels can overcome the inhibitory effect of erythroferrone on hepcidin production. They also imply that the decrease in Hamp1 expression in response to an erythropoietic stimulus is likely to be mediated by multiple signals.
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Affiliation(s)
- Cornel S G Mirciov
- Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Herston, Australia.,School of Medicine, The University of Queensland, St Lucia, Australia
| | - Sarah J Wilkins
- Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Herston, Australia
| | - Grace C C Hung
- Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Herston, Australia
| | - Sheridan L Helman
- Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Herston, Australia.,School of Biomedical Sciences, Queensland University of Technology, Gardens Point, Australia
| | - Gregory J Anderson
- Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Herston, Australia.,School of Medicine, The University of Queensland, St Lucia, Australia.,School of Chemistry and Molecular Bioscience, The University of Queensland, St Lucia, Australia
| | - David M Frazer
- Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Herston, Australia .,School of Medicine, The University of Queensland, St Lucia, Australia
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13
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In a Mouse Model of Sepsis, Hepcidin Ablation Ameliorates Anemia More Effectively than Iron and Erythropoietin Treatment. Shock 2018; 48:490-497. [PMID: 28452907 DOI: 10.1097/shk.0000000000000886] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Intensive care unit (ICU) anemia is an extreme version of anemia of inflammation that occurs commonly in critically ill patients and is associated with increased morbidity and mortality. Currently available therapies for ICU anemia have shown inconsistent efficacies in clinical trials. We conducted a systematic study of the effects of early versus delayed iron (Fe) and/or erythropoietin (EPO) therapy in our previously characterized mouse model of ICU anemia based on an injection of heat-killed Brucella abortus. To study the effects of ongoing inflammation on the response to therapy, inflamed wild-type (WT) and hepcidin knockout (HKO) mice were treated at either early (days 1 and 2) or delayed (days 7 and 8) time points after the inflammatory stimulus. In the early treatment group, Fe and/or EPO therapy did not increase hemoglobin (Hgb) levels or reticulocyte production in either the inflamed WT or HKO groups. In the delayed treatment group, combination Fe + EPO therapy did increase Hgb and reticulocyte production in WT mice (mean ΔHgb in WT saline group -9.2 g/dL vs. Fe/EPO -5.5 g/dL; P < 0.001). The HKO mice in the delayed treatment group did not improve their Hgb, but HKO mice in all treatment groups developed a milder anemia than the WT mice. Our findings indicate that combination Fe + EPO therapy is effective in partially reversing ICU anemia when administered after the phase of acute inflammation. Hepcidin ablation alone was more effective in attenuating ICU anemia than Fe + EPO therapy, which indicates the potential of antihepcidin therapeutics in treating ICU anemia.
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14
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McLachlan S, Page KE, Lee SM, Loguinov A, Valore E, Hui ST, Jung G, Zhou J, Lusis AJ, Fuqua B, Ganz T, Nemeth E, Vulpe CD. Hamp1 mRNA and plasma hepcidin levels are influenced by sex and strain but do not predict tissue iron levels in inbred mice. Am J Physiol Gastrointest Liver Physiol 2017; 313:G511-G523. [PMID: 28798083 PMCID: PMC5792216 DOI: 10.1152/ajpgi.00307.2016] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/18/2016] [Revised: 07/17/2017] [Accepted: 07/24/2017] [Indexed: 01/31/2023]
Abstract
Iron homeostasis is tightly regulated, and the peptide hormone hepcidin is considered to be a principal regulator of iron metabolism. Previous studies in a limited number of mouse strains found equivocal sex- and strain-dependent differences in mRNA and serum levels of hepcidin and reported conflicting data on the relationship between hepcidin (Hamp1) mRNA levels and iron status. Our aim was to clarify the relationships between strain, sex, and hepcidin expression by examining multiple tissues and the effects of different dietary conditions in multiple inbred strains. Two studies were done: first, Hamp1 mRNA, liver iron, and plasma diferric transferrin levels were measured in 14 inbred strains on a control diet; and second, Hamp1 mRNA and plasma hepcidin levels in both sexes and iron levels in the heart, kidneys, liver, pancreas, and spleen in males were measured in nine inbred/recombinant inbred strains raised on an iron-sufficient or high-iron diet. Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). However, liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice fed iron-sufficient or high-iron diets, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males. We also measured plasma erythroferrone, performed RNA-sequencing analysis of liver samples from six inbred strains fed the iron-sufficient, low-iron, or high-iron diets, and explored differences in gene expression between the strains with the highest and lowest hepcidin levels.NEW & NOTEWORTHY Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). Liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males.
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Affiliation(s)
- Stela McLachlan
- Centre for Population Health Sciences, Usher Institute of Population Health Sciences and Informatics, University of Edinburgh, Edinburgh, United Kingdom;
| | - Kathryn E. Page
- 2Department of Nutritional Science & Toxicology, University of California, Berkeley, California;
| | - Seung-Min Lee
- 3Department of Food and Nutrition, College of Human Ecology, Yonsei University, Seoul, Korea;
| | - Alex Loguinov
- 5Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida
| | - Erika Valore
- 4Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California; and
| | - Simon T. Hui
- 4Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California; and
| | - Grace Jung
- 4Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California; and
| | - Jie Zhou
- 5Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida
| | - Aldons J. Lusis
- 4Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California; and
| | - Brie Fuqua
- 4Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California; and
| | - Tomas Ganz
- 4Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California; and
| | - Elizabeta Nemeth
- 4Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California; and
| | - Chris D. Vulpe
- 2Department of Nutritional Science & Toxicology, University of California, Berkeley, California; ,5Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida
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15
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Latour C, Wlodarczyk MF, Jung G, Gineste A, Blanchard N, Ganz T, Roth MP, Coppin H, Kautz L. Erythroferrone contributes to hepcidin repression in a mouse model of malarial anemia. Haematologica 2016; 102:60-68. [PMID: 27658439 DOI: 10.3324/haematol.2016.150227] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2016] [Accepted: 09/14/2016] [Indexed: 01/10/2023] Open
Abstract
Malaria, a major global health challenge worldwide, is accompanied by a severe anemia secondary to hemolysis and increased erythrophagocytosis. Iron is an essential functional component of erythrocyte hemoglobin and its availability is controlled by the liver-derived hormone hepcidin. We examined the regulation of hepcidin during malarial infection in mice using the rodent parasite Plasmodium berghei K173. Mice infected with Plasmodium berghei K173 develop a severe anemia and die after 18 to 22 days without cerebral malaria. During the early phase of blood-stage infection (days 1 to 5), a strong inflammatory signature was associated with an increased production of hepcidin. Between days 7 and 18, while infection progressed, red blood cell count, hemoglobin and hematocrit dramatically decreased. In the late phase of malarial infection, hepcidin production was reduced concomitantly to an increase in the messenger RNA expression of the hepcidin suppressor erythroferrone in the bone marrow and the spleen. Compared with wild-type mice, Erfe-/- mice failed to adequately suppress hepcidin expression after infection with Plasmodium berghei K173. Importantly, the sustained production of hepcidin allowed by erythroferrone ablation was associated with decreased parasitemia, providing further evidence that transient iron restriction could be beneficial in the treatment of malaria.
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Affiliation(s)
- Chloé Latour
- IRSD, Université de Toulouse, INSERM U1220, INRA U1416, ENVT, UPS, Toulouse, France
| | - Myriam F Wlodarczyk
- CPTP, Université de Toulouse, CNRS U5282, Inserm U1043, UPS, Toulouse, France
| | - Grace Jung
- Department of Pathology, David Geffen School of Medicine, University of California, Los Angeles, USA
| | - Aurélie Gineste
- IRSD, Université de Toulouse, INSERM U1220, INRA U1416, ENVT, UPS, Toulouse, France
| | - Nicolas Blanchard
- CPTP, Université de Toulouse, CNRS U5282, Inserm U1043, UPS, Toulouse, France
| | - Tomas Ganz
- Department of Pathology, David Geffen School of Medicine, University of California, Los Angeles, USA.,Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, USA
| | - Marie-Paule Roth
- IRSD, Université de Toulouse, INSERM U1220, INRA U1416, ENVT, UPS, Toulouse, France
| | - Hélène Coppin
- IRSD, Université de Toulouse, INSERM U1220, INRA U1416, ENVT, UPS, Toulouse, France
| | - Léon Kautz
- IRSD, Université de Toulouse, INSERM U1220, INRA U1416, ENVT, UPS, Toulouse, France
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16
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Armitage AE, Lim PJ, Frost JN, Pasricha SR, Soilleux EJ, Evans E, Morovat A, Santos A, Diaz R, Biggs D, Davies B, Gileadi U, Robbins PA, Lakhal-Littleton S, Drakesmith H. Induced Disruption of the Iron-Regulatory Hormone Hepcidin Inhibits Acute Inflammatory Hypoferraemia. J Innate Immun 2016; 8:517-28. [PMID: 27423740 PMCID: PMC5322583 DOI: 10.1159/000447713] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2016] [Accepted: 06/20/2016] [Indexed: 12/15/2022] Open
Abstract
Withdrawal of iron from serum (hypoferraemia) is a conserved innate immune antimicrobial strategy that can withhold this critical nutrient from invading pathogens, impairing their growth. Hepcidin (Hamp1) is the master regulator of iron and its expression is induced by inflammation. Mice lacking Hamp1 from birth rapidly accumulate iron and are susceptible to infection by blood-dwelling siderophilic bacteria such as Vibrio vulnificus. In order to study the innate immune role of hepcidin against a background of normal iron status, we developed a transgenic mouse model of tamoxifen-sensitive conditional Hamp1 deletion (termed iHamp1-KO mice). These mice attain adulthood with an iron status indistinguishable from littermate controls. Hamp1 disruption and the consequent decline of serum hepcidin concentrations occurred within hours of a single tamoxifen dose. We found that the TLR ligands LPS and Pam3CSK4 and heat-killed Brucella abortus caused an equivalent induction of inflammation in control and iHamp1-KO mice. Pam3CSK4 and B. abortus only caused a drop in serum iron in control mice, while hypoferraemia due to LPS was evident but substantially blunted in iHamp1-KO mice. Our results characterise a powerful new model of rapidly inducible hepcidin disruption, and demonstrate the critical contribution of hepcidin to the hypoferraemia of inflammation.
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Affiliation(s)
- Andrew E Armitage
- Department of Biochemistry, Birmingham Heartlands Hospital, Heart of England NHS Foundation Trust, Birmingham, UK
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17
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Mandò C, Calabrese S, Mazzocco MI, Novielli C, Anelli GM, Antonazzo P, Cetin I. Sex specific adaptations in placental biometry of overweight and obese women. Placenta 2015; 38:1-7. [PMID: 26907375 DOI: 10.1016/j.placenta.2015.12.008] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/27/2015] [Revised: 11/30/2015] [Accepted: 12/14/2015] [Indexed: 12/19/2022]
Abstract
INTRODUCTION Placental biometry at birth has been shown to predict chronic disease in later life. We hypothesized that maternal overweight/obesity, a state of low-grade inflammation and risk factor for adverse pregnancy outcome, could negatively influence placental development and that differences would be sex-specific. METHODS 696 women (537 normal-weight, NW; 112 overweight, OW; 47 obese, OB) with singleton uncomplicated pregnancies were prospectively enrolled at term delivery. Gestational age, maternal (age, height, pre-pregnancy BMI, gestational weight gain -GWG, hemoglobin, hematocrit and glycemia), fetal (weight, length, ponderal index, cranial circumference) and placental (weight, diameters) data were collected. Placental area, thickness and efficiency (fetal/placental weight ratio, F/P) were calculated. RESULTS GWG was within standard recommendations in OB, while OW exceeded it. Placental weight was significantly higher in OW versus NW, but not in OB, leading to significantly higher placental thickness and lower F/P in this group. In the total population, a significant interaction effect between maternal BMI and fetal sex on placental weight and efficiency was found. Indeed, differences in placental parameters were present only in female offspring. DISCUSSION In our population of OW and OB uncomplicated pregnancies only OW women, presenting GWG over standard recommendations, had thicker and less efficient placentas. We also reported different placental adaptation depending on fetal sex, with significant changes only in female fetuses. This may be part of a female-specific strategy aiming to ensure survival if another adverse event occurs. Customized counseling according to maternal BMI and fetal sex should be evaluated in clinical care.
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Affiliation(s)
- Chiara Mandò
- Department of Mother and Child, Hospital L. Sacco, Department of Biomedical and Clinical Sciences L. Sacco, and Center for Fetal Research Giorgio Pardi, Università degli studi di Milano, Italy.
| | - Stefania Calabrese
- Department of Mother and Child, Hospital L. Sacco, Department of Biomedical and Clinical Sciences L. Sacco, and Center for Fetal Research Giorgio Pardi, Università degli studi di Milano, Italy
| | - Martina Ilaria Mazzocco
- Department of Mother and Child, Hospital L. Sacco, Department of Biomedical and Clinical Sciences L. Sacco, and Center for Fetal Research Giorgio Pardi, Università degli studi di Milano, Italy
| | - Chiara Novielli
- Department of Mother and Child, Hospital L. Sacco, Department of Biomedical and Clinical Sciences L. Sacco, and Center for Fetal Research Giorgio Pardi, Università degli studi di Milano, Italy
| | - Gaia Maria Anelli
- Department of Mother and Child, Hospital L. Sacco, Department of Biomedical and Clinical Sciences L. Sacco, and Center for Fetal Research Giorgio Pardi, Università degli studi di Milano, Italy
| | - Patrizio Antonazzo
- Department of Mother and Child, Hospital L. Sacco, Department of Biomedical and Clinical Sciences L. Sacco, and Center for Fetal Research Giorgio Pardi, Università degli studi di Milano, Italy
| | - Irene Cetin
- Department of Mother and Child, Hospital L. Sacco, Department of Biomedical and Clinical Sciences L. Sacco, and Center for Fetal Research Giorgio Pardi, Università degli studi di Milano, Italy
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18
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Kim A, Fung E, Parikh SG, Gabayan V, Nemeth E, Ganz T. Isocitrate treatment of acute anemia of inflammation in a mouse model. Blood Cells Mol Dis 2015; 56:31-6. [PMID: 26603720 DOI: 10.1016/j.bcmd.2015.09.007] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2015] [Revised: 09/24/2015] [Accepted: 09/24/2015] [Indexed: 12/21/2022]
Abstract
Acute and severe anemia of inflammation (AI) is a common complication of various clinical syndromes, including fulminant infections, critical illness with multiorgan failure, and exacerbations of autoimmune diseases. Building on recent data showing beneficial results with isocitrate treatment for chronic low-grade AI in a rat model, we used a mouse model of acute and severe AI induced by intraperitoneal heat-killed Brucella abortus to determine if isocitrate would be effective in this more stringent application. Inflamed mice treated with isocitrate developed an early but transient improvement in hemoglobin compared to solvent-treated controls, with a robust improvement on day 7, and only a trend towards improvement by day 14. Reticulocyte counts were increased in treated mice transiently, with no significant difference by day 21. Serum erythropoietin (EPO) levels were similar in treated versus control mice, indicating that isocitrate increased sensitivity to EPO. Serum and tissue iron levels showed no significant differences between the treated and control mice, ruling out improved iron availability as the cause of the increased response to endogenous EPO. Compared to the milder rat model, much higher doses of isocitrate were required for a relatively modest benefit.
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Affiliation(s)
- Airie Kim
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
| | - Eileen Fung
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
| | - Sona G Parikh
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
| | - Victoria Gabayan
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
| | - Elizabeta Nemeth
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
| | - Tomas Ganz
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA; Department of Pathology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
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19
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Cavey T, Ropert M, de Tayrac M, Bardou-Jacquet E, Island ML, Leroyer P, Bendavid C, Brissot P, Loréal O. Mouse genetic background impacts both on iron and non-iron metals parameters and on their relationships. Biometals 2015; 28:733-43. [PMID: 26041486 DOI: 10.1007/s10534-015-9862-8] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2015] [Accepted: 05/20/2015] [Indexed: 12/17/2022]
Abstract
Iron is reported to interact with other metals. In addition, it has been shown that genetic background may impact iron metabolism. Our objective was to characterize, in mice of three genetic backgrounds, the links between iron and several non-iron metals. Thirty normal mice (C57BL/6, Balb/c and DBA/2; n = 10 for each group), fed with the same diet, were studied. Quantification of iron, zinc, cobalt, copper, manganese, magnesium and rubidium was performed by ICP/MS in plasma, erythrocytes, liver and spleen. Transferrin saturation was determined. Hepatic hepcidin1 mRNA level was evaluated by quantitative RT-PCR. As previously reported, iron parameters were modulated by genetic background with significantly higher values for plasma iron parameters and liver iron concentration in DBA/2 and Balb/c strains. Hepatic hepcidin1 mRNA level was lower in DBA/2 mice. No iron parameter was correlated with hepcidin1 mRNA levels. Principal component analysis of the data obtained for non-iron metals indicated that metals parameters stratified the mice according to their genetic background. Plasma and tissue metals parameters that are dependent or independent of genetic background were identified. Moreover, relationships were found between plasma and tissue content of iron and some other metals parameters. Our data: (i) confirms the impact of the genetic background on iron parameters, (ii) shows that genetic background may also play a role in the metabolism of non-iron metals, (iii) identifies links between iron and other metals parameters which may have implications in the understanding and, potentially, the modulation of iron metabolism.
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20
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Lu S, Seravalli J, Harrison-Findik D. Inductively coupled mass spectrometry analysis of biometals in conditional Hamp1 and Hamp1 and Hamp2 transgenic mouse models. Transgenic Res 2015; 24:765-73. [PMID: 25904410 DOI: 10.1007/s11248-015-9879-3] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2014] [Accepted: 04/14/2015] [Indexed: 12/12/2022]
Abstract
Hepcidin, a circulatory antimicrobial peptide, is involved in iron homeostasis, inflammation, infection and metabolic signals. Humans express one hepcidin gene, HAMP but mice express two hepcidin genes, Hamp1 and Hamp2. Consecutive gene targeting events were performed to produce transgenic mice expressing conditional alleles of either Hamp1 or both Hamp1 and Hamp2 (Hamp1/2). The deletion of Hamp1 alleles elevated Hamp2 expression, particularly in males, which was reduced by endotoxin treatment. The tissue levels of iron and other biometals were quantified by inductively coupled mass spectrometry. The ubiquitous or liver-specific deletion of Hamp1 alleles yielded similar quantitative changes in iron levels in the liver, duodenum, spleen, kidney, heart and brain. The introduction of Hamp2 null allele did not exacerbate the iron-related phenotype of Hamp1 null allele. Besides iron, Hamp1 null allele significantly elevated the levels of selenium in the liver, manganese in the liver and duodenum, and copper in the brain. Mice with conditional Hamp alleles will be useful to determine the tissue-specific regulation and functions of Hamp1 and Hamp2 in biometal homeostasis and other biological processes.
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Affiliation(s)
- S Lu
- Department of Internal Medicine, University of Nebraska Medical Center, 95820 UNMC, DRC I, Omaha, NE, 68198-5820, USA
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21
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Cao C, Thomas CE, Insogna KL, O'Brien KO. Duodenal absorption and tissue utilization of dietary heme and nonheme iron differ in rats. J Nutr 2014; 144:1710-7. [PMID: 25332470 PMCID: PMC4195416 DOI: 10.3945/jn.114.197939] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
BACKGROUND Dietary heme contributes to iron intake, yet regulation of heme absorption and tissue utilization of absorbed heme remains undefined. OBJECTIVES In a rat model of iron overload, we used stable iron isotopes to examine heme- and nonheme-iron absorption in relation to liver hepcidin and to compare relative utilization of absorbed heme and nonheme iron by erythroid (RBC) and iron storage tissues (liver and spleen). METHODS Twelve male Sprague-Dawley rats were randomly assigned to groups for injections of either saline or iron dextran (16 or 48 mg Fe over 2 wk). After iron loading, rats were administered oral stable iron in the forms of (57)Fe-ferrous sulfate and (58)Fe-labeled hemoglobin. Expression of liver hepcidin and duodenal iron transporters and tissue stable iron enrichment was determined 10 d postdosing. RESULTS High iron loading increased hepatic hepcidin by 3-fold and reduced duodenal expression of divalent metal transporter 1 (DMT1) by 76%. Nonheme-iron absorption was 2.5 times higher than heme-iron absorption (P = 0.0008). Absorption of both forms of iron was inversely correlated with hepatic hepcidin expression (heme-iron absorption: r = -0.77, P = 0.003; nonheme-iron absorption: r = -0.80, P = 0.002), but hepcidin had a stronger impact on nonheme-iron absorption (P = 0.04). Significantly more (57)Fe was recovered in RBCs (P = 0.02), and more (58)Fe was recovered in the spleen (P = 0.01). CONCLUSIONS Elevated hepcidin significantly decreased heme- and nonheme-iron absorption but had a greater impact on nonheme-iron absorption. Differential tissue utilization of heme vs. nonheme iron was evident between erythroid and iron storage tissues, suggesting that some heme may be exported into the circulation in a form different from that of nonheme iron.
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Affiliation(s)
- Chang Cao
- Division of Nutritional Sciences, Cornell University, Ithaca, NY
| | - Carrie E. Thomas
- Department of Nutritional Sciences, University of Connecticut, Storrs, CT; and
| | - Karl L. Insogna
- Department of Internal Medicine, Yale University, New Haven, CT
| | - Kimberly O. O'Brien
- Division of Nutritional Sciences, Cornell University, Ithaca, NY;,To whom correspondence should be addressed. E-mail:
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22
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Kong WN, Niu QM, Ge L, Zhang N, Yan SF, Chen WB, Chang YZ, Zhao SE. Sex differences in iron status and hepcidin expression in rats. Biol Trace Elem Res 2014; 160:258-67. [PMID: 24962641 DOI: 10.1007/s12011-014-0051-3] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/02/2014] [Accepted: 06/16/2014] [Indexed: 02/08/2023]
Abstract
Studies have shown that men and women exhibit significant differences regarding iron status. However, the effects of sex on iron accumulation and distribution are not well established. In this study, female and male Sprague-Dawley rats were killed at 4 months of age. Blood samples were analyzed to determine the red blood cell (RBC) count, hemoglobin (Hb) concentration, hematocrit (Hct), and mean red blood cell volume (MCV). The serum samples were analyzed to determine the concentrations of serum iron (SI), transferrin saturation (TS), ferritin, soluble transferrin receptor (sTfR), and erythropoietin (EPO). The tissue nonheme iron concentrations were measured in the liver, spleen, bone marrow, kidney, heart, gastrocnemius, duodenal epithelium, lung, pallium, cerebellum, hippocampus, and striatum. Hepatic hepcidin expression was detected by real-time PCR analysis. The synthesis of ferroportin 1 (FPN1) in the liver, spleen, kidney, and bone marrow was determined by Western blot analysis. The synthesis of duodenal cytochrome B561 (DcytB), divalent metal transporter 1 (DMT1), FPN1, hephaestin (HP) in the duodenal epithelium was also measured by Western blot analysis. The results showed that the RBC, Hb, and Hct in male rats were higher than those in female rats. The SI and plasma TS levels were lower in male rats than in female rats. The levels of serum ferritin and sTfR were higher in male rats than in female rats. The EPO levels in male rats were lower than that in female rats. The nonheme iron contents in the liver, spleen, bone marrow, and kidney in male rats were also lower (56.7, 73.2, 60.6, and 61.4 % of female rats, respectively). Nonheme iron concentrations in the heart, gastrocnemius, duodenal epithelium, lung, and brain were similar in rats of both sexes. A moderate decrease in hepatic hepcidin mRNA content was also observed in male rats (to 56.0 % of female rats). The levels of FPN1 protein in the liver, spleen, and kidney were higher in male rats than in female rats. There was no significant change in FPN1 expression in bone marrow. Significant difference was also not found in DcytB, DMT1, FPN1, and HP protein levels in the duodenal epithelium between male and female rats. These data suggest that iron is distributed differently in male and female rats. This difference in iron distribution may be associated with the difference in the hepcidin level.
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Affiliation(s)
- Wei-Na Kong
- The 3rd Hospital of Hebei Medical University, Shijiazhuang, 050051, Hebei Province, People's Republic of China
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Pentoxifylline does not alter the concentration of hepcidin in chronic kidney disease patients undergoing hemodialysis. Int J Artif Organs 2014; 37:521-8. [PMID: 25044383 DOI: 10.5301/ijao.5000340] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/06/2014] [Indexed: 12/16/2022]
Abstract
INTRODUCTION Anemia is a frequent condition in patients with chronic kidney disease due to a reduction in the production of erythropoietin. Patients with inflammation respond less well to treatment with erythropoietin, possibly because the increased production of hepcidin reduces the availability of iron for hematopoiesis. Some studies suggest that pentoxifylline has anti-inflammatory properties and could be used as adjuvant therapy in the treatment of anemia. OBJECTIVE The aim of this study was to analyze the effect of pentoxifylline on serum hepcidin in chronic hemodialysis patients with inflammation. METHODS 71 adult patients on hemodialysis with C-reactive protein (CRP) ≥0.5 mg/dl in screening tests; patients were randomized to the treatment group (oral pentoxifylline 400 mg/thrice-weekly) or the control group for 3 months follow-up. RESULTS During the study, a decrease in hemoglobin, transferrin saturation, and hepcidin was observed in both groups. However, these reductions were related to the time and not to the drug. There was no difference in the concentrations of CRP, ferritin, and albumin over time in either group. CONCLUSIONS The use of this amount of pentoxifylline did not modify the serum levels of hepcidin in this population.
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24
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A mouse model of anemia of inflammation: complex pathogenesis with partial dependence on hepcidin. Blood 2014; 123:1129-36. [DOI: 10.1182/blood-2013-08-521419] [Citation(s) in RCA: 92] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Key Points
An injection of heat-killed Brucella abortus in mice causes prolonged anemia with features similar to human anemia of inflammation. Ablation of hepcidin ameliorates anemia of inflammation in this model and allows faster recovery.
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25
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Latour C, Kautz L, Besson-Fournier C, Island ML, Canonne-Hergaux F, Loréal O, Ganz T, Coppin H, Roth MP. Testosterone perturbs systemic iron balance through activation of epidermal growth factor receptor signaling in the liver and repression of hepcidin. Hepatology 2014; 59:683-94. [PMID: 23907767 DOI: 10.1002/hep.26648] [Citation(s) in RCA: 96] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/01/2013] [Accepted: 07/18/2013] [Indexed: 12/14/2022]
Abstract
UNLABELLED Gender-related disparities in the regulation of iron metabolism may contribute to the differences exhibited by men and women in the progression of chronic liver diseases associated with reduced hepcidin expression, e.g., chronic hepatitis C, alcoholic liver disease, or hereditary hemochromatosis. However, their mechanisms remain poorly understood. In this study we took advantage of the major differences in hepcidin expression and tissue iron loading observed between Bmp6-deficient male and female mice to investigate the mechanisms underlying this sexual dimorphism. We found that testosterone robustly represses hepcidin transcription by enhancing Egfr signaling in the liver and that selective epidermal growth factor receptor (Egfr) inhibition by gefitinib (Iressa) in males markedly increases hepcidin expression. In males, where the suppressive effects of testosterone and Bmp6-deficiency on hepcidin expression are combined, hepcidin is more strongly repressed than in females and iron accumulates massively not only in the liver but also in the pancreas, heart, and kidneys. CONCLUSION Testosterone-induced repression of hepcidin expression becomes functionally important during homeostatic stress from disorders that result in iron loading and/or reduced capacity for hepcidin synthesis. These findings suggest that novel therapeutic strategies targeting the testosterone/EGF/EGFR axis may be useful for inducing hepcidin expression in patients with iron overload and/or chronic liver diseases.
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Affiliation(s)
- Chloé Latour
- Inserm, U1043, Toulouse, France; CNRS, U5282, Toulouse, France; Université de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France
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26
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Brewer C, Otto-Duessel M, Wood RI, Wood JC. Sex differences and steroid modulation of cardiac iron in a mouse model of iron overload. Transl Res 2014; 163:151-9. [PMID: 24018182 PMCID: PMC3946637 DOI: 10.1016/j.trsl.2013.08.004] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/27/2013] [Revised: 08/07/2013] [Accepted: 08/16/2013] [Indexed: 12/26/2022]
Abstract
Iron cardiomyopathy is the leading cause of death in transfusional iron overload, and men have twice the mortality of women. Because the prevalence of cardiac iron overload increases rapidly during the second decade of life, we postulated that there are steroid-dependent sex differences in cardiac iron uptake. To test this hypothesis, we manipulated sex steroids in mice with constitutive iron absorption (homozygous hemojuvelin knockout); this model mimics the myocyte iron deposition observed in humans. At 4 weeks of age, female mice were ovariectomized (OVX) and male mice were castrated (OrchX). Female mice received an estrogen implant (OVX + E) or a cholesterol control (OVX), whereas male mice received an implant containing testosterone (OrchX + T), dihydrotestosterone (OrchX + DHT), estrogen (OrchX + E), or cholesterol (OrchX). All animals received a high-iron diet for 8 weeks. OrchX, OVX, and OVX + E mice all had similar cardiac iron loads. However, OrchX + E males had a significant increase in cardiac iron concentration compared with OrchX mice (P < 0.01), whereas the OrchX + T and OrchX + DHT groups only trended higher (P < 0.06 and P < 0.15, respectively). Hormone treatments did not impact liver iron concentration in either sex. When data were pooled across hormone therapies, liver iron concentration was 25% greater in males than females (P < 0.01). In summary, we found that estrogen increased cardiac iron loading in male mice, but not in females. Male mice loaded 25% more hepatic iron than female mice regardless of the hormone treatment.
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Affiliation(s)
- Casey Brewer
- Division of Pediatric Cardiology, Children's Hospital Los Angeles, Los Angeles, Calif
| | - Maya Otto-Duessel
- Division of Pediatric Cardiology, Children's Hospital Los Angeles, Los Angeles, Calif
| | - Ruth I Wood
- Department of Cell and Neurobiology, Keck School of Medicine of the University of Southern California, Los Angeles, Calif
| | - John C Wood
- Division of Pediatric Cardiology, Children's Hospital Los Angeles, Los Angeles, Calif.
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27
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Testing the iron hypothesis in a mouse model of atherosclerosis. Cell Rep 2013; 5:1436-42. [PMID: 24316081 DOI: 10.1016/j.celrep.2013.11.009] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2013] [Revised: 09/23/2013] [Accepted: 11/05/2013] [Indexed: 02/07/2023] Open
Abstract
Hepcidin, the iron-regulatory hormone and acute phase reactant, is proposed to contribute to the pathogenesis of atherosclerosis by promoting iron accumulation in plaque macrophages, leading to increased oxidative stress and inflammation in the plaque (the "iron hypothesis"). Hepcidin and iron may thus represent modifiable risk factors in atherosclerosis. We measured hepcidin expression in Apoe(-/-) mice with varying diets and ages. To assess the role of macrophage iron in atherosclerosis, we generated Apoe(-/-) mice with macrophage-specific iron accumulation by introducing the ferroportin ffe mutation. Macrophage iron loading was also enhanced by intravenous iron injection. Contrary to the iron hypothesis, we found that hepatic hepcidin expression was not increased at any stage of the atherosclerosis progression in Apoe(-/-) or Apoe/ffe mice and that the atherosclerotic plaque size was not increased in mice with elevated macrophage iron. Our results strongly argue against any significant role of macrophage iron in atherosclerosis progression in mice.
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28
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Hepcidin induction by pathogens and pathogen-derived molecules is strongly dependent on interleukin-6. Infect Immun 2013; 82:745-52. [PMID: 24478088 DOI: 10.1128/iai.00983-13] [Citation(s) in RCA: 93] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Hepcidin, the iron-regulatory hormone, is increased during infection or inflammation, causing hypoferremia. This response is thought to be a host defense mechanism that restricts iron availability to invading pathogens. It is not known if hepcidin is differentially induced by bacterial versus viral infections, whether the stimulation of pattern recognition receptors directly regulates hepcidin transcription, or which of the proposed signaling pathways are essential for hepcidin increase during infection. We analyzed hepcidin induction and its dependence on interleukin-6 (IL-6) in response to common bacterial or viral infections in mice or in response to a panel of pathogen-derived molecules (PAMPs) in mice and human primary hepatocytes. In wild-type (WT) mice, hepcidin mRNA was induced several hundred-fold both by a bacterial (Streptococcus pneumoniae) and a viral infection (influenza virus PR8) within 2 to 5 days. Treatment of mice and human primary hepatocytes with most Toll-like receptor ligands increased hepcidin mRNA within 6 h. Hepcidin induction by microbial stimuli was IL-6 dependent. IL-6 knockout mice failed to increase hepcidin in response to S. pneumoniae or influenza infection and had greatly diminished hepcidin response to PAMPs. In vitro, hepcidin induction by PAMPs in primary human hepatocytes was abolished by the addition of neutralizing IL-6 antibodies. Our results support the key role of IL-6 in hepcidin regulation in response to a variety of infectious and inflammatory stimuli.
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29
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Choi J, Masaratana P, Latunde-Dada GO, Arno M, Simpson RJ, McKie AT. Duodenal reductase activity and spleen iron stores are reduced and erythropoiesis is abnormal in Dcytb knockout mice exposed to hypoxic conditions. J Nutr 2012; 142:1929-34. [PMID: 22990466 DOI: 10.3945/jn.112.160358] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
Duodenal cytochrome b (Dcytb, Cybrd1) is a ferric reductase localized in the duodenum that is highly upregulated in circumstances of increased iron absorption. To address the contribution of Dcytb to total duodenal ferric reductase activity as well as its wider role in iron metabolism, we first measured duodenal ferric reductase activity in wild-type (WT) and Dcytb knockout (Dcytb(-/-)) mice under 3 conditions known to induce gut ferric reductase: dietary iron deficiency, hypoxia, and pregnancy. Dcytb(-/-) and WT mice were randomly assigned to control (iron deficiency experiment, 48 mg/kg dietary iron; hypoxia experiment, normal atmospheric pressure; pregnancy experiment, nonpregnant animals) or treatment (iron deficiency experiment, 2-3 mg/kg dietary iron; hypoxia experiment, 53.3 kPa pressure; pregnancy experiment, d 20 of pregnancy) groups and duodenal reductase activity measured. We found no induction of ferric reductase activity in Dcytb(-/-) mice under any of these conditions, indicating there are no other inducible ferric reductases present in the duodenum. To test whether Dcytb was required for iron absorption in conditions with increased erythropoietic demand, we also measured tissue nonheme iron levels and hematological indices in WT and Dcytb(-/-) mice exposed to hypoxia. There was no evidence of gross alterations in iron absorption, hemoglobin, or total liver nonheme iron in Dcytb(-/-) mice exposed to hypoxia compared with WT mice. However, spleen nonheme iron was significantly less (6.7 ± 1.0 vs. 12.7 ± 0.9 nmol · mg tissue(-1); P < 0.01, n = 7-8) in hypoxic Dcytb(-/-) compared with hypoxic WT mice and there was evidence of impaired reticulocyte hemoglobinization with a lower reticulocyte mean corpuscular hemoglobin (276 ± 1 vs. 283 ± 2 g · L(-1); P < 0.05, n = 7-8) in normoxic Dcytb(-/-) compared with normoxic WT mice. We therefore conclude that DCYTB is the primary iron-regulated duodenal ferric reductase in the gut and that Dcytb is necessary for optimal iron metabolism.
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Affiliation(s)
- Jeehyea Choi
- Diabetes and Nutritional Sciences Division, School of Biomedical and Health Sciences, King’s College, London, UK
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Zinc transporter ZIP14 functions in hepatic zinc, iron and glucose homeostasis during the innate immune response (endotoxemia). PLoS One 2012; 7:e48679. [PMID: 23110240 PMCID: PMC3480510 DOI: 10.1371/journal.pone.0048679] [Citation(s) in RCA: 118] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2012] [Accepted: 09/28/2012] [Indexed: 12/16/2022] Open
Abstract
ZIP14 (slc39A14) is a zinc transporter induced in response to pro-inflammatory stimuli. ZIP14 induction accompanies the reduction in serum zinc (hypozincemia) of acute inflammation. ZIP14 can transport Zn2+ and non-transferrin-bound Fe2+ in vitro. Using a Zip14−/− mouse model we demonstrated that ZIP14 was essential for control of phosphatase PTP1B activity and phosphorylation of c-Met during liver regeneration. In the current studies, a global screening of ZIP transporter gene expression in response to LPS-induced endotoxemia was conducted. Following LPS, Zip14 was the most highly up-regulated Zip transcript in liver, but also in white adipose tissue and muscle. Using ZIP14−/− mice we show that ZIP14 contributes to zinc absorption from the gastrointestinal tract directly or indirectly as zinc absorption was decreased in the KOs. In contrast, Zip14−/− mice absorbed more iron. The Zip14 KO mice did not exhibit hypozincemia following LPS, but do have hypoferremia. Livers of Zip14−/− mice had increased transcript abundance for hepcidin, divalent metal transporter-1, ferritin and transferrin receptor-1 and greater accumulation of iron. The Zip14−/− phenotype included greater body fat, hypoglycemia and higher insulin levels, as well as increased liver glucose and greater phosphorylation of the insulin receptor and increased GLUT2, SREBP-1c and FASN expression. The Zip14 KO mice exhibited decreased circulating IL-6 with increased hepatic SOCS-3 following LPS, suggesting SOCS-3 inhibited insulin signaling which produced the hypoglycemia in this genotype. The results are consistent with ZIP14 ablation yielding abnormal labile zinc pools which lead to increased SOCS-3 production through G-coupled receptor activation and increased cAMP production as well as signaled by increased pSTAT3 via the IL-6 receptor, which inhibits IRS 1/2 phosphorylation. Our data show the role of ZIP14 in the hepatocyte is multi-functional since zinc and iron trafficking are altered in the Zip14−/− mice and their phenotype shows defects in glucose homeostasis.
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Ikeda Y, Tajima S, Izawa-Ishizawa Y, Kihira Y, Ishizawa K, Tomita S, Tsuchiya K, Tamaki T. Estrogen regulates hepcidin expression via GPR30-BMP6-dependent signaling in hepatocytes. PLoS One 2012; 7:e40465. [PMID: 22792339 PMCID: PMC3394730 DOI: 10.1371/journal.pone.0040465] [Citation(s) in RCA: 95] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2012] [Accepted: 06/07/2012] [Indexed: 12/21/2022] Open
Abstract
Hepcidin, a liver-derived iron regulatory protein, plays a crucial role in iron metabolism. It is known that gender differences exist with respect to iron storage in the body; however, the effects of sex steroid hormones on iron metabolism are not completely understood. We focused on the effects of the female sex hormone estrogen on hepcidin expression. First, ovariectomized (OVX) and sham-operated mice were employed to investigate the effects of estrogen on hepcidin expression in an in vivo study. Hepcidin expression was decreased in the livers of OVX mice compared to the sham-operated mice. In OVX mice, bone morphologic protein-6 (BMP6), a regulator of hepcidin, was also found to be downregulated in the liver, whereas ferroportin (FPN), an iron export protein, was upregulated in the duodenum. Both serum and liver iron concentrations were elevated in OVX mice relative to their concentrations in sham-operated mice. In in vitro studies, 17β-estradiol (E(2)) increased the mRNA expression of hepcidin in HepG2 cells in a concentration-dependent manner. E(2)-induced hepatic hepcidin upregulation was not inhibited by ICI 182720, an inhibitor of the estrogen receptor; instead, hepcidin expression was increased by ICI 182720. E(2) and ICI 182720 exhibit agonist actions with G-protein coupled receptor 30 (GPR30), the 7-transmembrane estrogen receptor. G1, a GPR30 agonist, upregulated hepcidin expression, and GPR30 siRNA treatment abolished E(2)-induced hepcidin expression. BMP6 expression induced by E(2) was abolished by GPR30 silencing. Finally, both E(2) and G1 supplementation restored reduced hepatic hepcidin and BMP6 expression and reversed the augmentation of duodenal FPN expression in the OVX mice. In contrast, serum hepcidin was elevated in OVX mice, which was reversed in these mice with E(2) and G1. Thus, estrogen is involved in hepcidin expression via a GPR30-BMP6-dependent mechanism, providing new insight into the role of estrogen in iron metabolism.
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MESH Headings
- Animals
- Antimicrobial Cationic Peptides/blood
- Antimicrobial Cationic Peptides/genetics
- Antimicrobial Cationic Peptides/metabolism
- Bone Morphogenetic Protein 6/genetics
- Bone Morphogenetic Protein 6/metabolism
- Cation Transport Proteins/metabolism
- Duodenum/metabolism
- Estradiol/analogs & derivatives
- Estradiol/pharmacology
- Estradiol/physiology
- Estrogens/pharmacology
- Estrogens/physiology
- Female
- Fulvestrant
- Gene Knockdown Techniques
- Hep G2 Cells
- Hepatocytes/metabolism
- Hepcidins
- Humans
- Intestinal Absorption
- Iron/blood
- Iron/metabolism
- Liver/cytology
- Liver/metabolism
- Mice
- Mice, Inbred C57BL
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- RNA, Small Interfering/genetics
- Receptors, Estrogen/agonists
- Receptors, Estrogen/genetics
- Receptors, Estrogen/metabolism
- Receptors, G-Protein-Coupled/agonists
- Receptors, G-Protein-Coupled/genetics
- Receptors, G-Protein-Coupled/metabolism
- Signal Transduction
- Transcriptional Activation
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Affiliation(s)
- Yasumasa Ikeda
- Department of Pharmacology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.
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Abstract
Inappropriately low expression of the key iron regulator hepcidin (HAMP) causes iron overload in untransfused patients affected by β-thalassemia intermedia and Hamp modulation provides improvement of the thalassemic phenotype of the Hbb(th3/+) mouse. HAMP expression is activated by iron through the bone morphogenetic protein (BMP)-son of mothers against decapentaplegic signaling pathway and inhibited by ineffective erythropoiesis through an unknown "erythroid regulator." The BMP pathway is inactivated by the serine protease TMPRSS6 that cleaves the BMP coreceptor hemojuvelin. Here, we show that homozygous loss of Tmprss6 in Hbb(th3/+) mice improves anemia and reduces ineffective erythropoiesis, splenomegaly, and iron loading. All these effects are mediated by Hamp up-regulation, which inhibits iron absorption and recycling. Because Hbb(th3/+) mice lacking Tmprss6 show residual ineffective erythropoiesis, our results indicate that Tmprss6 is essential for Hamp inhibition by the erythroid regulator. We also obtained partial correction of the phenotype in Tmprss6 haploinsufficient Hbb(th3/+) male but not female mice and showed that the observed sex difference reflects an unequal balance between iron and erythropoiesis-mediated Hamp regulation. Our study indicates that preventing iron overload improves β-thalassemia and strengthens the essential role of Tmprss6 for Hamp suppression, providing a proof of concept that Tmprss6 manipulation can offer a novel therapeutic option in this condition.
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Abstract
Murine models have made valuable contributions to our understanding of iron metabolism. Investigation of mice with inherited forms of anemia has led to the discovery of novel proteins involved in iron homeostasis. A growing number of murine models are being developed to investigate mitochondrial iron metabolism. Mouse strains are available for the major forms of hereditary hemochromatosis. Findings in murine models support the concept that the pathogenesis of nearly all forms of hereditary hemochromatosis involves inappropriately low expression of hepcidin. The availability of mice with floxed iron-related genes allows the study of the in vivo consequences of cell-selective deletion of these genes.
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Affiliation(s)
- Robert E Fleming
- Departments of Pediatrics and Biochemistry & Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104, USA.
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34
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Mass spectrometry analysis of hepcidin peptides in experimental mouse models. PLoS One 2011; 6:e16762. [PMID: 21408141 PMCID: PMC3050808 DOI: 10.1371/journal.pone.0016762] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2010] [Accepted: 01/13/2011] [Indexed: 01/01/2023] Open
Abstract
The mouse is a valuable model for unravelling the role of hepcidin in iron homeostasis, however, such studies still report hepcidin mRNA levels as a surrogate marker for bioactive hepcidin in its pivotal function to block ferroportin-mediated iron transport. Here, we aimed to assess bioactive mouse Hepcidin-1 (Hep-1) and its paralogue Hepcidin-2 (Hep-2) at the peptide level. To this purpose, fourier transform ion cyclotron resonance (FTICR) and tandem-MS was used for hepcidin identification, after which a time-of-flight (TOF) MS-based methodology was exploited to routinely determine Hep-1 and -2 levels in mouse serum and urine. This method was biologically validated by hepcidin assessment in: i) 3 mouse strains (C57Bl/6; DBA/2 and BABL/c) upon stimulation with intravenous iron and LPS, ii) homozygous Hfe knock out, homozygous transferrin receptor 2 (Y245X) mutated mice and double affected mice, and iii) mice treated with a sublethal hepatotoxic dose of paracetamol. The results showed that detection of Hep-1 was restricted to serum, whereas Hep-2 and its presumed isoforms were predominantly present in urine. Elevations in serum Hep-1 and urine Hep-2 upon intravenous iron or LPS were only moderate and varied considerably between mouse strains. Serum Hep-1 was decreased in all three hemochromatosis models, being lowest in the double affected mice. Serum Hep-1 levels correlated with liver hepcidin-1 gene expression, while acute liver damage by paracetamol depleted Hep-1 from serum. Furthermore, serum Hep-1 appeared to be an excellent indicator of splenic iron accumulation. In conclusion, Hep-1 and Hep-2 peptide responses in experimental mouse agree with the known biology of hepcidin mRNA regulators, and their measurement can now be implemented in experimental mouse models to provide novel insights in post-transcriptional regulation, hepcidin function, and kinetics.
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Abstract
Hepcidin, the Fe-regulatory peptide, has been shown to inhibit Fe absorption and reticuloendothelial Fe recycling. The present study was conducted to explore the mechanism of in vivo Fe regulation through genetic disruption of hepcidin1 and acute effects of hepcidin treatment in hepcidin1 knockout (Hepc1-/-) and heterozygous mice. Hepcidin1 disruption resulted in significantly increased intestinal Fe uptake. Hepcidin injection inhibited Fe absorption in both genotypes, but the effects were more evident in the knockout mice. Hepcidin administration was also associated with decreased membrane localisation of ferroportin in the duodenum, liver and, most significantly, in the spleen of Hepc1-/- mice. Hypoferraemia was induced in heterozygous mice by hepcidin treatment, but not in Hepc1-/- mice, 4 h after injection. Interestingly, Fe absorption and serum Fe levels in Hepc1-/- and heterozygous mice fed a low-Fe diet were not affected by hepcidin injection. The present study demonstrates that hepcidin deficiency causes increased Fe absorption. The effects of hepcidin were abolished by dietary Fe deficiency, indicating that the response to hepcidin may be influenced by dietary Fe level or Fe status.
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Guo Xiaoqiang, Li Wenjie, Xin Qiliang, Ding Hui, Zhang Caiyun, Chang Yanzhong, Duan Xianglin. Vitamin C protective role for alcoholic liver disease in mice through regulating iron metabolism. Toxicol Ind Health 2010; 27:341-8. [PMID: 21078691 DOI: 10.1177/0748233710387007] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Alcoholic liver disease (ALD) is a major medical complication of drinking alcohol, and commonly accompanied with hepatic iron overload and liver injuries. Oxidative stress plays an important role in pathogenesis of ALD and also leads to iron-metabolic disorders. In this study, the effects of vitamin C (Vc) on iron metabolism-related genes expression and liver protection from drinking in mice were investigated. Twenty-four male kunming mice were divided into four groups (six mice per group): control (water drinking); alcohol group (20% alcohol drinking), alcohol + low Vc group (adding 50 mg/kg Vc daily) and alcohol + high Vc group (adding 100 mg/kg Vc daily). All these mice were sacrificed after 7 days. Vc can ameliorate the increase of sera alanine aminotransferase (ALT) activity and hepatic iron overload of drinking alcohol in mice. Vc increases the expression of the iron-regulated hormone hepcidin and decreases transferrin receptor 1 (TfR1) expression in liver. Vc also down-regulates the expression of ferroportin 1 (Fpn1) in the intestine and decreases the iron release to blood. In conclusion, Vc ameliorated the alcoholic liver injuries through regulating the iron metabolism-related genes expression.
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Affiliation(s)
- Guo Xiaoqiang
- Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University, Shijiazhuang, Hebei, People's Republic of China
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Intestinal ferritin H is required for an accurate control of iron absorption. Cell Metab 2010; 12:273-82. [PMID: 20816093 DOI: 10.1016/j.cmet.2010.08.003] [Citation(s) in RCA: 104] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/08/2010] [Revised: 05/08/2010] [Accepted: 06/16/2010] [Indexed: 12/26/2022]
Abstract
To maintain appropriate body iron levels, iron absorption by the proximal duodenum is thought to be controlled by hepcidin, a polypeptide secreted by hepatocytes in response to high serum iron. Hepcidin limits basolateral iron efflux from the duodenal epithelium by binding and downregulating the intestinal iron exporter ferroportin. Here, we found that mice with an intestinal ferritin H gene deletion show increased body iron stores and transferrin saturation. As expected for iron-loaded animals, the ferritin H-deleted mice showed induced liver hepcidin mRNA levels and reduced duodenal expression of DMT1 and DcytB mRNA. In spite of these feedback controls, intestinal ferroportin protein and (59)Fe absorption were increased more than 2-fold in the deleted mice. Our results demonstrate that hepcidin-mediated regulation alone is insufficient to restrict iron absorption and that intestinal ferritin H is also required to limit iron efflux from intestinal cells.
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Harrison-Findik DD. Gender-related variations in iron metabolism and liver diseases. World J Hepatol 2010; 2:302-10. [PMID: 21161013 PMCID: PMC2999297 DOI: 10.4254/wjh.v2.i8.302] [Citation(s) in RCA: 65] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/17/2010] [Revised: 06/30/2010] [Accepted: 07/07/2010] [Indexed: 02/06/2023] Open
Abstract
The regulation of iron metabolism involves multiple organs including the duodenum, liver and bone marrow. The recent discoveries of novel iron-regulatory proteins have brought the liver to the forefront of iron homeostasis. The iron overload disorder, genetic hemochromatosis, is one of the most prevalent genetic diseases in individuals of Caucasian origin. Furthermore, patients with non-hemochromatotic liver diseases, such as alcoholic liver disease, chronic hepatitis C or nonalcoholic steatohepatitis, often exhibit elevated serum iron indices (ferritin, transferrin saturation) and mild to moderate hepatic iron overload. Clinical data indicate significant differences between men and women regarding liver injury in patients with alcoholic liver disease, chronic hepatitis C or nonalcoholic steatohepatitis. The penetrance of genetic hemochromatosis also varies between men and women. Hepcidin has been suggested to act as a modifier gene in genetic hemochromatosis. Hepcidin is a circulatory antimicrobial peptide synthesized by the liver. It plays a pivotal role in the regulation of iron homeostasis. Hepcidin has been shown to be regulated by iron, inflammation, oxidative stress, hypoxia, alcohol, hepatitis C and obesity. Sex and genetic background have also been shown to modulate hepcidin expression in mice. The role of gender in the regulation of human hepcidin gene expression in the liver is unknown. However, hepcidin may play a role in gender-based differences in iron metabolism and liver diseases. Better understanding of the mechanisms associated with gender-related differences in iron metabolism and chronic liver diseases may enable the development of new treatment strategies.
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Affiliation(s)
- Duygu D Harrison-Findik
- Duygu D Harrison-Findik, Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198-5820, United States
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Sex-related differences in gene expression following Coxiella burnetii infection in mice: potential role of circadian rhythm. PLoS One 2010; 5:e12190. [PMID: 20730052 PMCID: PMC2921390 DOI: 10.1371/journal.pone.0012190] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2010] [Accepted: 07/22/2010] [Indexed: 12/20/2022] Open
Abstract
Background Q fever, a zoonosis due to Coxiella burnetii infection, exhibits sexual dimorphism; men are affected more frequently and severely than women for a given exposure. Here we explore whether the severity of C. burnetii infection in mice is related to differences in male and female gene expression profiles. Methodology/Principal Findings Mice were infected with C. burnetii for 24 hours, and gene expression was measured in liver cells using microarrays. Multiclass analysis identified 2,777 probes for which expression was specifically modulated by C. burnetti infection. Only 14% of the modulated genes were sex-independent, and the remaining 86% were differentially expressed in males and females. Castration of males and females showed that sex hormones were responsible for more than 60% of the observed gene modulation, and this reduction was most pronounced in males. Using functional annotation of modulated genes, we identified four clusters enriched in males that were related to cell-cell adhesion, signal transduction, defensins and cytokine/Jak-Stat pathways. Up-regulation of the IL-10 and Stat-3 genes may account for the high susceptibility of men with Q fever to C. burnetii infection and autoantibody production. Two clusters were identified in females, including the circadian rhythm pathway, which consists of positive (Clock, Arntl) and negative (Per) limbs of a feedback loop. We found that Clock and Arntl were down-modulated whereas Per was up-regulated; these changes may be associated with efficient bacterial elimination in females but not in males, in which an exacerbated host response would be prominent. Conclusion This large-scale study revealed for the first time that circadian rhythm plays a major role in the anti-infectious response of mice, and it provides a new basis for elucidating the role of sexual dimorphism in human infections.
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Arndt S, Maegdefrau U, Dorn C, Schardt K, Hellerbrand C, Bosserhoff AK. Iron-induced expression of bone morphogenic protein 6 in intestinal cells is the main regulator of hepatic hepcidin expression in vivo. Gastroenterology 2010; 138:372-82. [PMID: 19786029 DOI: 10.1053/j.gastro.2009.09.048] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/14/2009] [Revised: 08/12/2009] [Accepted: 09/17/2009] [Indexed: 01/07/2023]
Abstract
BACKGROUND & AIMS Recent studies identified bone morphogenic protein 6 (BMP6) as a key regulator of hepatic hepcidin expression and iron metabolism, but the cellular source of BMP6 and the reason for its specific effect on hepatocytes are unknown. METHODS BMP and hepcidin expression upon iron sensing were analyzed in vivo in BMP6(-/-) and BMP6(+/+) mice and ex vivo in tissue and in vitro in cells of the liver and the small intestine. RESULTS BMP6(-/-) mice developed severe hepatic iron accumulation and reduced hepcidin expression with increasing age. This phenotype could be triggered in younger BMP6(-/-) mice by dietary or parenteral iron application. Furthermore, both treatments induced a marked up-regulation of BMP6 expression in the small intestine of BMP6(+/+) mice. Ex vivo treatment of intestinal tissue of BMP6(+/+) mice with iron sulfate or holo-transferrin confirmed epithelial cells as an inducible source of BMP6. In contrast, iron overload did not promote a striking induction of BMP6 expression in hepatocytes or macrophages. Furthermore, iron-supplemented diet induced a compensatory up-regulation of BMP2, BMP4, and BMP9 in the small intestine of BMP6(-/-) mice that was apparently not sufficient to assure iron homeostasis. As a potential explanation, analysis of hepatocytes revealed an expression pattern of BMP receptor subunits preferentially used by BMP6, and treatment of hepatocytes with different recombinant BMPs identified BMP6 as the most potent stimulator of hepcidin expression. CONCLUSIONS Epithelial cells of the small intestine are the predominant cellular source of BMP6 upon iron sensing. Our findings reveal a previously unknown mechanism in which the small intestine controls iron homeostasis.
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Affiliation(s)
- Stephanie Arndt
- Institute of Pathology, University of Regensburg, Regensburg, Germany
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Bayele HK, Srai SKS. Genetic variation in hepcidin expression and its implications for phenotypic differences in iron metabolism. Haematologica 2009; 94:1185-8. [PMID: 19734411 DOI: 10.3324/haematol.2009.010793] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
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Wallace DF, Summerville L, Crampton EM, Frazer DM, Anderson GJ, Subramaniam VN. Combined deletion of Hfe and transferrin receptor 2 in mice leads to marked dysregulation of hepcidin and iron overload. Hepatology 2009; 50:1992-2000. [PMID: 19824072 DOI: 10.1002/hep.23198] [Citation(s) in RCA: 170] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
UNLABELLED Hepcidin is a central regulator of iron homeostasis. HFE and transferrin receptor 2 (TFR2) are mutated in adult-onset forms of hereditary hemochromatosis and regulate the expression of hepcidin in response to iron. Whether they act through the same or parallel pathways is unclear. To investigate this, we generated a mouse model with deletion of both Hfe and Tfr2 genes by crossing Hfe and Tfr2 null mice on a genetically identical background. Tissue and serum from wildtype, single-, and double-null mice were analyzed. Serum transferrin saturation and hepatic iron concentrations were determined. The expression of iron-related messenger RNA (mRNA) transcripts was analyzed by real-time polymerase chain reaction (PCR). Levels of the iron-related proteins Tfr1, Tfr2, ferritin, and prohepcidin, and the phosphorylation status of the cell signaling proteins extracellular signal-regulated kinase 1/2 (Erk1/2) and Smad1/5/8, were analyzed by immunoblotting. Double-null mice had more severe iron loading than mice lacking either Hfe or Tfr2; Tfr2 null mice had a greater iron burden than Hfe-null mice. Hepcidin expression relative to iron stores was reduced in the Hfe-null mice, with significantly lower values in the Tfr2-null mice. In the absence of both Hfe and Tfr2, hepcidin expression was reduced even further. A significant decrease in phospho-Erk1/2 in the livers of null mice and a reduction in phospho-Smad1/5/8 suggest that both the mitogen-activated protein kinase (MAPK) and bone morphogenetic protein / mothers against decapentaplegic homolog (BMP/SMAD) signaling pathways may be involved in Hfe- and Tfr2-mediated regulation of hepcidin. CONCLUSION These studies demonstrate that iron overload due to deletion of Tfr2 is more severe than that due to Hfe, and that loss of both molecules results in pronounced iron overload. Analysis of Hfe/Tfr2 double-null mice suggests that Hfe and Tfr2 regulate hepcidin through parallel pathways involving Erk1/2 and Smad1/5/8.
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Affiliation(s)
- Daniel F Wallace
- Membrane Transport Laboratory, Queensland Institute of Medical Research, Brisbane, Queensland, Australia
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Volke M, Gale DP, Maegdefrau U, Schley G, Klanke B, Bosserhoff AK, Maxwell PH, Eckardt KU, Warnecke C. Evidence for a lack of a direct transcriptional suppression of the iron regulatory peptide hepcidin by hypoxia-inducible factors. PLoS One 2009; 4:e7875. [PMID: 19924283 PMCID: PMC2773926 DOI: 10.1371/journal.pone.0007875] [Citation(s) in RCA: 74] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2009] [Accepted: 10/21/2009] [Indexed: 12/21/2022] Open
Abstract
Background Hepcidin is a major regulator of iron metabolism and plays a key role in anemia of chronic disease, reducing intestinal iron uptake and release from body iron stores. Hypoxia and chemical stabilizers of the hypoxia-inducible transcription factor (HIF) have been shown to suppress hepcidin expression. We therefore investigated the role of HIF in hepcidin regulation. Methodology/Principal Findings Hepcidin mRNA was down-regulated in hepatoma cells by chemical HIF stabilizers and iron chelators, respectively. In contrast, the response to hypoxia was variable. The decrease in hepcidin mRNA was not reversed by HIF-1α or HIF-2α knock-down or by depletion of the HIF and iron regulatory protein (IRP) target transferrin receptor 1 (TfR1). However, the response of hepcidin to hypoxia and chemical HIF inducers paralleled the regulation of transferrin receptor 2 (TfR2), one of the genes critical to hepcidin expression. Hepcidin expression was also markedly and rapidly decreased by serum deprivation, independent of transferrin-bound iron, and by the phosphatidylinositol 3 (PI3) kinase inhibitor LY294002, indicating that growth factors are required for hepcidin expression in vitro. Hepcidin promoter constructs mirrored the response of mRNA levels to interleukin-6 and bone morphogenetic proteins, but not consistently to hypoxia or HIF stabilizers, and deletion of the putative HIF binding motifs did not alter the response to different hypoxic stimuli. In mice exposed to carbon monoxide, hypoxia or the chemical HIF inducer N-oxalylglycine, liver hepcidin 1 mRNA was elevated rather than decreased. Conclusions/Significance Taken together, these data indicate that hepcidin is neither a direct target of HIF, nor indirectly regulated by HIF through induction of TfR1 expression. Hepcidin mRNA expression in vitro is highly sensitive to the presence of serum factors and PI3 kinase inhibition and parallels TfR2 expression.
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Affiliation(s)
- Melanie Volke
- Department of Nephrology and Hypertension, University Hospital Erlangen, University of Erlangen-Nuremberg, Erlangen, Germany
| | - Daniel P. Gale
- Department of Medicine, Rayne Institute, University College London, London, United Kingdom
| | - Ulrike Maegdefrau
- Institute of Pathology, University of Regensburg, Regensberg, Germany
| | - Gunnar Schley
- Department of Nephrology and Hypertension, University Hospital Erlangen, University of Erlangen-Nuremberg, Erlangen, Germany
| | - Bernd Klanke
- Department of Nephrology and Hypertension, University Hospital Erlangen, University of Erlangen-Nuremberg, Erlangen, Germany
| | | | - Patrick H. Maxwell
- Department of Medicine, Rayne Institute, University College London, London, United Kingdom
| | - Kai-Uwe Eckardt
- Department of Nephrology and Hypertension, University Hospital Erlangen, University of Erlangen-Nuremberg, Erlangen, Germany
| | - Christina Warnecke
- Department of Nephrology and Hypertension, University Hospital Erlangen, University of Erlangen-Nuremberg, Erlangen, Germany
- * E-mail:
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Sow FB, Alvarez GR, Gross RP, Satoskar AR, Schlesinger LS, Zwilling BS, Lafuse WP. Role of STAT1, NF-kappaB, and C/EBPbeta in the macrophage transcriptional regulation of hepcidin by mycobacterial infection and IFN-gamma. J Leukoc Biol 2009; 86:1247-58. [PMID: 19652026 DOI: 10.1189/jlb.1208719] [Citation(s) in RCA: 65] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Hepcidin is an antimicrobial peptide involved in regulating iron homeostasis. It is induced by iron overload and decreased by hypoxia and anemia. Hepcidin regulates iron metabolism by inhibiting iron absorption by the duodenum and by inhibiting macrophage iron recycling. Hepcidin is induced in hepatocytes during the acute-phase response by IL-6. Previously, we have shown that hepcidin is not induced in macrophages by IL-6 but is induced by the synergistic interaction of IFN-gamma and Mycobacterium tuberculosis infection. In the present study, we examined the pathways involved in inducing macrophage hepcidin expression. We show that TLRs TLR2 and TLR4 and the transcription factor STAT1 are required for induction of hepcidin mRNA. Hepcidin promoter activity is also synergistically induced in RAW264.7 macrophages by IFN-gamma and M. tuberculosis. NF-kappaB and C/CEBP binding sites are required for promoter activity. Binding of NF-kappaB (p50/p65) to the NF-kappaB site and STAT1 and C/EBPbeta to the C/CEBP site was confirmed by EMSA. Knockdown of STAT1 and C/EBPbeta expression in RAW264.7 cells with siRNA plasmids inhibited hepcidin promoter activity induced by IFN-gamma and M. tuberculosis. Together, these studies demonstrate that macrophage hepcidin expression is induced by the activation of STAT1 and NF-kappaB and the induction of C/EBPbeta expression.
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Affiliation(s)
- Fatoumata B Sow
- Department of Microbiology, The Ohio State University, Columbus, Ohio, USA
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Viatte L, Gröne HJ, Hentze MW, Galy B. In vivo role(s) of the iron regulatory proteins (IRP) 1 and 2 in aseptic local inflammation. J Mol Med (Berl) 2009; 87:913-21. [DOI: 10.1007/s00109-009-0494-8] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2009] [Revised: 05/05/2009] [Accepted: 06/02/2009] [Indexed: 01/24/2023]
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Regulatory variation in hepcidin expression as a heritable quantitative trait. Biochem Biophys Res Commun 2009; 384:22-7. [DOI: 10.1016/j.bbrc.2009.04.032] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2009] [Accepted: 04/09/2009] [Indexed: 01/24/2023]
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Lee PL, Beutler E. Regulation of hepcidin and iron-overload disease. ANNUAL REVIEW OF PATHOLOGY-MECHANISMS OF DISEASE 2009; 4:489-515. [PMID: 19400694 DOI: 10.1146/annurev.pathol.4.110807.092205] [Citation(s) in RCA: 142] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Hepcidin, a 25-amino-acid antimicrobial peptide, is the central regulator of iron homeostasis. Hepcidin transcription is upregulated by inflammatory cytokines, iron, and bone morphogenetic proteins and is downregulated by iron deficiency, ineffective erythropoiesis, and hypoxia. The iron transporter ferroportin is the cognate receptor of hepcidin and is destroyed as a result of interaction with the peptide. Except for inherited defects of ferroportin and hepcidin itself, all forms of iron-storage disease appear to arise from hepcidin dysregulation. Studies using multiple approaches have begun to delineate the molecular mechanisms that regulate hepcidin expression, particularly at the transcriptional level. Knowledge of the regulation of hepcidin by inflammation, iron, erythropoiesis, and hypoxia will lead to an understanding of the pathogenesis of primary hemochromatosis, secondary iron overload, and anemia of inflammatory disease.
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Affiliation(s)
- Pauline L Lee
- Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
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Tseng HH, Chang JG, Hwang YH, Yeh KT, Chen YL, Yu HS. Expression of hepcidin and other iron-regulatory genes in human hepatocellular carcinoma and its clinical implications. J Cancer Res Clin Oncol 2009; 135:1413-20. [PMID: 19387685 DOI: 10.1007/s00432-009-0585-5] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2008] [Accepted: 04/01/2009] [Indexed: 02/07/2023]
Abstract
PURPOSE We aimed to assess expression of ten iron-regulatory genes in hepatocellular carcinoma (HCC) and its clinical implications. METHODS We used real-time polymerase chain reaction to measure ten iron-regulatory genes' mRNA and Perls' stain to assess iron stores in 50 HCCs and adjacent nontumor specimens. We compared the differences of gene expression and iron stores between tumor and nontumor specimens, and analyzed the relationships of gene expression with hepatic iron stores, patients' hemoglobin levels and clinicopathologic parameters. RESULTS Hepcidin, ceruloplasmin, transferrin, and transferrin receptor 2 were downregulated, while transferrin receptor 1 was upregulated in HCC. Hepcidin was markedly decreased in HCC but still correlated with hepatic iron stores. Iron-regulatory genes varied in their relationships of expression with clinicopathologic parameters. CONCLUSIONS Altered expression of iron-regulatory genes in HCC may disturb patient's iron balance. Hepcidin may play a role in defending the body against HCC.
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Affiliation(s)
- Hsi-Huang Tseng
- Institute of Occupational Medicine and Industrial Hygiene, College of Public Health, National Taiwan University, Taipei, Taiwan
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Lesnikov V, Gorden N, Fausto N, Spaulding E, Campbell J, Shulman H, Fleming RE, Deeg HJ. Transferrin fails to provide protection against Fas-induced hepatic injury in mice with deletion of functional transferrin-receptor type 2. Apoptosis 2008; 13:1005-12. [PMID: 18561026 DOI: 10.1007/s10495-008-0233-6] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
We reported previously that Fas-induced hepatic failure in normal mice was attenuated or prevented by exogenous transferrin (Tf), particularly apoTf. Here we show in C57BL6J/129 mice with genetic inactivation of transferrin receptor 2 (TfR2(Y245X)), that Fas-induced hepatotoxicity (apoptosis; rise in plasma aspartate aminotransferase (AST) levels) was comparable to that in wild-type mice, but was not modified by pretreatment with Tf. Rises in plasma AST were preceded by a decline in serum iron levels. AST elevations and iron declines were more profound in female than in male mice. Female mice also showed higher baseline levels of Bcl-xL in hepatocytes, which declined significantly upon treatment with agonistic anti-Fas antibody. These data confirm the cytoprotective function of Tf, and show a novel property of TfR2. Both apoptotic Fas responses and cytoprotective effects of Tf were associated with significant shifts in plasma iron levels, which quantitatively differed between male and female mice.
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Affiliation(s)
- Vladimir Lesnikov
- Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, D1-100, P.O. Box 19024, Seattle, WA 98109-1024, USA
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Iron regulates phosphorylation of Smad1/5/8 and gene expression of Bmp6, Smad7, Id1, and Atoh8 in the mouse liver. Blood 2008; 112:1503-9. [PMID: 18539898 DOI: 10.1182/blood-2008-03-143354] [Citation(s) in RCA: 352] [Impact Index Per Article: 20.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Although hepcidin expression was shown to be induced by the BMP/Smad signaling pathway, it is not yet known how iron regulates this pathway and what its exact molecular targets are. We therefore assessed genome-wide liver transcription profiles of mice of 2 genetic backgrounds fed iron-deficient, -balanced, or -enriched diets. Among 1419 transcripts significantly modulated by the dietary iron content, 4 were regulated similarly to the hepcidin genes Hamp1 and Hamp2. They are coding for Bmp6, Smad7, Id1, and Atoh8 all related to the Bmp/Smad pathway. As shown by Western blot analysis, variations in Bmp6 expression induced by the diet iron content have for functional consequence similar changes in Smad1/5/8 phosphorylation that leads to formation of heteromeric complexes with Smad4 and their translocation to the nucleus. Gene expression variations induced by secondary iron deficiency or iron overload were compared with those consecutive to Smad4 and Hamp1 deficiency. Iron overload developed by Smad4- and Hamp1-deficient mice also increased Bmp6 transcription. However, as shown by analysis of mice with liver-specific disruption of Smad4, activation of Smad7, Id1, and Atoh8 transcription by iron requires Smad4. This study points out molecules that appear to play a critical role in the control of systemic iron balance.
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