Increased Ferroportin Expression Research Articles

Targeted autophagy disruption reveals the central role of macrophage iron metabolism in systemic iron homeostasis

AbstractIron homeostasis depends on both intracellular control through iron-responsive proteins and the systemic level of iron through hepcidin-ferroportin axis. Indeed, the hormone hepcidin downregulates the ferroportin iron exporter to control iron recycling from macrophages and iron uptake from enterocytes. Here, we focused on the role of autophagy in macrophage iron metabolism and systemic iron homeostasis. Mice deficient for autophagy in macrophages (LysM-Atg5−/−) mimicked a primary iron overload phenotype, resulting in high ferroportin expression in both macrophages and enterocytes that correlated with marked parenchymal iron overload. Furthermore, LysM-Atg5−/− mice exhibited increased hematopoietic activity with no sign of anemia but correlating with rather high plasma iron level. Compared with wild-type cells, bone marrow–derived macrophages from LysM-Atg5−/− mice had significantly increased ferroportin expression and decreased iron content, confirming high iron export. In erythrophagocytic macrophages, autophagy regulates hemosiderin storage mechanisms as well as degradation of ferroportin and subsequently its plasma membrane localization and iron export; furthermore, ferroportin colocalization with hepcidin indicates hepcidin autocrine activity. Relatively high hepatic hepcidin expression and decreased hepcidin level in the spleen of LysM-Atg5−/− mice, correlating with low hemosiderin iron storage, as well as in erythrophagocytic Atg5−/− macrophages were evidenced. Therefore, our results highlight the critical role of autophagy in macrophages for iron trafficking and systemic iron homeostasis. We propose that in macrophages, autophagy restricts ferroportin level and iron export, resulting in hepcidin expression with an autocrine-paracrine effect that plays a role in the regulation of ferroportin expression in duodenal enterocytes.

Cobalt accumulation and iron-regulatory protein profile expression in immature mouse brain after perinatal exposure to cobalt chloride

Developing brain is very sensitive to the influence of environmental factors during gestation and the neonatal period. The aim of the study is to assess cobalt and iron accumulation in the brain as well as changes in the expression of iron-regulatory proteins transferrin receptor 1, hepcidin, and ferroportin in suckling mice. Perinatal exposure to cobalt chloride increased significantly cobalt content in brain tissue homogenates of 18-day-old (d18) and 25-day-old (d25) mice inducing alterations in brain iron homeostasis. Higher degree of transferrin receptor 1 expression was demonstrated in cobalt chloride-exposed mice with no substantial changes between d18 and d25 mice. A weak ferroportin expression was found in 18-day-old control and cobalt-treated mouse brain. Cobalt exposure of d25 mice resulted in increased ferroportin expression in brain compared to the untreated age-matched control group. Hepcidin level in cobalt-exposed groups was decreased in d18 mice and slightly increased in d25 mice. The obtained data contribute for the better understanding of metal toxicity impact on iron homeostasis in the developing brain with further possible implications in neurodegeneration.

Ferroportin in Erythrocytes: Importance for Iron Homeostasis and its Role in Infection

Ferroportin (FPN), the only known vertebrate iron exporter, transports iron from intestinal, splenic, and hepatic cells into the blood to provide iron to other tissues and cells in vivo. Most of the circulating iron is consumed by erythroid cells to synthesize hemoglobin. Recently, we found that erythroid cells not only consume large amounts of iron, but also return significant amounts of iron to the blood. Erythroblast-specific Fpn knockout (Fpn KO) mice developed lower serum iron levels in conjunction with tissue iron overload and increased FPN expression in spleen and liver without changing hepcidin levels. Our results also showed that Fpn KO mice, which suffer from mild hemolytic anemia, were sensitive to phenylhydrazine-induced oxidative stress but were able to tolerate iron deficiency upon exposure to a low-iron diet and phlebotomy, supporting that the anemia of Fpn KO mice resulted from erythrocytic iron overload and resulting oxidative injury rather than a red blood cell (RBC) production defect. Moreover, we found that the mean corpuscular volume (MCV) values of gain-of-function FPN mutation patients were positively associated with serum transferrin saturations, whereas MCVs of loss-of-function FPN mutation patients were not, supporting that erythroblasts donate iron to blood through FPN in response to serum iron levels. Our results indicate that FPN of erythroid cells has an unexpectedly essential role in maintaining systemic iron homeostasis and protecting RBCs from oxidative stress, providing insight into the pathophysiology of FPN diseases. When malaria parasites invade red blood cells (RBCs), they consume copious amounts of hemoglobin, and severely disrupt iron regulation in humans. Anemia often accompanies malaria disease; however, iron supplementation therapy inexplicably exacerbates malarial infections. We recently found that the iron exporter ferroportin (FPN) was highly abundant in RBCs, and iron supplementation suppressed its activity. Conditional deletion of the Fpn gene in erythroid cells resulted in accumulation of excess intracellular iron, cellular damage, hemolysis, and increased fatality in malaria-infected mice. In humans, a prevalent FPN mutation,Q248H (glutamine to histidine at position 248), prevented hepcidin-induced degradation of FPN and protected against severe malaria disease. FPNQ248H appears to have been positively selected in African populations in response to the impact of malaria disease. Thus, FPN protects RBCs against oxidative stress and malaria infection. Zhang DL, Wu J, Shah BN et al. Erythrocytic ferroportin reduces intracellular iron accumulation, hemolysis, and malaria risk. Science. 2018;359 (6383):1520-1523. Zhang DL, Ghosh MC, Ollivierre H, Li Y, Rouault TA. Ferroportin deficiency in erythroid cells causes serum iron deficiency and promotes hemolysis due to oxidative stress. Blood. 2018;132 (19):2078-2087. Zhang DL, Rouault TA. How does hepcidin hinder ferroportin activity. Blood. 2018;131 (8):840-842. Disclosures No relevant conflicts of interest to declare.

PartiallyHydrolyzedGuarGumIncreases FerroportinExpressionintheColonofAnemic GrowingRats.

Studies have reported a positive effect of prebiotics on the bioavailability of iron. This study evaluated the effect of partially hydrolyzed guar gum (PHGG) on iron absorption mechanisms in anemic rats. Male Wistar rats were fed 75g American Institute of Nutrition Rodent Diets for growth, pregnancy and lactation (AIN93-G) without iron for three weeks in order to induce iron deficiency anemia. Then they were fed a control diet (n = 12; without fiber) or a diet with 7.5% of PHGG (n = 12), both without iron. Food intake, body growth and the feed efficiency coefficient (FEC) were measured. The animals were euthanized after two weeks of treatment. The weight of the organs, the pH of the cecal content, and the hepatic iron and ferroportin expression in the cecum, duodenum, and liver were assessed. The intake of PHGG reduced food intake without affecting body growth, and there was a difference between the groups regarding the FEC (p = 0.026), with the highest value found in the PHGG group. The weight of the cecal content increased (p ≤ 0.001) and the pH of the cecal content was significantly lower in the PHGG group. The intake of PHGG significantly increased ferroportin expression in the cecum;however, the difference was not significant in the duodenum and the liver. PHGG seems to have a positive influence on iron absorption through transporter expression, and structural and physiological changes in the colon of anemic growing animals.

Acidic Polysaccharide from Angelica sinensis Reverses Anemia of Chronic Disease Involving the Suppression of Inflammatory Hepcidin and NF-κB Activation.

Anemia of chronic disease (ACD) is the second most prevalent anemia and frequently occurs in patients with acute or chronic immune activation. In the current study, we evaluated the therapeutic efficacy of Angelica sinensis polysaccharide (ASP) against ACD in rats and the potential mechanisms involved. The results showed that ASP inhibited inflammatory hepcidin in both HepG2 cells and ACD rats by blocking the IL-6/STAT3 and BMP/SMAD pathways. In ACD rats, the administration of ASP increased ferroportin expression, mobilized iron from the liver and spleen, increased serum iron levels, caused an elevation of serum EPO, and effectively relieved the anemia. Furthermore, ASP inhibited NF-κB p65 activation via the IκB kinases- (IKKs-) IκBα pathway, thereby reducing the secretion of interleukin-6 (IL-6) and TNF-α, which is known to inhibit erythropoiesis. Our findings indicate that ASP is a potential treatment option for patients suffering from ACD.

Corrigendum] Naotaifang extract treatment results in increased ferroportin expression in the hippocampus of rats subjected to cerebral ischemia.

Mol Med Rep 11: [Related article:] 4047–4052, 2015; DOI: 10.3892/mmr.2015.3309 Following the publication of this article, an interested reader drew to our attention anomalies associated with the presentation of Figs. 1 and 4. The image selected for Fig. 1A, the data pertaining to '2 h', was inadvertently selected for Fig. 4A, the image labeled 'Sham'. Additionally, in Fig. 4, the same source image had inadvertently been used to provide the images for the 'Low dose group' and 'Model' panels (although the view presented differed in these panels). On re-examining our data, we realized that these errors had occurred during the compilation of Fig. 4, and that the images were correctly selected for Fig. 1. An amended version of Fig. 4 is presented below, featuring images which correctly show the data for the 'Low dose', 'Sham' and 'Model' groups. The immunohistochemical results suggested that, following treatment with 27 g/kg naotaifang extract, the expression of Fpn increased significantly compared with the other treatment doses (P<0.05), whereas significant changes were not observed among the other groups (P>0.05). The errors made in the selection of certain images for Fig. 4A did not affect the overall conclusions reported in the present study. We sincerely apologize for this mistake, and thank the reader of our article who drew this matter to our attention. Furthermore, we regret any inconvenience this mistake has caused.

Hepcidin Mitigates Renal Ischemia-Reperfusion Injury by Modulating Systemic Iron Homeostasis.

Iron-mediated oxidative stress is implicated in the pathogenesis of renal ischemia-reperfusion injury. Hepcidin is an endogenous acute phase hepatic hormone that prevents iron export from cells by inducing degradation of the only known iron export protein, ferroportin. In this study, we used a mouse model to investigate the effect of renal ischemia-reperfusion injury on systemic iron homeostasis and determine if dynamic modulation of iron homeostasis with hepcidin has therapeutic benefit in the treatment of AKI. Renal ischemia-reperfusion injury induced hepatosplenic iron export through increased ferroportin expression, which resulted in hepatosplenic iron depletion and an increase in serum and kidney nonheme iron levels. Exogenous hepcidin treatment prevented renal ischemia-reperfusion-induced changes in iron homeostasis. Hepcidin also decreased kidney ferroportin expression and increased the expression of cytoprotective H-ferritin. Hepcidin-induced restoration of iron homeostasis was accompanied by a significant reduction in ischemia-reperfusion-induced tubular injury, apoptosis, renal oxidative stress, and inflammatory cell infiltration. Hepcidin -: deficient mice demonstrated increased susceptibility to ischemia-reperfusion injury compared with wild-type mice. Reconstituting hepcidin-deficient mice with exogenous hepcidin induced hepatic iron sequestration, attenuated the reduction in renal H-ferritin and reduced renal oxidative stress, apoptosis, inflammation, and tubular injury. Hepcidin-mediated protection was associated with reduced serum IL-6 levels. In summary, renal ischemia-reperfusion injury results in profound alterations in systemic iron homeostasis. Hepcidin treatment restores iron homeostasis and reduces inflammation to mediate protection in renal ischemia-reperfusion injury, suggesting that hepcidin-ferroportin pathway holds promise as a novel therapeutic target in the treatment of AKI.

Naotaifang extract treatment results in increased ferroportin expression in the hippocampus of rats subjected to cerebral ischemia.

The expression of Ferroportin (Fpn) was examined at different time points in rats following focal cerebral ischemia treated with or without the traditional Chinese medicine Naotaifang. Initially, rats were randomly divided into 2, 6, 12, 24 and 72 h groups following middle cerebral artery occlusion (MCAO) and the mRNA and protein level of Fpn was detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) at the above time points. Secondly, the rats were randomly divided into five groups as follows: Sham surgery group, model group, low-dose group (3 g/kg NTE), medium dose group (9 g/kg NTE) and the high-dose group (27 g/kg NTE). After 3 days of corresponding therapy by intragastric administration once a day, the regional cerebral ischemia model was reproduced by the MCAO suture method. On the third day, the neurological behavior of the rats was analyzed by neurobehavioral assessment. Fpn in the hippocampal CA2 region was measured by immunohistochemistry and the mRNA level of Fpn was detected by RT-PCR. Expression of Fpn in the hippocampal CA2 region reached a peak 12 h after surgery (P<0.05, compared with the model group). The high-dose group (27 g/kg NTE) exhibited a lower neurological behavior score (P<0.05) and a higher level of expression of Fpn at the mRNA and protein level compared with the sham surgery group and model group (P<0.05). Dysregulation of intracellular iron balance is possibly a new mechanism underlying cerebral ischemia. NTE can protect the neuronal population in the hippocampal CA2 region by adjusting the expression of Fpn to balance iron levels following cerebral ischemia.

The role of vitamin D in regulating the iron-hepcidin-ferroportin axis in monocytes

Chronic kidney disease affects 40% of adults aged 65 and older. Anemia of CKD is present in 30% of patients with CKD and is associated with increased cardiovascular risk, decreased quality of life, and increased mortality. Hepcidin-25 (hepcidin), the key iron regulating hormone, prevents iron egress from macrophages and thus prevents normal recycling of the iron needed to support erythropoiesis. Hepcidin levels are increased in adults and children with CKD. Vitamin D insufficiency is highly prevalent in CKD and is associated with erythropoietin hyporesponsiveness. Recently, hepcidin levels were found to be inversely correlated with vitamin D status in CKD. The aim of this study was to investigate the role of vitamin D in the regulation of hepcidin expression in vitro and in vivo. This study reports that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the hormonally active form of vitamin D, is associated with decreased hepcidin and increased ferroportin expression in lipopolysaccharide (LPS) stimulated THP-1 cells. 1,25(OH)2D3 also resulted in a dose-dependent decrease in pro-hepcidin cytokines, IL-6 and IL-1β, release in vitro. Further, we show that high-dose vitamin D therapy impacts systemic hepcidin levels in subjects with early stage CKD. These data suggest that improvement in vitamin D status is associated with lower systemic concentrations of hepcidin in subjects with CKD. In conclusion, vitamin D regulates the hepcidin-ferroportin axis in macrophages which may facilitate iron egress. Improvement in vitamin D status in patients with CKD may reduce systemic hepcidin levels and may ameliorate anemia of CKD.

Abstract 8857: Iron Restriction Prevents the Progression of Diabetic Nephropathy in db/db Mice

Background: Iron is well-known as an essential trace metal for all living body. On the other hand, excess iron causes oxidative stress through catalyzing highly toxic hydroxyl radical via Fenton/Haber-Weiss reaction. Recent studies have shown that iron reduction ameliorates diabetic condition and prevents development of adipocyte hypertrophy. In the present study, we studied the protective effect of iron reduction against diabetic nephropathy, a diabetic complication. Methods and Results: We employed male db/db mice, a model of diabetic nephropathy and male db/m mice as control mice. db/db mice were divided into 2 groups, normal diet group (ND)(Fe 100mg/kg food) and iron-restricted diet group (ID)(Fe 10mg/kg food). Urinary albumin excretion was increasing in db/db mice with ND compared with db/m mice with ND, which was suppressed in db/db mice with ID. In renal histological analysis, increased glomerular volume and mesangial area expansion in ND-fed db/db mice were reduced in ID-fed db/db mice. Deposition of extracelluar matrix such as collagen IV and fibronectin was decreased in db/db mice with ID. Upregulated glomerular desmin expression in db/db mice with ND was diminished in db/db mice with ID. In terms with oxidative stress, ID diminished increased superoxide production in kidney of ND-fed db/db mice. Renal expression of NADPH oxidase components, p22phox and NOX4, were augmented in db/db mice with ND-fed, which was abolished by ID-fed. In terms with renal iron transporter, there were no differences of expression of transferrin receptor and divalent metal transporter-1 between db/m mice and db/db mice, increased ferroportin expression in db/db mice with ND- was reversed by ID to the same level as db/m mice. Urinary iron excretion was significantly higher in ND-fed db/db mice, which was reduced with ID administration. Conclusion: These findings suggest that dietary iron restriction exert preventive effect against development of diabetic nephropathy via the reduction of oxidative stress.

Hemoglobin Directs Macrophage Differentiation and Prevents Foam Cell Formation in Human Atherosclerotic Plaques

The purpose of this study was to examine selective macrophage differentiation occurring in areas of intraplaque hemorrhage in human atherosclerosis. Macrophage subsets are recognized in atherosclerosis, but the stimulus for and importance of differentiation programs remain unknown. We used freshly isolated human monocytes, a rabbit model, and human atherosclerotic plaques to analyze macrophage differentiation in response to hemorrhage. Macrophages characterized by high expression of both mannose and CD163 receptors preferentially exist in atherosclerotic lesions at sites of intraplaque hemorrhage. These hemoglobin (Hb)-stimulated macrophages, M(Hb), are devoid of neutral lipids typical of foam cells. In vivo modeling of hemorrhage in the rabbit model demonstrated that sponges exposed to red cells showed an increase in mannose receptor-positive macrophages only when these cells contained Hb. Cultured human monocytes exposed to Hb:haptoglobin complexes, but not interleukin-4, expressed the M(Hb) phenotype and were characterized by their resistance to cholesterol loading and up-regulation of ATP-binding cassette (ABC) transporters. M(Hb) demonstrated increased ferroportin expression, reduced intracellular iron, and reactive oxygen species (ROS). Degradation of ferroportin using hepcidin increased ROS and inhibited ABCA1 expression and cholesterol efflux to apolipoprotein A-I, suggesting reduced ROS triggers these effects. Knockdown of liver X receptor alpha (LXRα) inhibited ABC transporter expression in M(Hb) and macrophages differentiated in the antioxidant superoxide dismutase. Last, LXRα luciferase reporter activity was increased in M(Hb) and significantly reduced by overnight treatment with hepcidin. Collectively, these data suggest that reduced ROS triggers LXRα activation and macrophage reverse cholesterol transport. Hb is a stimulus for macrophage differentiation in human atherosclerotic plaques. A decrease in macrophage intracellular iron plays an important role in this nonfoam cell phenotype by reducing ROS, which drives transcription of ABC transporters through activation of LXRα. Reduction of macrophage intracellular iron may be a promising avenue to increase macrophage reverse cholesterol transport.

Glutaredoxin 5 deficiency causes sideroblastic anemia by specifically impairing heme biosynthesis and depleting cytosolic iron in human erythroblasts

Glutaredoxin 5 (GLRX5) deficiency has previously been identified as a cause of anemia in a zebrafish model and of sideroblastic anemia in a human patient. Here we report that GLRX5 is essential for iron-sulfur cluster biosynthesis and the maintenance of normal mitochondrial and cytosolic iron homeostasis in human cells. GLRX5, a mitochondrial protein that is highly expressed in erythroid cells, can homodimerize and assemble [2Fe-2S] in vitro. In GLRX5-deficient cells, [Fe-S] cluster biosynthesis was impaired, the iron-responsive element-binding (IRE-binding) activity of iron regulatory protein 1 (IRP1) was activated, and increased IRP2 levels, indicative of relative cytosolic iron depletion, were observed together with mitochondrial iron overload. Rescue of patient fibroblasts with the WT GLRX5 gene by transfection or viral transduction reversed a slow growth phenotype, reversed the mitochondrial iron overload, and increased aconitase activity. Decreased aminolevulinate delta, synthase 2 (ALAS2) levels attributable to IRP-mediated translational repression were observed in erythroid cells in which GLRX5 expression had been downregulated using siRNA along with marked reduction in ferrochelatase levels and increased ferroportin expression. Erythroblasts express both IRP-repressible ALAS2 and non-IRP-repressible ferroportin 1b. The unique combination of IRP targets likely accounts for the tissue-specific phenotype of human GLRX5 deficiency.

Iron uptake from plasma transferrin by a transferrin receptor 2 mutant mouse model of haemochromatosis

Hereditary haemochromatosis type 3 is caused by mutations in transferrin receptor (TFR) 2. TFR2 has been shown to mediate iron transport in vitro and regulate iron homeostasis. The aim of this study was to determine the role of Tfr2 in iron transport in vivo using a Tfr2 mutant mouse. Tfr2 mutant and wild-type mice were injected intravenously with (59)Fe-transferrin and tissue (59)Fe uptake was measured. Tfr1, Tfr2 and ferroportin expression was measured by real-time PCR and Western blot. Cellular localisation of ferroportin was determined by immunohistochemistry. Transferrin-bound iron uptake by the liver and spleen in Tfr2 mutant mice was reduced by 20% and 65%, respectively, whilst duodenal and renal uptake was unchanged compared with iron-loaded wild-type mice. In Tfr2 mutant mice, liver Tfr2 protein was absent, whilst ferroportin protein was increased in non-parenchymal cells and there was a low level of expression in hepatocytes. Tfr1 expression was unchanged compared with iron-loaded wild-type mice. Splenic Tfr2 protein expression was absent whilst Tfr1 and ferroportin protein expression was increased in Tfr2 mutant mice compared with iron-loaded wild-type mice. A small reduction in hepatic transferrin-bound iron uptake in Tfr2 mutant mice suggests that Tfr2 plays a minor role in liver iron transport and its primary role is to regulate iron metabolism. Increased ferroportin expression due to decreased hepcidin mRNA levels is likely to be responsible for impaired splenic iron uptake in Tfr2 mutant mice.

Copper deficiency increases ferroportin expression in rats

Ferroportin is a transmembrane iron‐export protein that plays an important role in dietary iron absorption and iron recycling from senescent red blood cells. Ferroportin expression increases with tissue iron loading but decreases in response to hepcidin, the circulating iron‐regulatory hormone that binds to ferroportin, causing its internalization and degradation. Hepcidin expression increases with iron loading but decreases during anemia. We examined ferroportin and hepcidin expression in copper‐deficient (CuD) rats, which have elevated liver iron levels but are anemic. We compared CuD and copper‐adequate (CuA) animals (n=4/group) after one month of treatment postweaning. Hemoglobin levels were lower in CuD than in CuA rats (101 vs. 159 g/L). Western blot analysis revealed that ferroportin levels increased in CuD rats relative to CuA rats by 4‐ and 1.6‐fold in liver and spleen, respectively (P&lt;0.02). Increased ferroportin expression in CuD rat tissues was associated with increased ferroportin transcript abundance, as measured by qRT‐PCR. Copy number of hepatic hepcidin mRNA was more than 50‐fold lower in CuD than CuA rats despite a doubling of liver iron. We conclude that tissue ferroportin levels increase in CuD rats because of diminished hepcidin levels and increased ferroportin mRNA abundance.

Potential role of bone morphogenetic protein (BMP) signalling as a potential therapeutic target for modification of iron balance

This important paper by Babitt et al. outlines a novel method of regulating hepcidin expression that could potentially lead to novel methods of improving iron utilization amongst patients with anaemia of chronic kidney disease (CKD). Hepcidin is the master regulator of systemic iron homeostasis acting by causing the internalization and degradation of the iron exporter channel, ferroportin. This blocks the efficient transfer of iron to plasma and therefore functionally blocks iron absorption [1,2,3]. Hepcidin also blocks the transfer of iron from the reticuloendothelial system to plasma in chronic inflammatory disease states [4]. Increased levels of hepcidin thus contribute to anaemia of inflammation by shunting iron away from erythropoiesis and sequestrating it in organs such as the liver and spleen [5]. Hepcidin deficiency itself can lead to haemochromatosis [6]. Babitt et al. outline a novel method of regulating hepcidin expression using BMP signalling. Hepcidin expression was initially increased by the injection of purified BMP-2 at a dose of 1 mg/kg. retroorbitally into mice, and 4 h postinjection, hepatic hepcidin mRNA levels increased 1.8fold and serum iron levels decreased. Subsequently, soluble haemojuvelin (HJV.Fc) a selective inhibitor of BMP induction of hepcidin expression in vitro decreases hepcidin expression, leading to increased ferroportin expression, mobilization of splenic iron stores and increased serum iron levels in vivo. This work further demonstrates the key role of hepcidin in iron haemostasis, supporting the pharmacological use of hepcidin agonists, and antagonists in various disorders of iron haemostasis [7,8].