Iron Export Protein Ferroportin Research Articles

Monocyte MRI Relaxation Rates Are Regulated by Extracellular Iron and Hepcidin.

Many chronic inflammatory conditions are mediated by an increase in the number of monocytes in peripheral circulation, differentiation of monocytes to macrophages, and different macrophage subpopulations during pro- and anti-inflammatory stages of tissue injury. When hepcidin secretion is stimulated during inflammation, the iron export protein ferroportin is targeted for degradation on a limited number of cell types, including monocytes and macrophages. Such changes in monocyte iron metabolism raise the possibility of non-invasively tracking the activity of these immune cells using magnetic resonance imaging (MRI). We hypothesized that hepcidin-mediated changes in monocyte iron regulation influence both cellular iron content and MRI relaxation rates. In response to varying conditions of extracellular iron supplementation, ferroportin protein levels in human THP-1 monocytes decreased two- to eightfold, consistent with paracrine/autocrine regulation of iron export. Following hepcidin treatment, ferroportin protein levels further decreased two- to fourfold. This was accompanied by an approximately twofold increase in total transverse relaxation rate, R2*, compared to non-supplemented cells. A positive correlation between total cellular iron content and R2* improved from moderate to strong in the presence of hepcidin. These findings suggest that hepcidin-mediated changes detected in monocytes using MRI could be valuable for in vivo cell tracking of inflammatory responses.

Reprogramming of pancreatic adenocarcinoma immunosurveillance by a microbial probiotic siderophore

There is increasing evidence suggesting the role of microbiome alterations in relation to pancreatic adenocarcinoma and tumor immune functionality. However, molecular mechanisms of the interplay between microbiome signatures and/or their metabolites in pancreatic tumor immunosurveillance are not well understood. We have identified that a probiotic strain (Lactobacillus casei) derived siderophore (ferrichrome) efficiently reprograms tumor-associated macrophages (TAMs) and increases CD8 + T cell infiltration into tumors that paralleled a marked reduction in tumor burden in a syngeneic mouse model of pancreatic cancer. Interestingly, this altered immune response improved anti-PD-L1 therapy that suggests promise of a novel combination (ferrichrome and immune checkpoint inhibitors) therapy for pancreatic cancer treatment. Mechanistically, ferrichrome induced TAMs polarization via activation of the TLR4 pathway that represses the expression of iron export protein ferroportin (FPN1) in macrophages. This study describes a novel probiotic based molecular mechanism that can effectively induce anti-tumor immunosurveillance and improve immune checkpoint inhibitors therapy response in pancreatic cancer.

Novel Regulatory Role of SIRT3 on Cardiomyocyte Mitochondrial Frataxin and Ferroptosis

AIMSSirtuin 3 (SIRT3) has been shown to contribute to the mitochondrial cardiomyopathy of Friedreich’s Ataxia (FRDA), which is characterized by a deficiency in mitochondrial frataxin along with mitochondrial hyperacetylation. Using a cardiomyocyte‐specific SIRT3 knockout (SIRT3cKO) mouse model, we addressed two key questions: (1) whether SIRT3‐induced acetylation of proteins is specific to the mitochondria; and (2) if mitochondrial SIRT3 is a key regulator of mitochondrial frataxin which impacts mitochondrial iron homeostasis and ferroptosis.METHODS AND RESULTSThe mitochondrial and cytosolic fractions were isolated from the ventricles of the hearts of SIRT3cKO mice. The mitochondrial and cytosolic proteins and mitochondrial iron levels were analyzed by comparison to SIRT3‐Loxp wild‐type (WT) control mice. Cardiac function study showed that SIRT3cKO mice developed heart failure as evidenced by reduction of ejection fraction (EF) and fraction shortening (FS) and increased isovolumic relaxation time (IVRT) and myocardial performance index (MPI) when compared to WT controls. Comparison of the mitochondrial and cytosolic fractions of the SIRT3cKO model to those of the WT control shows that, upon loss of SIRT3, mitochondrial, but not cytosolic, total lysine acetylation was significantly increased in the heart. Similarly, acetylated p53 (p53ace) was significantly upregulated only in the mitochondria, while levels of p53 were not altered in either compartment. These data demonstrate that SIRT3 is the primary mitochondrial deacetylase, while acetylation in the cytosol is independent of SIRT3 in cardiomyocytes.Most importantly, loss of SIRT3 in the mitochondria resulted in significant reduction of the protein frataxin and the Iron (II) export protein ferroportin (FPN). Furthermore, levels of glutathione peroxidase 4 (GPX4) were also downregulated in the mitochondria. This was accompanied by a significant increase in levels of 4‐hydroxynonenal (4‐HNE), an indicator of lipid peroxidation, and suggestive of upregulated mitochondrial ferroptosis in SIRT3cKO mouse hearts. Additionally, mitophagy marker beclin‐1 expression was significantly reduced in the mitochondria of SIRT3cKO mice. Levels of glucose transporter‐1 (GLUT1), which functions to deliver the antioxidant Vitamin C to the mitochondria and reduce ROS production, were also diminished in the mitochondria. Mechanistically, mitochondrial levels of hypoxia inducible factor‐2α (HIF‐2α) were downregulated, while those of HIF‐1α remained unchanged in SIRT3cKO mouse hearts. Treatment with ferroptosis inhibitor ferrostatin‐1 for 14 days significantly reduced 4‐HNE levels and rescued preexisting impaired cardiac function in SIRT3cKO mice.CONCLUSIONSFor the first time, we have demonstrated that cardiomyocyte SIRT3 deficiency causes mitochondrion‐specific acetylation and impairment of frataxin and ferroportin potentially via downregulation of HIF‐2α. Our results suggest that the SIRT3‐ferroptosis pathway may be a novel target for the mitochondrial cardiomyopathy of FRDA.

The biology of mammalian multi-copper ferroxidases.

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

HDAC inhibition induces EMT and alterations in cellular iron homeostasis to augment ferroptosis sensitivity in SW13 cells

Epithelial-to-mesenchymal transition (EMT) is an essential mechanism for development and wound healing, but in cancer it also mediates the progression and spread of aggressive tumors while increasing therapeutic resistance. Adoption of a mesenchymal state is also associated with increased iron uptake, but the relationship between EMT and the key regulators of cellular iron metabolism remains undefined. In this regard, the human adrenal cortical carcinoma SW13 cell line represents an invaluable research model as HDAC inhibitor treatment can convert them from an epithelial-like (SW13-) cell type to a mesenchymal-like (SW13+) subtype. In this study we establish SW13 cells as a model for exploring the link between iron and EMT. Increased iron accumulation following HDAC inhibitor mediated EMT is associated with decreased expression of the iron export protein ferroportin, enhanced ROS production, and reduced expression of antioxidant response genes. As availability of redox active iron and loss of lipid peroxide repair capacity are hallmarks of ferroptosis, a form of iron-mediated cell death, we next examined whether HDAC inhibitor treatment could augment ferroptosis sensitivity. Indeed, HDAC inhibitor treatment synergistically increased cell death following induction of ferroptosis. The exact mechanisms by which HDAC inhibition facilitates cell death following ferroptosis induction requires further study. As several HDAC inhibitors are already in use clinically for the treatment of certain cancer types, the findings from these studies have immediate implications for improving iron-targeted chemotherapeutic strategies.

Pumping iron in the kidney

Iron balance is tightly controlled to provide adequate amounts of this essential nutrient, but to limit the adverse effects of excess iron. Key mediators of systemic iron homeostasis are the iron regulatory hormone hepcidin and its receptor, the iron export protein ferroportin. A new study by Mohammad etal. demonstrates the functional role of the hepcidin-ferroportin axis in the kidney, and how this contributes to kidney iron levels and the systemic iron economy.

Fetal liver hepcidin secures iron stores in utero

AbstractIn the adult, the liver-derived hormone hepcidin (HAMP) controls systemic iron levels by blocking the iron-exporting protein ferroportin (FPN) in the gut and spleen, the sites of iron absorption and recycling, respectively. Impaired HAMP expression or FPN responsiveness to HAMP result in iron overload. HAMP is also expressed in the fetal liver but its role in controlling fetal iron stores is not understood. To address this question in a manner that safeguards against the confounding effects of altered maternal iron homeostasis, we generated fetuses harboring a paternally-inherited ubiquitous knock-in of the HAMP-resistant fpnC326Y. Additionally, to safeguard against any confounding effects of altered placental iron homeostasis, we generated fetuses with a liver-specific knock-in of fpnC326Y or knockout of the hamp gene. These fetuses had reduced liver iron stores and hemoglobin, and markedly increased FPN in the liver, but not in the placenta. Thus, fetal liver HAMP operates cell-autonomously to increase fetal liver iron stores. Our findings also suggest that FPN in the placenta is not actively regulated by fetal liver HAMP under normal physiological conditions.

Nrf2 knockout dysregulates iron metabolism and increases the hemolysis through ROS in aging mice

AimsDysregulation of iron homeostasis in the body causes a variety of diseases. Iron deficiency leads to anemia, whereas iron overload aggravates cellular oxidative stress. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a protein that is activated in the nucleus and turns on the production of antioxidant enzymes, protecting cell against oxidative damage. This study aimed to investigate whether Nrf2 gene knockout influences iron homeostasis in aging mice. Materials and methodsIron content and iron metabolism-related proteins were assessed in different organs and blood serum of the 18 month-old Nrf2 knockout (Nrf2−/−) mice in comparison with the wild-type (WT) mice. Key findingsResults showed that the iron contents in spleen and liver all increased, and expression levels of iron transporters were altered in Nrf2−/− mice. In particularly, we found that the expression of iron export protein ferroportin 1 (Fpn1) in liver, spleen and small intestine all decreased in Nrf2−/− mice, which might account for the deposition of iron in different organs and the increased ROS. Surprisingly, we found that the serum iron level of Nrf2−/− mice did not decrease, but increased significantly even when the iron absorption at small intestine decreased. Our further investigation revealed that the increase of serum iron was due to the release of iron from the hemolysis of erythrocytes, which caused by the increased ROS level in red blood cells of the Nrf2−/− mice. SignificanceThese findings provide a more comprehensive understanding of the important role of Nrf2 in the regulation of systemic iron metabolism.

Increased Hepcidin Levels During a Period of High Training Load Do Not Alter Iron Status in Male Elite Junior Rowers.

The liver-derived hormone hepcidin plays a key role in iron metabolism by mediating the degradation of the iron export protein ferroportin 1 (FPN1). Circulating levels of hepcidin and the iron storage protein ferritin are elevated during the recovery period after acute endurance exercise, which can be interpreted as an acute phase reaction to intense exercise with far-reaching consequences for iron metabolism and homeostasis. Since absolute and functional iron deficiency (ID) potentially lead to a loss of performance and well-being, it is surprising that the cumulative effects of training stress on hepcidin levels and its interplay with cellular iron availability are not well described. Therefore, the aim of this study was to determine serum levels of hepcidin at six time points during a 4-week training camp of junior world elite rowers preparing for the world championships and to relate the alterations in training load to overall iron status determined by serum ferritin, transferrin, iron, and soluble transferrin receptor (sTfR). Serum hepcidin levels increased significantly (p = 0.02) during the initial increase in training load (23.24 ± 2.43 ng/ml) at day 7 compared to the start of training camp (11.47 ± 3.92 ng/ml) and turned back on day 13 (09.51 ± 3.59 ng/ml) already, meeting well the entrance level of hepcidin at day 0. Serum ferritin was significantly higher at day 7 compared to all other timepoints with exception of the subsequent time point at day 13 reflecting well the time course pattern of hepcidin. Non-significant changes between training phases were found for serum iron, transferrin, and sTfR levels as well as for transferrin saturation, and ferritin-index (sTfR/log ferritin). Our findings indicate that hepcidin as well as ferritin, both representing acute phase proteins, are sensitive to initial increases in training load. Erythropoiesis was unaffected by iron compartmentalization through hepcidin. We conclude that hepcidin is sensitive to rigorous changes in training load in junior world elite rowers without causing short-term alterations in functional iron homeostasis.

Abstract 102: Ferrichrome suppresses pancreatic tumor growth via targeting TAMs and enhancing CD8+ T-cells infiltration

Abstract Background: Pancreatic cancer (PanCa) is one of the most lethal malignancy with a very poor survival rate in patients due to inadequate treatment options. Accumulating evidences suggest that tumor associated macrophages (TAMs) and reduce infiltration of CD8+ T-cell population provide tumor suppressive environment leading to advanced growth and metastasis of PanCa. Thus, targeting TAMs and enhancing infiltration of CD8+ T-cells to the tumor site by non-toxic agents could be an effective therapeutic approach for the management of PanCa. In this study, we demonstrate that a novel probiotic-derived agent (ferrichrome) inhibits pancreatic tumor growth in syngeneic mouse model via modulating TAMs and increasing infiltration of CD8+ T-cells. Methods: RAW264.7 cells, murine peritoneal macrophages, mouse pancreatic cancer cells (UN-KC-6141) were used for this study. Effect of ferrichrome on the expression of pro (IL-12, p40, iNOS, IL-6), anti-inflammatory cytokines (MRC1 and Arginase-1) and iron metabolism markers (ferritin and ferroportin) in macrophages and co-culture systems was analyzed by qPCR. Effect of ferrichrome on metastatic phenotypes of PanCa cells was analyzed in a co-culture model of RAW264.7 and UN-KC-6141 cells. Therapeutic efficacy of ferrichrome was determined in syngeneic mouse model. Tumor immune infiltrating cells were analyzed in excised tumors by FACS, double immunofluorescence and immunohistochemistry analyses. Results: Ferrichrome treatment reversed the polarization of M2 macrophages towards the M1 phenotype as observed by increase expression of IL-12p40, iNOS, IL-6 and decrease expression of MRC1 and Arginase-1. Ferrichrome significantly (P<0.01) reduced the RAW264.7 cells induced migration and invasive potential of UN-KC-6141 cells. We further investigated the molecular mechanisms of ferrichrome induced M1 macrophage phenotype. Our results revealed that ferrichrome-induced M1 polarization of macrophages via upregulation of iron-sequestration protein (ferritin) and downregulation of iron-export protein (ferroportin). Ferrichrome administration (50 µg) intratumorally for 5 days per week for 26 days significantly (P<0.01) inhibited growth of xenograft tumors. We observed an increase of CD8+ T-cell and iNOS+F4/80+ macrophages infiltration and a decrease in the expression of CD163+ macrophages in resected tumors of ferrichrome treated mice. Conclusion: Our results strongly suggest that ferrichrome inhibits the tumor growth of PanCa via targeting TAMs and enhancing CD8+ T-cell infiltration. Ferrichrome could be used alone or in combination with current therapeutic regimens for the treatment of advanced PanCa. Citation Format: Mehdi Chaib, Bilal Bin Hafeez, Sonam Kumari, Mohammed Sikander, Hassan Mandil, Liza Makowski, Ajeeth Kumar Pingili, Elham Hatami, Advait Shetty, Dan Nirnoy, Murali Mohan Yallapu, Meena Jaggi, Subhash Chauhan. Ferrichrome suppresses pancreatic tumor growth via targeting TAMs and enhancing CD8+ T-cells infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 102.

Intracellular iron deficiency in pulmonary arterial smooth muscle cells induces pulmonary arterial hypertension in mice

Iron deficiency augments hypoxic pulmonary arterial pressure in healthy individuals and exacerbates pulmonary arterial hypertension (PAH) in patients, even without anemia. Conversely, iron supplementation has been shown to be beneficial in both settings. The mechanisms underlying the effects of iron availability are not known, due to lack of understanding of how cells of the pulmonary vasculature respond to changes in iron levels. The iron export protein ferroportin (FPN) and its antagonist peptide hepcidin control systemic iron levels by regulating release from the gut and spleen, the sites of absorption and recycling, respectively. We found FPN to be present in pulmonary arterial smooth muscle cells (PASMCs) and regulated by hepcidin cell autonomously. To interrogate the importance of this regulation, we generated mice with smooth muscle-specific knock in of the hepcidin-resistant isoform fpn C326Y. While retaining normal systemic iron levels, this model developed PAH and right heart failure as a consequence of intracellular iron deficiency and increased expression of the vasoconstrictor endothelin-1 (ET-1) within PASMCs. PAH was prevented and reversed by i.v. iron and by the ET receptor antagonist BQ-123. The regulation of ET-1 by iron was also demonstrated in healthy humans exposed to hypoxia and in PASMCs from PAH patients with mutations in bone morphogenetic protein receptor type II. Such mutations were further associated with dysregulation of the HAMP/FPN axis in PASMCs. This study presents evidence that intracellular iron deficiency specifically within PASMCs alters pulmonary vascular function. It offers a mechanistic underpinning for the known effects of iron availability in humans.

Dietary iron absorption during early postnatal life.

Inadequate iron levels during early life can have adverse consequences for the developing infant. Iron deficiency during this critical period of growth can affect brain development and cognitive function, problems that can be lifelong despite subsequent correction of the iron deficit. Therefore, it is critical that the suckling infant has sufficient iron for their developmental needs. Much of the iron used in the immediate post-natal period is stored iron that was acquired from the mother in the final trimester of pregnancy, however, despite having low iron levels, breast milk can also make a significant contribution to infant iron needs. This reflects the ability of the suckling infant to absorb dietary iron far more efficiently than is possible after weaning. The mechanisms underlying this enhanced iron absorption are poorly understood. The iron export protein ferroportin is essential for this process, as it is in adults, however, the role of other molecules normally involved in iron absorption following weaning is less clear. The composition and distribution of iron in breast milk may be important, as could the contribution of more distal parts of the gastrointestinal tract. This review discusses the potential role of each of the above components in intestinal iron absorption during suckling and highlights the need for further research into this important process.

Post Translational Modulation of \u03b2-Amyloid Precursor Protein Trafficking to the Cell Surface Alters Neuronal Iron Homeostasis

Cell surface β-Amyloid precursor protein (APP) is known to have a functional role in iron homeostasis through stabilising the iron export protein ferroportin (FPN). Mechanistic evidence of this role has previously only been provided through transcriptional or translational depletion of total APP levels. However, numerous post-translational modifications of APP are reported to regulate the location and trafficking of this protein to the cell surface. Stable overexpressing cell lines were generated that overexpressed APP with disrupted N-glycosylation (APPN467K and APPN496K) or ectodomain phosphorylation (APPS206A); sites selected for their proximity to the FPN binding site on the E2 domain of APP. We hypothesise that impaired N-glycosylation or phosphorylation of APP disrupts the functional location on the cell surface or binding to FPN to consequentially alter intracellular iron levels through impaired cell surface FPN stability. Outcomes confirm that these post-translational modifications are essential for the correct location of APP on the cell surface and highlight a novel mechanism by which the cell can modulate iron homeostasis. Further interrogation of other post-translational processes to APP is warranted in order to fully understand how each modification plays a role on regulating intracellular iron levels in health and disease.

Role of hepcidin and iron metabolism in the onset of prostate cancer.

The present study aimed to understand the roles of hepcidin and iron metabolism in the onset of prostate cancer. The prostate cancer LNCap, PC3 and DU145 cell lines were transfected with small interfering RNA (siRNA) targeting hepcidin to knockdown hepcidin expression in LNCap, PC3 and DU145 cells. The expression levels of hepcidin in prostate cancer and normal prostate RWPE-1cells were detected by western blot analysis. Exogenous hepcidin was added into the hepcidin-silenced cell lines. Intracellular iron levels were detected using a fluorescence assay, and the proliferative and migratory capacities of cells were detected using the MTT and wound-healing assays, respectively. The apoptotic rate was measured using flow cytometry, and changes in the expression of the iron-export protein ferroportin on the cell membrane were detected by western blot analysis. Hepcidin expression in prostate cancer cells was significantly higher than that of normal prostate cells (P<0.05). Furthermore, the iron levels of hepcidin-silenced cells (hepcidin-ve groups) were significantly lower than those in the cells treated with exogenous hepcidin (hepcidin+ve groups) (P<0.05). The proliferative capacity of the hepcidin+ve cells significantly exceeded those of the hepcidin-ve groups (P<0.05) and increased over time. In the wound-healing assay, the number of hepcidin+ve cells present within the scratch sites increased compared with hepcidin-ve cells, indicating a higher migration rate. Additionally, the expression of ferroportin in the hepcidin-ve groups significantly exceeded that in the hepcidin+ve groups (P<0.05). Hepcidin is involved in the onset of prostate cancer, most likely by reducing ferroportin expression and increasing intracellular iron levels to enhance the proliferation, migration and anti-apoptotic capacities of cancer cells.

Hepcidin interaction with ferroportin in the Xenopus oocyte expression system

The cellular iron‐export protein ferroportin (Fpn), expressed in enterocytes and macrophages, is essential for iron homeostasis. Fpn operates under the control of the iron‐regulatory hormone hepcidin. The canonical mechanism by which hepcidin suppresses Fpn activity comprises hepcidin binding by Fpn and the subsequent internalization and ubiquitin‐dependent degradation of the transport protein [Drakesmith H et al (2015) Cell Metab 22, 777–787]. Here we explored hepcidin inhibition of 55Fe efflux from RNA‐injected Xenopus oocytes expressing human Fpn. Hepcidin treatment induced a rapid inhibition (t½ = 8.2 ± [SEM] 0.5 min; r2 &gt; 0.99, P &lt; 0.001) of Fpn‐mediated 55Fe efflux that was not dependent on endocytosis, a conclusion based on the following four observations: (i) Inhibition of Fpn by hepcidin was only modestly temperature‐dependent (Q10 = 2.5 ± [SEM] 1.4 over the range 17–27 °C) whereas endocytosis is expected to be substantially more temperature‐dependent, Q10 in the order of 5–17 [Weigel PH and Oka JA (1981) J Biol Chem 256, 2615–2617]. (ii) Inhibition of Fpn by hepcidin proceeded even in the presence of blockers of clathrin‐mediated (Dynasore) and clathrin‐independent (filipin, genistein) endocytosis. (iii) Hepcidin treatment inhibited 55Fe efflux in oocytes expressing the K8R mutant, in which 8 Lys residues required for ubiquination and endocytosis of Fpn were mutated to Arg, to the same degree as in wildtype Fpn. (iv) Confocal LSM imaging of wildtype Fpn‐GFP and K8R‐Fpn‐GFP expression in oocytes revealed no detectable endocytosis of either protein after 30‐min treatment with hepcidin. Our data reveal that hepcidin can directly block Fpn activity independent of Fpn internalization. We speculate that the Xenopus oocyte lacks the specific signal by which hepcidin triggers Fpn internalization. The Xenopus oocyte is therefore an ideal model system in which to study the mechanism by which hepcidin directly blocks Fpn activity.Support or Funding InformationNIH–NIDDK grant R01 DK107309This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cystathionine β‐synthase is required for body iron homeostasis

Our findings point to a critical role of CBS in iron homeostasis of the body, and the liver in particular; it is likely that a hemochromatosis-like phenotype in patients can be induced by aberration not only in the expression of key molecules in the hepcidin pathway but also of those related to CBS. (Hepatology 2018;67:21-35).

Epigallocatechin Gallate Has a Neurorescue Effect in a Mouse Model of Parkinson Disease

Background: Parkinson disease (PD) is a neurodegenerative disorder that has been associated with many factors, including oxidative stress, inflammation, and iron accumulation. The antioxidant, anti-inflammatory, and iron-chelating properties of epigallocatechin gallate (EGCG), a major polyphenol in green tea, may offer protection against PD.Objective: We sought to determine the neurorescue effects of EGCG and the role of iron in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD.Methods: We evaluated the neurorescue effect of EGCG (25 mg/kg, 7 d, oral administration) against MPTP-induced (20 mg/kg, 3 d, intraperitoneal injection) neurodegeneration in C57 male black mice. Thirty mice weighing ∼25 g were divided into 3 groups: control, MPTP, and MPTP + EGCG. The neurorescue effect of EGCG was assessed with the use of motor behavior tests, neurotransmitter analysis, oxidative stress indicators, and iron-related protein expression.Results: Compared with the control group, MPTP treatment shortened the mice's latency to fall from the rotarod by 16% (P < 0.05), decreased the striatal dopamine concentration by 58% (P < 0.001) and dihydroxyphenylacetic acid by 35% (P < 0.05), and increased serum protein carbonyls by 71% (P = 0.07). However, EGCG rescued MPTP-induced neurotoxicity by increasing the rotational latency by 17% (P < 0.05) to a value similar to the control group. Striatal dopamine concentrations were 40% higher in the MPTP + EGCG group than in the MPTP group (P < 0.05), but the values were significantly lower than in the control group. Compared with the MPTP and control groups, mice in the MPTP + EGCG group had higher substantia nigra ferroportin expression (44% and 35%, respectively) (P < 0.05) but not hepcidin and divalent metal transporter 1 expression.Conclusion: Overall, our study demonstrated that EGCG regulated the iron-export protein ferroportin in substantia nigra, reduced oxidative stress, and exerted a neurorescue effect against MPTP-induced functional and neurochemical deficits in mice.

Iron Enhances Hepatic Fibrogenesis and Activates Transforming Growth Factor-β Signaling in Murine Hepatic Stellate Cells

BackgroundAlthough excess iron induces oxidative stress in the liver, it is unclear whether it directly activates the hepatic stellate cells (HSC). Materials and MethodsWe evaluated the effects of excess iron on fibrogenesis and transforming growth factor beta (TGF-β) signaling in murine HSC. Cells were treated with holotransferrin (0.005-5g/L) for 24 hours, with or without the iron chelator deferoxamine (10µM). Gene expressions (α-SMA, Col1-α1, Serpine-1, TGF-β, Hif1-α, Tfrc and Slc40a1) were analyzed by quantitative real time-polymerase chain reaction, whereas TfR1, ferroportin, ferritin, vimentin, collagen, TGF-β RII and phospho-Smad2 proteins were evaluated by immunofluorescence, Western blot and enzyme-linked immunosorbent assay. ResultsHSC expressed the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-export protein ferroportin. Holotransferrin upregulated TfR1 expression by 1.8-fold (P < 0.03) and ferritin accumulation (iron storage) by 2-fold (P < 0.01), and activated HSC with 2-fold elevations (P < 0.03) in α-SMA messenger RNA and collagen secretion, and a 1.6-fold increase (P < 0.01) in vimentin protein. Moreover, holotransferrin activated the TGF-β pathway with TGF-β messenger RNA elevated 1.6-fold (P = 0.05), and protein levels of TGF-β RII and phospho-Smad2 increased by 1.8-fold (P < 0.01) and 1.6-fold (P < 0.01), respectively. In contrast, iron chelation decreased ferritin levels by 30% (P < 0.03), inhibited collagen secretion by 60% (P < 0.01), repressed fibrogenic genes α-SMA (0.2-fold; P < 0.05) and TGF-β (0.4-fold; P < 0.01) and reduced levels of TGF-β RII and phospho-Smad2 proteins. ConclusionsHSC express iron-transport proteins. Holotransferrin (iron) activates HSC fibrogenesis and the TGF-β pathway, whereas iron depletion by chelation reverses this, suggesting that this could be a useful adjunct therapy for patients with fibrosis. Further studies in primary human HSC and animal models are necessary to confirm this.

On both sides of the ocean.

On both sides of the ocean.

Cardiac ferroportin regulates cellular iron homeostasis and is important for cardiac function

Iron is essential to the cell. Both iron deficiency and overload impinge negatively on cardiac health. Thus, effective iron homeostasis is important for cardiac function. Ferroportin (FPN), the only known mammalian iron-exporting protein, plays an essential role in iron homeostasis at the systemic level. It increases systemic iron availability by releasing iron from the cells of the duodenum, spleen, and liver, the sites of iron absorption, recycling, and storage respectively. However, FPN is also found in tissues with no known role in systemic iron handling, such as the heart, where its function remains unknown. To explore this function, we generated mice with a cardiomyocyte-specific deletion of Fpn. We show that these animals have severely impaired cardiac function, with a median survival of 22 wk, despite otherwise unaltered systemic iron status. We then compared their phenotype with that of ubiquitous hepcidin knockouts, a recognized model of the iron-loading disease hemochromatosis. The phenotype of the hepcidin knockouts was far milder, with normal survival up to 12 mo, despite far greater iron loading in the hearts. Histological examination demonstrated that, although cardiac iron accumulates within the cardiomyocytes of Fpn knockouts, it accumulates predominantly in other cell types in the hepcidin knockouts. We conclude, first, that cardiomyocyte FPN is essential for intracellular iron homeostasis and, second, that the site of deposition of iron within the heart determines the severity with which it affects cardiac function. Both findings have significant implications for the assessment and treatment of cardiac complications of iron dysregulation.