Ferroportin Down-regulation Research Articles

JWH133 attenuates behavior deficits and iron accumulation in 6-OHDA-induced Parkinson's disease model rats.

Growing evidence indicates that activation of cannabinoid type 2 (CB2) receptors protects dopamine neurons in the pathogenesis of Parkinson's disease (PD). However, the mechanisms underlying neuroprotection mediated by CB2 receptors are still elusive. In this study, we investigated the effects of CB2 receptor activation on 6-hydroxydopamine (6-OHDA)-induced dopamine neuron degeneration and iron accumulation in the substantia nigra (SN) of rats. We found that treatment with JWH133, a selective CB2 receptor agonist, significantly improved the apomorphine (APO)-induced rotational behavior in 6-OHDA-treated rats. The decreased numbers of tyrosine hydroxylase (TH)-positive neurons and reduced TH protein expression in the lesioned SN of rats were effectively restored by JWH133. Moreover, we found that JWH133 inhibited the increase of iron-staining cells in the lesioned SN of rats. To explore the protective mechanisms of activation of CB2 receptors on dopamine neurons, we further observed the effect of JWH133 on 1-methyl-4-phenylpyridinium (MPP+)-treated primary cultured ventral mesencephalon (VM) neurons from rats. We found that JWH133 significantly inhibited the increase of intracellular reactive oxygen species (ROS), the activation of Caspase-3, the decrease of mitochondrial transmembrane potential (ΔΨm), and the decrease of Bcl-2/Bax protein expression caused by MPP+ treatment. JWH133 also inhibited the MPP+-induced upregulation of divalent metal transporter-1 (DMT1) and downregulation of ferroportin 1 (FPN1). Furthermore, JWH133 also suppressed the MPP+-accelerated iron influx in the VM neurons. These results suggest that activation of CB2 receptor suppresses MPP+-induced cellular iron accumulation and prevents neurodegeneration.NEW & NOTEWORTHY Expression of cannabinoid type 2 receptors (CB2Rs) was discovered on dopamine neurons in recent years. The role of CB2R expressed on dopamine neurons in the pathogenesis of Parkinson's disease (PD) has not been fully elucidated. The content of iron accumulation in the brain is closely related to the progress of PD. We verified the inhibitory effect of CB2R on iron deposition in dopamine neurons through experiments, which provided a new idea for the treatment of PD.

Defective ferritinophagy and imbalanced iron metabolism in PBDE-47-triggered neuronal ferroptosis and salvage by Canolol

The brominated flame retardant 2,2′,4,4′-tetrabromodiphenyl ether (PBDE-47) is a ubiquitous environmental pollutant that causes neurotoxicity. However, incomplete understanding of the underlying mechanisms has hampered the development of effective intervention strategies. Oxidative stress and related cell death are the modes of action for PBDE-47 neurotoxicity, which are also the characteristics of ferroptosis. Nonetheless, the role of ferroptosis in PBDE-47-induced neurotoxicity remains unclear. In the present study, we found that PBDE-47 triggered ferroptosis in neuron-like PC12 cells, as evidenced by intracellular iron overload, lipid peroxidation, and mitochondrial damage. This was confirmed by ferroptosis inhibitors including the lipid reactive oxygen species scavenger ferrostatin-1 and iron chelator deferoxamine mesylate. Mechanistically, PBDE-47 impaired ferritinophagy by disrupting nuclear receptor coactivator 4-mediated lysosomal degradation of the iron storage protein ferritin. Moreover, PBDE-47 disturbed iron metabolism by increasing cellular iron import via upregulation of transferrin receptor 1 and decreasing cellular iron export via downregulation of ferroportin 1 (FPN1). Intriguingly, rescuing lysosomal function by overexpressing cathepsin B (CatB) mitigated PBDE-47-induced ferroptosis by partially restoring dysfunctional ferritinophagy and enhancing iron excretion via the upregulation of FPN1. However, FPN1 knockdown reversed the beneficial effects of CatB overexpression on the PBDE-47-induced iron overload. Finally, network pharmacology integrated with experimental validation revealed that Canolol, the main phenolic compound in canola oil, protected against PBDE-47-evoked iron overload, resulting in ferroptosis by restoring defective ferritinophagy and improving abnormal iron metabolism via lowering iron uptake and facilitating iron excretion. Overall, these data suggest that ferroptosis is a novel mechanism of PBDE-47-induced neuronal death and that manipulation of ferritinophagy and iron metabolism via Canolol represents a promising therapeutic strategy.

Quercetin Inhibits Hephaestin Expression and Iron Transport in Intestinal Cells: Possible Role of PI3K Pathway

Previous studies demonstrated that quercetin, a polyphenolic compound, inhibits the transport of iron by down-regulation of ferroportin (FPN1), an iron export protein. We have previously demonstrated that activation of the PI3K signaling pathway by zinc stimulates the intestinal iron uptake and transport by stimulating the expression of iron regulatory protein 2 (IRP2) dependent divalent metal iron transporter 1 (DMT1, apical iron transporter) expression and caudal-related homeobox transcription factor 2 (CDX2) dependent hephaestin (HEPH, basolateral ferroxidase required for iron oxidation) expression, respectively. Since polyphenols are antagonists of the PI3K pathway, we hypothesized that quercetin might inhibit basolateral iron transport via the down-regulation of hephaestin (HEPH). Here in we investigated the effect of quercetin on iron uptake, transport, and expression of iron transporters in intestinal cells. In differentiated Caco-2 cells grown on permeable supports, quercetin inhibited the basolateral iron transport while increasing the iron uptake, possibly due to higher cellular retention. Further, quercetin down-regulated the protein and mRNA expression of HEPH and FPN1 but not that of IRP2 or DMT1. In addition, quercetin also abrogated the zinc-induced Akt, CDX2 phosphorylation, and HEPH expression. Together these results suggest that inhibition of iron transport by quercetin is mediated via the down-regulation of CDX2-dependent HEPH expression via inhibition of the PI3K pathway.

Vulnerability of Triple-Negative Breast Cancer to Saponin Formosanin C-Induced Ferroptosis.

Targeting ferritin via autophagy (ferritinophagy) to induce ferroptosis, an iron- and reactive oxygen species (ROS)-dependent cell death, provides novel strategies for cancer therapy. Using a ferroptosis-specific inhibitor and iron chelator, the vulnerability of triple-negative breast cancer (TNBC) MDA-MB-231 cells to ferroptosis was identified and compared to that of luminal A MCF-7 cells. Saponin formosanin C (FC) was revealed as a potent ferroptosis inducer characterized by superior induction in cytosolic and lipid ROS formation as well as GPX4 depletion in MDA-MB-231 cells. The FC-induced ferroptosis was paralleled by downregulation of ferroportin and xCT expressions. Immunoprecipitation and electron microscopy demonstrated the involvement of ferritinophagy in FC-treated MDA-MB-231 cells. The association of FC with ferroptosis was strengthened by the results that observed an enriched pathway with differentially expressed genes from FC-treated cells. FC sensitized cisplatin-induced ferroptosis in MDA-MB-231 cells. Through integrated analysis of differentially expressed genes and pathways using the METABRIC patients’ database, we confirmed that autophagy and ferroptosis were discrepant between TNBC and luminal A and that TNBC was hypersensitive to ferroptosis. Our data suggest a therapeutic strategy by ferroptosis against TNBC, an aggressive subtype with a poor prognosis.

Interplay of Ferritin Accumulation and Ferroportin Loss in Ageing Brain: Implication for Protein Aggregation in Down Syndrome Dementia, Alzheimer's, and Parkinson's Diseases.

Iron accumulates in the ageing brain and in brains with neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and Down syndrome (DS) dementia. However, the mechanisms of iron deposition and regional selectivity in the brain are ill-understood. The identification of several proteins that are involved in iron homeostasis, transport, and regulation suggests avenues to explore their function in neurodegenerative diseases. To uncover the molecular mechanisms underlying this association, we investigated the distribution and expression of these key iron proteins in brain tissues of patients with AD, DS, PD, and compared them with age-matched controls. Ferritin is an iron storage protein that is deposited in senile plaques in the AD and DS brain, as well as in neuromelanin-containing neurons in the Lewy bodies in PD brain. The transporter of ferrous iron, Divalent metal protein 1 (DMT1), was observed solely in the capillary endothelium and in astrocytes close to the ventricles with unchanged expression in PD. The principal iron transporter, ferroportin, is strikingly reduced in the AD brain compared to age-matched controls. Extensive blood vessel damage in the basal ganglia and deposition of punctate ferritin heavy chain (FTH) and hepcidin were found in the caudate and putamen within striosomes/matrix in both PD and DS brains. We suggest that downregulation of ferroportin could be a key reason for iron mismanagement through disruption of cellular entry and exit pathways of the endothelium. Membrane damage and subsequent impairment of ferroportin and hepcidin causes oxidative stress that contributes to neurodegeneration seen in DS, AD, and in PD subjects. We further propose that a lack of ferritin contributes to neurodegeneration as a consequence of failure to export toxic metals from the cortex in AD/DS and from the substantia nigra and caudate/putamen in PD brain.

Melatonin Alleviates Acute Sleep Deprivation-Induced Memory Loss in Mice by Suppressing Hippocampal Ferroptosis.

Objectives: Memory decline caused by insufficient sleep is a critical public health issues and currently lacks effective treatments. This study objective was to explore alleviative effect of melatonin on sleep deprivation (SD)-induced deficiencies in learning and memory. Materials and Methods: A continuous 72 h SD mouse model, with or without melatonin or Fer-1 supplementation were established. The changes of cognitive function, iron homeostasis, lipid peroxidation and intracellular signal pathways in mice were detected by Morris water maze, antioxidant assay, immunohistochemistry, western blot, RT-PCR and Prussian blue staining. In vitro, we treated HT-22 cells with ferroptosis inducer (Erastin) to further explore the specific mechanism of melatonin in ferroptosis. Results: Mice subjected to SD had significantly elevated latency and path length to reach hidden platform, as well as a decrease in number of entries and time spent in the target zone when the hidden platform was removed (p < 0.05). Nevertheless, supplementation with ferroptosis inhibitor (Fer-1) mitigated the memory impairment associated with SD. Further evaluation revealed an up-regulation of intracellular iron accumulation, transferrin receptor 1 and divalent metal transporter 1 expression and ROS and MDA production, and a down-regulation of ferroportin and antioxidant enzyme (GPX4 and SOD) expression in SD mice. SD decreased expression of MT2 receptor rather than of MT1, and inhibited ERK/Nrf2 signaling activation in the hippocampus (p < 0.05). In contrast, the aforementioned SD-inductions were reversed by supplementation using 20 and 40 mg/kg melatonin in SD mice. In vitro, melatonin pretreatment reversed Erastin-induced ferroptosis, abnormalities in iron transporter protein and antioxidant enzyme expression and suppression of ERK/Nrf2 signaling in HT-22 cells, however this protective effect of melatonin was blocked by MT2-, ERK- and Nrf2-specific antagonists (p < 0.05). Conclusion: Our finding suggested SD may induce ferroptosis, in turn leading to cognitive deficits. Melatonin alleviated memory loss and hippocampal ferroptosis caused by acute SD through binding to the MT2 receptor to activate ERK/Nrf2 signaling.

Gene Expression Changes in Epinephelus marginatus (Teleostei, Serranidae) Liver as Molecular Biomarker of Iron Ore Contamination.

The aim of this study was to determine selected molecular biomarkers of iron ore contamination in Epinephelus marginatus. Molecular biomarkers were tested by checking the relative expression of genes involved in oxidative stress, trace element regulation, and cellular damage, by RT-qPCR. Iron ore exposure caused the downregulation of ferroportin (FP) gene expression and a significant upregulation in superoxide dismutase (SOD) and cytochrome P450 1A (CYP1A) genes. Iron ore affects gene expression in E. marginatus liver, indicating that it can potentially induce toxic effects in fish. Moreover, this altered gene expression pattern may be applied in monitoring iron ore contamination in marine environments.

Apolipoprotein E deficiency induces a progressive increase in tissue iron contents with age in mice

Association of both iron/hepcidin and apolipoprotein E (ApoE) with development of Alzheimer disease (AD) and atherosclerosis led us to hypothesize that ApoE might be required for body iron homeostasis. Here, we demonstrated that ApoE knock-out (KO) induced a progressive accumulation of iron with age in the liver and spleen of mice. Subsequent investigations showed that the increased iron in the liver and spleen was due to phosphorylated extracellular regulated protein kinases (pERK) mediated up-regulation of transferrin receptor 1 (TfR1), and nuclear factor erythroid 2-related factor-2 (Nrf2)-dependent down-regulation of ferroportin 1. Furthermore, replenishment of ApoE could partially reverse the iron-related phenotype in ApoE KO mice. The findings imply that ApoE may be essential for body iron homeostasis and also suggest that clinical late-onset diseases with unexplained iron abnormality may partly be related to deficiency or reduced expression of ApoE.

Ferroportin downregulation promotes cell proliferation by modulating the Nrf2\u2013miR-17-5p axis in multiple myeloma

Recent findings demonstrate that aberrant downregulation of the iron-exporter protein, ferroportin (FPN1), is associated with poor prognosis and osteoclast differentiation in multiple myeloma (MM). Here, we show that FPN1 was downregulated in MM and that clustered regularly interspaced short palindromic repeat (CRISPR)-mediated FPN1 knockout promoted MM cell growth and survival. Using a microRNA target-scan algorithm, we identified miR-17-5p as an FPN1 regulator that promoted cell proliferation and cell cycle progression, and inhibited apoptosis—both in vitro and in vivo. miR-17-5p inhibited retarded tumor growth in a MM xenograft model. Moreover, restoring FPN1 expression at least partially abrogated the biological effects of miR-17-5p in MM cells. The cellular iron concentration regulated the expression of the iron-regulatory protein (IRP) via the 5′-untranslated region of IRP messenger RNA and modulated the post-transcriptional stability of FPN1. Bioinformatics analysis with subsequent chromatin immunoprecipitation-polymerase chain reaction and luciferase activity experiments revealed that the transcription factor Nrf2 drove FPN1 transcription through promoter binding and suppressed miR-17-5p (which also increased FPN1 expression). Nrf2-mediated FPN1 downregulation promoted intracellular iron accumulation and reactive oxygen species. Our study links FPN1 transcriptional and post-transcriptional regulation with MM cell growth and survival, and validates the prognostic value of FPN1 and its utility as a novel therapeutic target in MM.

Role of the hepcidin-ferroportin axis in pathogen-mediated intracellular iron sequestration in human phagocytic cells

AbstractUpon infection, pathogen and host compete for the same iron pool, because this trace metal is a crucial micronutrient for all living cells. Iron dysregulation in the host is strongly associated with poor outcomes in several infectious diseases, including tuberculosis, AIDS, and malaria, and inefficient iron scavenging by pathogens severely affects their virulence. Hepcidin is the master regulator of iron homeostasis in vertebrates, responsible for diminishing iron export from macrophages during iron overload or infection. Hepcidin regulation in hepatocytes is well characterized and mostly dependent on interleukin-6 signaling during inflammation, although in myeloid cells, hepcidin induction and the mechanisms leading to intracellular iron regulation remain elusive. Here we show that activation of different Toll-like receptors (TLRs) by their respective ligands leads to increased iron sequestration in macrophages. By measuring the transcriptional levels of iron-related proteins (eg, hepcidin, ferroportin, and ferritin), we observed that TLR signaling can induce intracellular iron sequestration in macrophages through 2 independent but redundant mechanisms. Interestingly, TLR2 ligands or infection with Listeria monocytogenes lead to direct ferroportin transcriptional downregulation, whereas TLR4 ligands, such as lipopolysaccharide, induce hepcidin expression. Infection with Mycobacterium bovis Bacillus Calmette-Guerin promotes intracellular iron sequestration through both hepcidin upregulation and ferroportin downregulation. This is the first study in which TLR1-9–mediated iron homeostasis in human macrophages was evaluated, and the outcome of this study elucidates the mechanism of iron dysregulation in macrophages during infection.

Cell iron status influences macrophage polarization.

Macrophages play crucial roles in innate immune response and in the priming of adaptive immunity, and are characterized by their phenotypic heterogeneity and plasticity. Reprogramming intracellular metabolism in response to microenvironmental signals is required for M1/M2 macrophage polarization and function. Here we assessed the influence of iron on the polarization of the immune response in vivo and in vitro. Iron-enriched diet increased M2 marker Arg1 and Ym1 expression in liver and peritoneal macrophages, while iron deficiency decreased Arg1 expression. Under LPS-induced inflammatory conditions, low iron diet exacerbated the proinflammatory response, while the IL-12/IL-10 balance decreased with iron-rich diet, thus polarizing toward type 2 response. Indeed, in vitro macrophage iron loading reduced the basal percentage of cells expressing M1 co-stimulatory CD86 and MHC-II molecules. Further, iron loading of macrophages prevented the pro-inflammatory response induced by LPS through reduction of NF-κB p65 nuclear translocation with decreased iNOS, IL-1β, IL-6, IL-12 and TNFα expression. The increase of intracellular iron also reduced LPS-induced hepcidin gene expression and abolished ferroportin down-regulation in macrophages, in line with macrophage polarization. Thus, iron modulates the inflammatory response outcome, as elevated iron levels increased M2 phenotype and negatively regulated M1 proinflammatory LPS-induced response.

Whole-body iron transport and metabolism: Mechanistic, multi-scale model to improve treatment of anemia in chronic kidney disease.

Iron plays vital roles in the human body including enzymatic processes, oxygen-transport via hemoglobin and immune response. Iron metabolism is characterized by ~95% recycling and minor replenishment through diet. Anemia of chronic kidney disease (CKD) is characterized by a lack of synthesis of erythropoietin leading to reduced red blood cell (RBC) formation and aberrant iron recycling. Treatment of CKD anemia aims to normalize RBC count and serum hemoglobin. Clinically, the various fluxes of iron transport and accumulation are not measured so that changes during disease (e.g., CKD) and treatment are unknown. Unwanted iron accumulation in patients is known to lead to adverse effects. Current whole-body models lack the mechanistic details of iron transport related to RBC maturation, transferrin (Tf and TfR) dynamics and assume passive iron efflux from macrophages. Hence, they are not predictive of whole-body iron dynamics and cannot be used to design individualized patient treatment. For prediction, we developed a mechanistic, multi-scale computational model of whole-body iron metabolism incorporating four compartments containing major pools of iron and RBC generation process. The model accounts for multiple forms of iron in vivo, mechanisms involved in iron uptake and release and their regulation. Furthermore, the model is interfaced with drug pharmacokinetics to allow simulation of treatment dynamics. We calibrated our model with experimental and clinical data from peer-reviewed literature to reliably simulate CKD anemia and the effects of current treatment involving combination of epoietin-alpha and iron dextran. This in silico whole-body model of iron metabolism predicts that a year of treatment can potentially lead to 90% downregulation of ferroportin (FPN) levels, 15-fold increase in iron stores with only a 20% increase in iron flux from the reticulo-endothelial system (RES). Model simulations quantified unmeasured iron fluxes, previously unknown effects of treatment on FPN-level and iron stores in the RES. This mechanistic whole-body model can be the basis for future studies that incorporate iron metabolism together with related clinical experiments. Such an approach could pave the way for development of effective personalized treatment of CKD anemia.

Identification of Antibody and Small Molecule Antagonists of Ferroportin-Hepcidin Interaction.

The iron exporter ferroportin and its ligand, the hormone hepcidin, control fluxes of stored and recycled iron for use in a variety of essential biochemical processes. Inflammatory disorders and malignancies are often associated with high hepcidin levels, leading to ferroportin down-regulation, iron sequestration in tissue macrophages and subsequent anemia. The objective of this research was to develop reagents to characterize the expression of ferroportin, the interaction between ferroportin and hepcidin, as well as to identify novel ferroportin antagonists capable of maintaining iron export in the presence of hepcidin. Development of investigative tools that enabled cell-based screening assays is described in detail, including specific and sensitive monoclonal antibodies that detect endogenously-expressed human and mouse ferroportin and fluorescently-labeled chemically-synthesized human hepcidin. Large and small molecule antagonists inhibiting hepcidin-mediated ferroportin internalization were identified, and unique insights into the requirements for interaction between these two key iron homeostasis molecules are provided.

Hamp1 but not Hamp2 regulates ferroportin in fish with two functionally distinct hepcidin types

Hepcidin is a small cysteine rich peptide that regulates the sole known cellular iron exporter, ferroportin, effectively controlling iron metabolism. Contrary to humans, where a single hepcidin exists, many fish have two functionally distinct hepcidin types, despite having a single ferroportin gene. This raises the question of whether ferroportin is similarly regulated by the iron regulator Hamp1 and the antimicrobial Hamp2. In sea bass (Dicentrarchus labrax), iron overload prompted a downregulation of ferroportin, associated with an upregulation of hamp1, whereas an opposite response was observed during anemia, with no changes in hamp2 in either situation. During infection, ferroportin expression decreased, indicating iron withholding to avoid microbial proliferation. In vivo administration of Hamp1 but not Hamp2 synthetic peptides caused significant reduction in ferroportin expression, indicating that in teleost fish with two hepcidin types, ferroportin activity is mediated through the iron-regulator Hamp1, and not through the dedicated antimicrobial Hamp2. Additionally, in vitro treatment of mouse macrophages with fish Hamp1 but not Hamp2 caused a decrease in ferroportin levels. These results raise questions on the evolution of hepcidin and ferroportin functional partnership and open new possibilities for the pharmaceutical use of selected fish Hamp2 hepcidins during infections, with no impact on iron homeostasis.

Iron- and Hepcidin-Independent Downregulation of the Iron Exporter Ferroportin in Macrophages during Salmonella Infection.

Retention of iron in tissue macrophages via upregulation of hepcidin (HAMP) and downregulation of the iron exporter ferroportin (FPN) is thought to participate in the establishment of anemia of inflammation after infection. However, an upregulation of FPN has been proposed to limit macrophages iron access to intracellular pathogens. Therefore, we studied the iron homeostasis and in particular the regulation of FPN after infection with Salmonella enterica serovar Typhimurium in mice presenting tissue macrophages with high iron (AcB61), basal iron (A/J and wild-type mice), or low iron (Hamp knock out, Hamp−/−) levels. The presence of iron in AcB61 macrophages due to extravascular hemolysis and strong erythrophagocytosis activity favored the proliferation of Salmonella in the spleen and liver with a concomitant decrease of FPN protein expression. Despite systemic iron overload, no or slight increase in Salmonella burden was observed in Hamp−/− mice compared to controls. Importantly, FPN expression at both mRNA and protein levels was strongly decreased during Salmonella infection in Hamp−/− mice. The repression of Fpn mRNA was also observed in Salmonella-infected cultured macrophages. In addition, the downregulation of FPN was associated with decreased iron stores in both the liver and spleen in infected mice. Our findings show that during Salmonella infection, FPN is repressed through an iron and hepcidin-independent mechanism. Such regulation likely provides the cellular iron indispensable for the growth of Salmonella inside the macrophages.

A novel inflammatory pathway mediating rapid hepcidin-independent hypoferremia

AbstractRegulation of iron metabolism and innate immunity are tightly interlinked. The acute phase response to infection and inflammation induces alterations in iron homeostasis that reduce iron supplies to pathogens. The iron hormone hepcidin is activated by such stimuli causing degradation of the iron exporter ferroportin and reduced iron release from macrophages, suggesting that hepcidin is the crucial effector of inflammatory hypoferremia. Here, we report the discovery of an acute inflammatory condition that is mediated by Toll-like receptors 2 and 6 (TLR2 and TLR6) and which induces hypoferremia in mice injected with TLR ligands. Stimulation of TLR2/TLR6 triggers profound decreases in ferroportin messenger RNA and protein expression in bone marrow–derived macrophages, liver, and spleen of mice without changing hepcidin expression. Furthermore, C326S ferroportin mutant mice with a disrupted hepcidin/ferroportin regulatory circuitry respond to injection of the TLR2/6 ligands FSL1 or PAM3CSK4 by ferroportin downregulation and a reduction of serum iron levels. Our findings challenge the prevailing role of hepcidin in hypoferremia and suggest that rapid hepcidin-independent ferroportin downregulation in the major sites of iron recycling may represent a first-line response to restrict iron access for numerous pathogens.

SAPP modulates iron efflux from brain microvascular endothelial cells by stabilizing the ferrous iron exporter ferroportin.

A sequence within the E2 domain of soluble amyloid precursor protein (sAPP) stimulates iron efflux. This activity has been attributed to a ferroxidase activity suggested for this motif. We demonstrate that the stimulation of efflux supported by this peptide and by sAPPα is due to their stabilization of the ferrous iron exporter, ferroportin (Fpn), in the plasma membrane of human brain microvascular endothelial cells (hBMVEC). The peptide does not bind ferric iron explaining why it does not and thermodynamically cannot promote ferrous iron autoxidation. This peptide specifically pulls Fpn down from the plasma membrane of hBMVEC; based on these results, FTP, for ferroportin-targeting peptide, correctly identifies the function of this peptide. The data suggest that in stabilizing Fpn via the targeting due to the FTP sequence, sAPP will increase the flux of iron into the cerebral interstitium. This inference correlates with the observation of significant iron deposition in the amyloid plaques characteristic of Alzheimer's disease.

Impact of D181V and A69T on the function of ferroportin as an iron export pump and hepcidin receptor

Mutations in the only known mammalian iron exporter ferroportin cause a rare iron overload disorder termed ferroportin disease. Two distinct clinical phenotypes are caused by different disease mechanisms: mutations in ferroportin either cause loss of iron export function or gain of function due to resistance to hepcidin, the peptide hormone that normally downregulates ferroportin. The aim of the present study was to examine the disease mechanisms of the thus far unclassified A69T and D181V ferroportin mutations. We overexpressed wild-type and mutant ferroportin fused to green fluorescent protein in human embryonic kidney cells and used a 59Fe-assay, intracellular ferritin concentrations, confocal microscopy and flow cytometry to study iron export function, subcellular localization and the responsiveness to hepcidin. While the A69T ferroportin mutation seems not to affect the iron export function it causes dose-dependent hepcidin resistance. We further found that D181V mutated ferroportin is iron export defective and hepcidin resistant, similar to the loss of function mutations A77D and C367X. This indicates that intact iron export might be necessary for hepcidin-induced downregulation of ferroportin. This hypothesis was investigated by studying the hepcidin response under modulation of iron availability. Incubation of wild-type ferroportin overexpressing cells with holo-transferrin increases the hepcidin effect whereas chelating extracellular ferrous iron causes hepcidin resistance. In this study we present data that postulates to classify the D181V ferroportin mutation as loss of function and the A69T mutation as dose-dependent hepcidin resistant and outline a possible causal link between iron export function and the hepcidin effect.

A-Type Proanthocyanidins Prevent Engraftment Of Primary Acute Myelogenous Leukemia Cells In Mice and Exhibit Potentially Novel Anti-Leukemia Mechanisms

Acute myelogenous leukemia (AML) is often a fatal disease where after strong induction therapy most patients relapse and die. AML originates and is maintained by leukemia stem cells (LSCs). Failure to eliminate LSCs by chemotherapy is likely to result in disease relapse. Therefore, it is a priority to identify new therapies that eliminate blasts while ablating LSCs and preventing a relapse.We have found that a unique class of compounds in cranberries (Vaccinium macrocarponAit.), known as A-type proanthocyanidins (A-PACs), were effective against several leukemia cell lines and primary AML samples in vitro. A-PACs consist of monomeric epicatechin units attached to one another by a carbon-carbon bond and a distinctive ether bond that differentiates these compounds from other proanthocyanidins found in nature. Moreover, A-PACs possess ortho-hydroxyl phenolic groups that have the potential to bind to iron and alter redox status.Preliminary work showed that pre-treatment with antioxidants or holo-transferrin (iron-saturated transferrin) partially protected AML cells from A-PAC induced cell death (p<0.01). A-PACs were also found to selectively ablate leukemia stem and progenitor cells, with minimal effects on normal hematopoetic stem cells. Furthermore, AML engraftment of cells treated ex vivo with 62.5 µg/ml A-PACs was decreased (90.6%, n=3, p<0.001), while normal CD34+ cells retained engraftment capability in immunodeficient mice. It was also found that a fraction of A-PACs of up to 7 degree of polymerization was more effective than individual A-PACs. This information prompted us to investigate the in vivo anti-leukemia effects of A-PACs in xenotransplanted mice with primary AML samples, and to further investigate the mechanisms associated with these compounds.Primary AML cells were injected in sub-lethally irradiated NOD/SCID mice. Four weeks after injections, when human leukemia cells have engrafted, intraperitoneal injections of cytarabine (AraC) at 60 mg/kg were given to the mice for 1 week everyday or A-PACs (100 mg/kg dose every 3 days for A-PACs) and vehicle control (1% DMSO in PBS every 3 days) were injected for 2.5 weeks. Mice were sacrificed and leukemia engraftment evaluated using anti-human CD45 and CD33. Moreover, primary cells treated with A-PACs were assessed for effects on iron metabolism, ROS, and survival pathways either by gene expression analysis, flow cytometry or mass spectrometry.Administration of A-PACs to NOD-SCID mice bearing AML tumors reduced tumor burden. Mice that were treated with the vehicle control had engraftment of AML primary cells equivalent to 16.1% (95% CI: -6.0, 38.37; n=4), whereas the mice treated with the A-PACs and AraC showed a level of engraftment of 4.9% (95% CI: 2, 8; n=5) and 5.8% (95% CI: -1.1, 12.7; n=5), respectively. No significant changes in hemoglobin or weight were found between the different treatment groups. Moreover, qPCR analysis of sensitive leukemia cell lines treated with A-PACs showed changes in gene expression of several iron metabolism genes in sensitive leukemia cell lines (up-regulation of ferritin and transferrin receptors 1 and down-regulation of ferroportin) and several ROS-relevant genes (down-regulation of nuclear factor erythroid-2-related factor 2 and glutamate-cysteine ligase regulatory subunit). Mass spectrometry also confirmed that A-PACs bind iron.The results indicate that A-PACs not only target primary AML cells in vitro but are also effective in vivo. Secondary transplants are also being performed to determine the effects on LSC activity. Some of the anti-leukemia mechanisms under investigation include effects related to iron metabolism, ROS or inhibition of survival pathways. Understanding the unique structure and biological effects of A-PACs may provide novel information about pathways involved in the survival of LSCs and provide crucial information in preparation for clinical trials and/or optimal combination drug therapies. Disclosures:Rivella:Novartis: Consultancy; Bayer: Consultancy; Isis: Consultancy, Research Funding; Merganser: Equity Ownership, Research Funding; Biomarin: Consultancy; Alexion: Consultancy; Imago: Consultancy.

Pro-inflammatory cytokine-mediated ferroportin down-regulation contributes to the nigral iron accumulation in lipopolysaccharide-induced Parkinsonian models

Pro-inflammatory cytokines induced by inflammation and iron accumulation in the substantia nigra (SN) have been implicated in the pathogenesis of Parkinson’s disease (PD). In the present study, we aimed to investigate the relationship between inflammation and iron accumulation in a lipopolysaccharide (LPS)-induced Parkinsonian rat model. The activation of glial cells and elevated levels of pro-inflammatory cytokines were observed in the SN of LPS models, accompanied by iron deposits in the same region. Moreover, ferroportin (Fpn), the only channel for iron export, was down-regulated. SH-SY5Y dopaminergic cells were pre-incubated with conditioned media enriched in pro-inflammatory cytokines, and abnormal iron deposits and a drop of Fpn were observed. The expression of heme oxygenase-1 (HO-1) was also upregulated in vivo and in vitro. These results suggested that pro-inflammatory cytokines might induce Fpn downregulation, which leads to iron accumulation and dopaminergic neurons’ degeneration in PD. HO-1 may also contribute to the iron accumulation in neurons, but its mechanism needs to be further investigated.