Intracellular Iron Availability Research Articles

Ferritinophagy Is a Druggable Vulnerability of Quiescent Leukemic Stem Cells

Acute myeloid leukemia (AML) poses significant challenges in terms of prognosis and treatment options. Leukemic stem cells (LSCs) contribute to therapy resistance, relapse and adverse outcomes. This study aimed to explore one of the major features of LSCs (e.g. quiescence) and its underlying molecular mechanisms, as well as identify potential targets for therapeutic intervention. Using in vivo cell labeling in patient-derived xenograft (PDX) assays, we identified a quiescent cell subpopulation with high LSC potential. Transcriptomics experiments conducted separately on quiescent and cycling cell subsets revealed gene signatures characteristic of quiescent (Q_AML_UP) and cycling cells (C_AML_UP). Using single-cell RNA sequencing, we identified clusters of quiescent leukemic cell that were highly enriched in gene signatures related to LSCs (Figure 1A). Through a machine learning approach, we identified a gene set highly specific of quiescent cells (named the QS35 gene signature) that emerged as a significant prognostic factor for poor outcomes in independent cohorts of AML patients (Figure 1B). Next, we aimed to identify new vulnerabilities of LSCs by investigating the quiescent subset of leukemic cells. Transcriptomics and proteomics approaches on quiescent vs cycling cells revealed enhanced autophagy within the quiescent subset. Additionally, the transferrin receptor TFRC and ferritin, two key regulators of iron metabolism, exhibited lower expression in the quiescent subpopulations. As intracellular iron availability is tightly regulated by ferritinophagy, a specific form of autophagy leading to the degradation of ferritin, we investigated the coordinated regulation of autophagy and iron metabolism in the quiescent LSC subset. Intriguingly, the intracellular iron pool was lower in quiescent LSCs, particularly in the CD34+CD38- compartment compared to more mature leukemic cells. Moreover, autophagy inhibition selectively induced cytotoxicity in the CD34+CD38- compartment and reduced the frequency of long-term culture-initiating cells (LTC-IC), and these effects were rescued by exogenous iron supply, underscoring the critical role of autophagy in iron bioavailability within the quiescent LSC subpopulation. To target ferritinophagy in this compartment, we depleted NCOA4, the main carrier in ferritinophagy responsible for delivering ferritin-bound iron to the autophagosome, using shRNAs in primary AML samples. Interestingly, NCOA4 was found more expressed in quiescent compared to cycling AML cells. In ex vivo assays, NCOA4 downregulation significantly reduced the frequency of LTC-IC and inhibited the clonogenic capacities of leukemic cells. Furthermore, NCOA4 invalidation abrogated leukemia initiation and self-renewal in serial transplantation experiments in vivo, particularly within the quiescent LSC population (Figure 2A). Additionally, treatment with a novel small molecule inhibitor named compound 9a that disrupts the NCOA4-ferritin interaction, depleted intracellular iron in primary AML cells, reduced their clonogenic potential and LTC-IC frequency ex vivo, and induced cell death specifically within the quiescent CD34+CD38- subset. Importantly, the viability of LSCs treated with compound 9a was rescued by the addition of iron, demonstrating that the cytotoxic effects of this compound were dependent on iron depletion. Notably, compound 9a exhibited minimal toxicity to normal hematopoietic cells ex vivo. In PDX models, in vivo short-term treatment with compound 9a decreased tumor burden and induced cell death within the CD34+CD38- LSC population (Figure 2B). Mechanistically, genetic of chemical inhibition of NCOA4 led to the accumulation of dysfunctional mitochondria, and to the generation of reactive oxygen species especially within the LSCs population. Furthermore, inhibition of NCOA4 by compound 9a decreased mitophagy, strongly suggesting that NCOA4 coordinates ferritinophagy and mitophagy pathways to increase intracellular iron, thereby specifically promoting the survival of LSCs. These findings highlight the critical vulnerability of quiescent LSCs to ferritinophagy. Thus, NCOA4 inhibition represents an innovative therapeutic approach for AML patients.

The effects of intracellular iron availability on the outcome of Toxoplasma gondii infection in mice.

Toxoplasma gondii (T. gondii) is a parasite that obtains the iron it needs for its own metabolism from the host-cell iron pool. In this work, we aimed to investigate if iron supplementation or deficiency affected the course of T. gondii infection. Eighty mice were divided into four groups, each with 20 animals: Group (I): Uninfected control group. Group (II): Infected control group: injected with Phosphate buffered saline. Group (III): Infected group: received iron sucrose treatment. Group (IV): Infected group: treated with deferoxamine. Quantitative PCR studies were performed on days 3 and 8 post-infection to detect the expression of iron metabolism genes (hamp and ferroprotin) and immune-histochemical analysis to study the percentage of TNF-α and TGF-β tissue expression. Iron supplementation induced progressions of infection evident by increased tissue expression of pro-inflammatory cytokine TNF-α and downregulation of TGF-β which is mostly linked to suppression of the inflammatory process caused by T. gondii. Increased expression of TGF-β and decreased expression of TNF-α was noticed when iron deprivation occurred. On day 3, we noticed increased expression in the hamp gene with iron supplementation while it decreases when the iron supply is low. On the contrary, iron deficiency increased ferroprotin gene expression whereas supplementing decreased it. On day 8, the level of expression of these genes returned to normal levels. These observations document the potential role of iron in controlling toxoplasmosis infection and indicate that the transcription of hamp and ferroprotin in T. gondii-infected cells appears to be regulated by a sophisticated indirect mechanism.

PTEN-induced kinase PINK1 supports colorectal cancer growth by regulating the labile iron pool

Mitophagy is a cargo-specific autophagic process that recycles damaged mitochondria to promote mitochondrial turnover. PTEN-induced putative kinase 1 (PINK1) mediates the canonical mitophagic pathway. However, the role of PINK1 in diseases where mitophagy has been purported to play a role, such as colorectal cancer, is unclear. Our results here demonstrate that higher PINK1 expression is positively correlated with decreased colon cancer survival, and mitophagy is required for colon cancer growth. We show that doxycycline-inducible knockdown (KD) of PINK1 in a panel of colon cancer cell lines inhibited proliferation, whereas disruption of other mitophagy receptors did not impact cell growth. We observed that PINK KD led to a decrease in mitochondrial respiration, membrane hyperpolarization, accumulation of mitochondrial DNA, and depletion of antioxidant glutathione. In addition, mitochondria are important hubs for the utilization of iron and synthesizing iron-dependent cofactors such as heme and iron sulfur clusters. We observed an increase in the iron storage protein ferritin and a decreased labile iron pool in the PINK1 KD cells, but total cellular iron or markers of iron starvation/overload were not affected. Finally, cellular iron storage and the labile iron pool are maintainedviaautophagic degradation of ferritin (ferritinophagy). We found overexpressing nuclear receptor coactivator 4, a key adaptor for ferritinophagy, rescued cell growth and the labile iron pool in PINK1 KD cells. These results indicate that PINK1 integrates mitophagy and ferritinophagy to regulate intracellular iron availability and is essential for maintaining intracellular iron homeostasis to support survival and growth in colorectal cancer cells.

The Burkholderia cenocepacia iron starvation σ factor, OrbS, possesses an on-board iron sensor.

Burkholderia cenocepacia is an opportunistic pathogen that causes severe infections of the cystic fibrosis (CF) lung. To acquire iron, B. cenocepacia secretes the Fe(III)-binding compound, ornibactin. Genes for synthesis and utilisation of ornibactin are served by the iron starvation (IS) extracytoplasmic function (ECF) σ factor, OrbS. Transcription of orbS is regulated in response to the prevailing iron concentration by the ferric uptake regulator (Fur), such that orbS expression is repressed under iron-sufficient conditions. Here we show that, in addition to Fur-mediated regulation of orbS, the OrbS protein itself responds to intracellular iron availability. Substitution of cysteine residues in the C-terminal region of OrbS diminished the ability to respond to Fe(II) in vivo. Accordingly, whilst Fe(II) impaired transcription from and recognition of OrbS-dependent promoters in vitro by inhibiting the binding of OrbS to core RNA polymerase (RNAP), the cysteine-substituted OrbS variant was less responsive to Fe(II). Thus, the cysteine residues within the C-terminal region of OrbS contribute to an iron-sensing motif that serves as an on-board ‘anti-σ factor’ in the presence of Fe(II). A model to account for the presence two regulators (Fur and OrbS) that respond to the same intracellular Fe(II) signal to control ornibactin synthesis and utilisation is discussed.

Cysteine Toxicity Drives Age-Related Mitochondrial Decline by Altering Iron Homeostasis

Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease. Despite the long-standing connection between these organelles, the function(s) of lysosomes required to sustain mitochondrial health remains unclear. Here, working in yeast, we show that the lysosome-like vacuole maintains mitochondrial respiration by spatially compartmentalizing amino acids. Defects in vacuole function result in a breakdown in intracellular amino acid homeostasis, which drives age-related mitochondrial decline. Among amino acids, we find that cysteine is most toxic for mitochondria and show that elevated non-vacuolar cysteine impairs mitochondrial respiration by limiting intracellular iron availability through an oxidant-based mechanism. Cysteine depletion or iron supplementation restores mitochondrial health in vacuole-impaired cells and prevents mitochondrial decline during aging. These results demonstrate that cysteine toxicity is a major driver of age-related mitochondrial deterioration and identify vacuolar amino acid compartmentation as a cellular strategy to minimize amino acid toxicity.

Intracellular iron availability modulates the requirement for Leishmania Iron Regulator 1 (LIR1) during macrophage infections

The Leishmania plasma membrane transporter Leishmania Iron Regulator 1 (LIR1) facilitates iron export and is required for parasite virulence. By modulating macrophage iron content, we investigated the host site where LIR1 regulates Leishmania amazonensis infectivity. In bone marrow-derived macrophages, LIR1 null mutants demonstrated a paradoxical increase in virulence during infections in heme-depleted media, while wild-type growth was inhibited under the same conditions. Loading the endocytic pathway of macrophages with cationized ferritin prior to infection reversed the effect of heme depletion on both strains. Thus, LIR1 contributes to Leishmania virulence by protecting the parasites from toxicity resulting from iron accumulation inside parasitophorous vacuoles.

Susceptibility of human villous (BeWo) and extravillous (HTR-8/SVneo) trophoblast cells to Toxoplasma gondii infection is modulated by intracellular iron availability.

Congenital toxoplasmosis is a serious health problem that can lead to miscarriage. HTR-8/SVneo is a first trimester extravillous trophoblast, while BeWo is a choriocarcinoma with properties of villous trophoblast cells. In the placenta, iron is taken up from Fe-transferrin through the transferrin receptor being the ion an important nutrient during pregnancy and also for Toxoplasma gondii proliferation. The aim of this study was to evaluate the role of iron in T. gondii proliferation in BeWo and HTR-8/SVneo cells and in human chorionic villous explants. The cells were infected with T. gondii, iron supplemented or deprived by holo-transferrin or deferoxamine, respectively, and parasite proliferation and genes related to iron balance were analyzed. It was verified that the addition of holo-transferrin increased, and DFO decreased the parasite multiplication in both trophoblastic cells, however, in a more expressive manner in HTR-8/SVneo, indicating that the parasite depends on iron storage in trophoblastic cells for its growth. Also, tachyzoites pretread with DFO proliferate normally in trophoblastic cells demonstrating that DFO itself does not interfere with parasite proliferation. Additionally, T. gondii infection induced enhancement in transferrin receptor mRNA expression levels in trophoblastic cells, and the expression was higher in HTR-8/SVneo compared with BeWo. Finally, DFO-treatment was able to reduce the parasite replication in villous explants. Thus, the iron supplementation can be a double-edged sword; in one hand, it could improve the supplement of an essential ion to embryo/fetus development, and on the other hand, could improve the parasite proliferation enhancing the risk of congenital infection.

FPN1, Targeted By Mir-17-5p, Regulates Cellular Iron Homeostasis Via activating Nrf2/Mir-17-5p/FPN1 Axis in Preclinical Model of Multiple Myeloma

AbstractMultiple myeloma (MM) is a hematologic malignancy with lytic bone lessions, including dysregulation of iron homeostasis. Ferroportin (FPN1), the only known cellular iron exporter, has been demonstrated to have a dysregulated expression and to be associated with poor clinical outcome. However, the detailed mechanism of FPN1 in iron transportion is not clear. Our current study showed that miR-17-5p was another new FPN1 microRNA target, which had a negative correlation with FPN1 expression level in MM. We also identified that nuclear factor erythroid 2-related factor 2 (Nrf2) induced the transcription of miR-17-5p, which in turn suppressed the protein level of FPN1. Moreover, chromatin immunopreciptation (ChIP) and luciferase reporter assay revealed that Nrf2 directly inhibited the transcription of FPN1. Here, we investigated the mechanism of unbalanced Nrf2/miR-17-5p/FPN1 axis and CRISPR-mediated knockout of FPN1 function involved in iron metabolism of MM.To investigate the potential role of miR-17-5p in MM, several public datasets were analyzed, and we identified that miR-17-5p was significantly overexpressed in the progression of MM. We next examined the functional effect on cell proliferation and apoptosis, ARP1 and OCI-MY5 cells were transiently transfected with miR-17-5p mimics or inhibitors. The experiment revealed that miR-17-5p promoted proliferation, cell cycle and apoptosis resistance both in vitro and in vivo. Besides, we explored whether FPN1 could rescue the effect on cells growth by overexpressing FPN1 in MM cells co-transfecting with miR-17-5p mimics or scramble. Our results showed that FPN1 restoration partially induced an inhibitory effect on myeloma cells, confirming that miR-17-5p accumulation favored cell growth and survival by targeting FPN1.Furthermore, to investigate the regulatory mechanism of FPN1 in iron transportion, several transcription factor targeting predication algorithms were exploited, and we revealed that the FPN1 and miR-17-5p common target gene Nrf2, was significantly relative overexpressed in myeloma. We also analyzed the MM patient public datasets, including paired samples and different stages of disease, and all showed Nrf2 overexpression in relapsed MM with poor prognosis. Luciferase activity analysis verified that FPN1 was significantly inhibited when co-transfected, including truncated bodies of FPN1 promotor sequence. Meanwhile, ChIP-PCR showed that there was an interaction between Nrf2 and FPN1. Additionally, the direct interactions of Nrf2 with the miR-17-5p promoter were also confirmed, suggesting that Nrf2 bound to the miR-17-5p promoter region, thus regulating the transcription of miR-17-5p. These data indicateed that Nrf2 activation could modulate FPN1 levels directly or through miR-17-5p.To more comprehensively evaluate FPN1 in regulating iron exportation in MM, we introduced single guide (sg) RNA targeting FPN1 into MM cells stably expressing Cas9. Of note, CRISPR-mediated FPN1 knockout promoted the growth and increased intracellular iron levels. In addition, to test the requirement of iron for the growth of myeloma, two sgFPN1 depleted MM cells were treated with iron supplement Fecl3, or the iron chelator deferoxamine (DFO). Fecl3 had significantly increased intracellular iron while the effect treated with DFO was opposite. Besides, MM cell proliferation was significantly inhibited by DFO treatment, further confirming the role of intracellular iron in MM cell growth. We also analyzed the mRNA levels of signature genes involved in maintaining cellular iron homeostasis and the level of reactive oxygen species (ROS) generation during the iron-deficient or iron-abundant state, and our results showed that these genes involved in iron acquisition or iron storage were differentially expressed and influenced intracellular iron availability and ROS levels.In conclusion, our data suggested that the miR-17-5p/FPN1 axis induced by Nrf2 regulated intracellular liable iron pool, thus providing surplus iron for metabolic processes like DNA synthesis, the proliferation and growth of myeloma cells. Understanding the mechanisms on iron metabolism is important to develop new therapeutic strategies for clinical application to the treatment of MM.FundingThis study was supported by grants from the National Natural Science Foundation of China (Nos. 81570190; 81670194 and 81529001). DisclosuresNo relevant conflicts of interest to declare.

Heparin inhibits intracellular Mycobacterium tuberculosis bacterial replication by reducing iron levels in human macrophages

Iron is a crucial micronutrient for both mammals and their associated pathogens, and extensive literature has shown that Mycobacterium tuberculosis (Mtb) bacilli inhibited from acquiring iron from the host are severely attenuated. In contrast, increased dietary iron concentrations or patients with hemochromatosis have long been associated with a more severe tuberculosis (TB) disease outcome. We have observed that upon macrophage infection, Mtb bacilli strongly promote intracellular iron sequestration, both through increased expression of hepcidin, a key mammalian iron regulatory protein, and downregulation of the iron exporter protein, ferroportin. Heparin is a highly sulfated glycosaminoglycan released by mast cells and basophils at sites of tissue injury. During Mtb infection, heparin alters intracellular trafficking in alveolar epithelial cells and decreases extrapulmonary dissemination but recently, heparin also has been reported to inhibit hepcidin expression in hepatocytes, decreasing intracellular iron availability. In this report, we demonstrate that heparin significantly reduces hepcidin expression in macrophages infected with Mtb bacilli. Heparin-treated macrophages have higher ferroportin expression compared to untreated macrophages, promoting iron export and decreasing iron availability to intracellular bacilli. Thus, here we describe a novel immunomodulatory effect and potential therapeutic role for heparin against mycobacterial infection in human macrophages.

A CqFerritin protein inhibits white spot syndrome virus infection via regulating iron ions in red claw crayfish Cherax quadricarinatus

It is well known that iron is an essential element for all living organism. The intracellular iron availability is also important for the host's innate immune response to various pathogens, in which the iron homeostasis can be regulated by ferritin due to its iron storage property. In this study, a full-length cDNA sequence of ferritin (named as CqFerritin) was identified with 1410 bp from red claw crayfish Cherax quadricarinatus, which contained an open reading frame of 513 bp, encoding 170 amino acids with a conserved ferritin domain. Tissue distribution analysis demonstrated that CqFerritin was widely expressed in various tissues with high presence in haemocyte, haematopoietic tissue (Hpt) and heart, while lowest expression in hepatopancreas. In addition, loss-of-function of CqFerritin by gene silencing resulted in significantly higher expression of an envelope protein VP28 of white spot syndrome virus (WSSV) in red claw crayfish Hpt cell cultures, indicating the potential antiviral response of CqFerritin. To further explore the effect on WSSV replication by CqFerritin, recombinant CqFerritin protein (rCqFerritin) was transfected into Hpt cells followed by WSSV infection. Importantly, the replication of WSSV was obviously decreased in Hpt cells if transfected with rCqFerritin protein, suggesting that CqFerritin had clearly negative effect on WSSV infection. Furthermore, intracellular accumulation of iron ions was found to promote the WSSV replication in a dose-dependent manner, illustrating that the iron level regulated by CqFerritin was likely to be vital for WSSV infection in red claw crayfish. Taken together, these data suggest that CqFerritin plays an important role in immune defense against WSSV infection in a crustacean C. quadricarinatus.

Sequential induction of Fur-regulated genes in response to iron limitation in Bacillus subtilis

Bacterial cells modulate transcription in response to changes in iron availability. The ferric uptake regulator (Fur) senses intracellular iron availability and plays a central role in maintaining iron homeostasis in Bacillus subtilis Here we utilized FrvA, a high-affinity Fe2+ efflux transporter from Listeria monocytogenes, as an inducible genetic tool to deplete intracellular iron. We then characterized the responses of the Fur, FsrA, and PerR regulons as cells transition from iron sufficiency to deficiency. Our results indicate that the Fur regulon is derepressed in three distinct waves. First, uptake systems for elemental iron (efeUOB), ferric citrate (fecCDEF), and petrobactin (fpbNOPQ) are induced to prevent iron deficiency. Second, B. subtilis synthesizes its own siderophore bacillibactin (dhbACEBF) and turns on bacillibactin (feuABC) and hydroxamate siderophore (fhuBCGD) uptake systems to scavenge iron from the environment and flavodoxins (ykuNOP) to replace ferredoxins. Third, as iron levels decline further, an "iron-sparing" response (fsrA, fbpAB, and fbpC) is induced to block the translation of abundant iron-utilizing proteins and thereby permit the most essential iron-dependent enzymes access to the limited iron pools. ChIP experiments demonstrate that in vivo occupancy of Fur correlates with derepression of each operon, and the graded response observed here results, at least in part, from higher-affinity binding of Fur to the "late"-induced genes.

JMJD6-mediated histone demethylation in preeclampsia is compromised due to reduced intracellular iron availability

In this paper, a novel dynamic voltage–frequency scaling-aware (DVFS-aware) bandwidth- efficient motion estimation (ME) scheme is presented for mobile application processor (AP) systems. Under volatile operating performance conditions due to the power management mechanism, we model the coding bandwidth (BW) and coding performance for the video processor as a convex function of the working frequency. In this paper, we present a bandwidth–rate–distortion (B–R–D) optimized framework that will guarantee the smallest possible rate–distortion cost among coding BW constraints applied in video coding design. By formulating the coding bandwidth-constrained ME problem as an optimization problem, known convex optimization theory can be applied to yield optimal resource-constrained compression. Using varied CIF (352×288)- and HP (1280×720)-sized video sequences with different motion activities over our proposed DVFS-aware video coding approach, the excellent results in terms of coding performance and coding bandwidth savings are obtained. With negligible quality loss, the proposed scheme under coding BW constraints achieves 45–65% coding BW usage reduction over HD-sized 30 frame/s video coding.

Inhibition of prostate cancer proliferation by Deferiprone.

Cancer growth and proliferation rely on intracellular iron availability. We studied the effects of Deferiprone (DFP), a chelator of intracellular iron, on three prostate cancer cell lines: murine, metastatic TRAMP-C2; murine, non-metastatic Myc-CaP; and human, non-metastatic 22rv1. The effects of DFP were evaluated at different cellular levels: cell culture proliferation and migration; metabolism of live cells (time-course multi-nuclear magnetic resonance spectroscopy cell perfusion studies, with 1-13 C-glucose, and extracellular flux analysis); and expression (Western blot) and activity of mitochondrial aconitase, an iron-dependent enzyme. The 50% and 90% inhibitory concentrations (IC50 and IC90 , respectively) of DFP for the three cell lines after 48h of incubation were within the ranges 51-67μM and 81-186μM, respectively. Exposure to 100μM DFP led to: (i) significant inhibition of cell migration after different exposure times, ranging from 12h (TRAMP-C2) to 48h (22rv1), in agreement with the respective cell doubling times; (ii) significantly decreased glucose consumption and glucose-driven tricarboxylic acid cycle activity in metastatic TRAMP-C2 cells, during the first 10h of exposure, and impaired cellular bioenergetics and membrane phospholipid turnover after 23h of exposure, consistent with a cytostatic effect of DFP. At this time point, all cell lines studied showed: (iii) significant decreases in mitochondrial functional parameters associated with the oxygen consumption rate, and (iv) significantly lower mitochondrial aconitase expression and activity. Our results indicate the potential of DFP to inhibit prostate cancer proliferation at clinically relevant doses and plasma concentrations.

Iron-regulatory proteins secure iron availability in cardiomyocytes to prevent heart failure.

Iron deficiency (ID) is associated with adverse outcomes in heart failure (HF) but the underlying mechanisms are incompletely understood. Intracellular iron availability is secured by two mRNA-binding iron-regulatory proteins (IRPs), IRP1 and IRP2. We generated mice with a cardiomyocyte-targeted deletion of Irp1 and Irp2 to explore the functional implications of ID in the heart independent of systemic ID and anaemia. Iron content in cardiomyocytes was reduced in Irp-targeted mice. The animals were not anaemic and did not show a phenotype under baseline conditions. Irp-targeted mice, however, were unable to increase left ventricular (LV) systolic function in response to an acute dobutamine challenge. After myocardial infarction, Irp-targeted mice developed more severe LV dysfunction with increased HF mortality. Mechanistically, the activity of the iron-sulphur cluster-containing complex I of the mitochondrial electron transport chain was reduced in left ventricles from Irp-targeted mice. As demonstrated by extracellular flux analysis in vitro, mitochondrial respiration was preserved at baseline but failed to increase in response to dobutamine in Irp-targeted cardiomyocytes. As shown by 31P-magnetic resonance spectroscopy in vivo, LV phosphocreatine/ATP ratio declined during dobutamine stress in Irp-targeted mice but remained stable in control mice. Intravenous injection of ferric carboxymaltose replenished cardiac iron stores, restored mitochondrial respiratory capacity and inotropic reserve, and attenuated adverse remodelling after myocardial infarction in Irp-targeted mice but not in control mice. As shown by electrophoretic mobility shift assays, IRP activity was significantly reduced in LV tissue samples from patients with advanced HF and reduced LV tissue iron content. ID in cardiomyocytes impairs mitochondrial respiration and adaptation to acute and chronic increases in workload. Iron supplementation restores cardiac energy reserve and function in iron-deficient hearts.

Effects of GlyT1 inhibition on erythropoiesis and iron homeostasis in rats.

Glycine is a key rate-limiting component of heme biosynthesis in erythropoietic cells, where the high intracellular glycine demand is primarily supplied by the glycine transporter 1 (GlyT1). The impact of intracellular glycine restriction after GlyT1 inhibition on hematopoiesis and iron regulation is not well established. We investigated the effects of a potent and selective inhibitor of GlyT1, bitopertin, on erythropoiesis and iron homeostasis in rats. GlyT1 inhibition significantly affected erythroid heme biosynthesis, manifesting as microcytic hypochromic regenerative anemia with a 20% steady-state reduction in hemoglobin. Reduced erythropoietic iron utilization was characterized by down-regulation of the transferrin receptor 1 (TfR1) on reticulocytes and modest increased iron storage in the spleen. Hepatic hepcidin expression was not affected. However, under the condition of reduced heme biosynthesis with reduced iron reutilization and increased storage iron, hepcidin at the lower and higher range of normal showed a striking role in tissue distribution of iron. Rapid formation of iron-positive inclusion bodies (IBs) was observed in circulating reticulocytes, with an ultrastructure of iron-containing polymorphic mitochondrial remnants. IB or mitochondrial iron accumulation was absent in bone marrow erythroblasts. In conclusion, GlyT1 inhibition in rats induced a steady-state microcytic hypochromic regenerative anemia and a species-specific accumulation of uncommitted mitochondrial iron in reticulocytes. Importantly, this glycine-restricted anemia provides no feedback signal for increased systemic iron acquisition and the effects reported are pathogenetically distinct from systemic iron-overload anemias and erythropoietic disorders such as acquired sideroblastic anemia.

Iron, oxygen, and the pulmonary circulation.

The human pulmonary vasculature vasoconstricts in response to a reduction in alveolar oxygen tension, a phenomenon termed hypoxic pulmonary vasoconstriction (HPV). This review describes the time course of this behavior, which occurs in distinct phases, and then explores the importance for HPV of the hypoxia-inducible factor (HIF) pathway. Next, the HIF-hydroxylase enzymes that act as molecular oxygen sensors within the HIF pathway are discussed. These enzymes are particularly sensitive to intracellular iron availability, which confers iron-sensing properties on the HIF pathway. Human studies of iron chelation and supplementation are then reviewed. These demonstrate that the iron sensitivity of the HIF pathway evident from in vitro experiments is relevant to human pulmonary vascular physiology. Next, the importance of iron status in high-altitude illness and chronic cardiopulmonary disease is explored, and the therapeutic potential of intravenous iron discussed. The review concludes by highlighting some further complexities that arise from interactions between the HIF pathway and other intracellular iron-sensing mechanisms.

Differentiation‐dependent Regulation of Intestinal Iron Absorption: Cell and Molecular Mechanisms

Differentiation of intestinal epithelial cells is associated with up‐ and down‐regulation of a variety of genes, but little is known about the differentiation‐dependent regulation of genes encoding proteins involved in intestinal iron absorption. We have addressed this issue using human‐derived intestinal epithelial Caco‐2 cells and crypt/villus epithelial cells isolated from wild‐type mice as models. We examined expression of divalent metal‐ion transporter‐1 (DMT‐1; brush border iron importer), ferroportin (FPN; basolateral iron exporter) and hephaestin (HEPH; basolateral iron oxidase). Differentiation of Caco‐2 cells was associated with a significant up‐regulation of iron uptake. This up‐regulation was associated with a significant increase in the expression of DMT‐1 and HEPH mRNA and protein, as well as increased expression of FPN mRNA, but not FPN protein. Similarly, iron uptake and the expression of DMT‐1, HEPH, and FPN mRNAs was higher in mouse villous cells than crypt cells. Promoter‐reporter constructs were used to show that the activity of the promoters of DMT‐1 and HEPH increased with differentiation of Caco‐2 cells. These studies demonstrate that differentiation of intestinal epithelial cells is associated with up‐regulation of the iron uptake process and increased expression of DMT‐1, HEPH, and FPN. The expression of all these proteins of iron transport can be mediated at the post‐transcriptional level according to intracellular iron availability, but our results indicate that up‐regulation of DMT‐1 and HEPH during differentiation appears to be mediated at least in part via transcriptional regulatory mechanisms. (Supported by funds from the Department of Veterans Affairs.)

Association of Pro-Inflammatory Cytokines and Iron Regulatory Protein 2 (IRP2) with Leishmania Burden in Canine Visceral Leishmaniasis

Leishmania infantum infection in humans and dogs can evolve with a wide range of clinical presentations, varying from asymptomatic infections to visceral leishmaniasis. We hypothesized that the immune response elicited by L. infantum infection could modulate whether the host will remain asymptomatic or progress to disease. A total of 44 dogs naturally infected with L. infantum were studied. Leishmania burden was estimated in the blood and spleen by qPCR. The expression of IFN-γ, TNF-α, IL-10 and Iron Regulatory Protein 2 (IRP2) were determined in the spleen by quantitative PCR. Sera cytokines were evaluated by ELISA. Dogs were grouped in quartiles according parasite burden. Increased expression of IFN-γ and TNF-α was associated with reduced Leishmania burden, whereas increased IL-10 and IRP2 expressions were associated with higher Leishmania load. Increased plasma albumin and IFN-γ expression explained 22.8% of the decrease in parasite burden in the spleen. These data confirm that lower IFN-γ response and higher IL-10 correlated with increased parasite load and severity of the visceral leishmaniasis in dogs. The balance between the branches of immune response and the intracellular iron availability could determine, in part, the course of Leishmania infection.

Iron Deprivation in Cancer––Potential Therapeutic Implications

Iron is essential for normal cellular function. It participates in a wide variety of cellular processes, including cellular respiration, DNA synthesis, and macromolecule biosynthesis. Iron is required for cell growth and proliferation, and changes in intracellular iron availability can have significant effects on cell cycle regulation, cellular metabolism, and cell division. Perhaps not surprisingly then, neoplastic cells have been found to have higher iron requirements than normal, non-malignant cells. Iron depletion through chelation has been explored as a possible therapeutic intervention in a variety of cancers. Here, we will review iron homeostasis in non-malignant and malignant cells, the widespread effects of iron depletion on the cell, the various iron chelators that have been explored in the treatment of cancer, and the tumor types that have been most commonly studied in the context of iron chelation.

Clearance mechanism of protoporphyrin IX from mouse skin after application of 5-aminolevulinic acid

5-Aminolevulinic acid (ALA) or its esters mediated photodynamic therapy (PDT) is the most widely practiced form of PDT in dermatology. One of its advantages is that undesirable photosensitization lasts only for 24-48 h. In order to optimize ALA-PDT it is necessary to understand the mechanisms of intracellular production and clearance of PpIX (efflux from cells into blood stream and/or its conversion into haem). The aim of this study is to investigate the factors controlling the clearance of intracellular PpIX from healthy skin of mice. PpIX was induced in mouse skin by topical or systemic application of ALA or by topical application of the iron chelator ethylenediaminetetraacetic acid (EDTA). Fluorescence spectroscopy was used to study PpIX kinetics in alive and dead skin. Topical application of ALA or EDTA leads to porphyrin production in living skin, but not in excised skin. The clearance rates of PpIX from alive and dead skin were the same in the absence of an intracellular ALA pool. The clearance half-life of EDTA-induced PpIX was 4-7 times longer than that of PpIX after application of ALA. Skin temperature and intracellular iron availability strongly affect PpIX clearance, while ALA application mode (topical versus systemic) and skin viability (dead versus alive) have no influence on PpIX decay. These results demonstrate that the clearance kinetics of PpIX from skin are determined mostly by the conversion of PpIX into haem, while the cellular efflux of PpIX into blood plays a minor role.