Single-cell transcriptomics identifies CD44 as a marker and regulator of endothelial to haematopoietic transition.

The endothelial to haematopoietic transition (EHT) is the process whereby haemogenic endothelium differentiates into haematopoietic stem and progenitor cells (HSPCs). The intermediary steps of this process are unclear, in particular the identity of endothelial cells that give rise to HSPCs is unknown. Using single-cell transcriptome analysis and antibody screening, we identify CD44 as a marker of EHT enabling us to isolate robustly the different stages of EHT in the aorta-gonad-mesonephros (AGM) region. This allows us to provide a detailed phenotypical and transcriptional profile of CD44-positive arterial endothelial cells from which HSPCs emerge. They are characterized with high expression of genes related to Notch signalling, TGFbeta/BMP antagonists, a downregulation of genes related to glycolysis and the TCA cycle, and a lower rate of cell cycle. Moreover, we demonstrate that by inhibiting the interaction between CD44 and its ligand hyaluronan, we can block EHT, identifying an additional regulator of HSPC development.

Iron deficiency disrupts embryonic haematopoiesis but not the endothelial to haematopoietic transition.

In this study, we aimed to explore how cellular iron status affects embryonic haematopoiesis. For this purpose, we used a model of mouse embryonic stem cell differentiation into embryonic haematopoietic progenitors. We modulated the iron status by adding either the iron chelator Deferoxamine (DFO) for iron deficiency, or ferric ammonium citrate for iron excess, and followed the emergence of developing haematopoietic progenitors. Interestingly, we found that iron deficiency did not block the endothelial to haematopoietic transition, the first step of haematopoiesis. However, it did reduce the proliferation, survival and clonogenic capacity of haematopoietic progenitors. Surprisingly, iron deficiency affected erythro-myeloid progenitors significantly more than the primitive erythroid ones. Erythro-myeloid progenitors expressed less transferrin-receptor on the cell surface and had less labile iron compared to primitive erythroid progenitors, which could reduce their capacity to compete for scarce iron and survive iron deficiency. In conclusion, we show that iron deficiency could disturb haematopoiesis at an early embryonic stage by compromising more severely the survival, proliferation and differentiation of definitive haematopoietic progenitors compared to restricted erythroid progenitors.

The actin-binding protein profilin 2 is a novel regulator of iron homeostasis.

Cellular iron homeostasis is controlled by the iron regulatory proteins (IRPs) 1 and 2 that bind cis-regulatory iron-responsive elements (IRE) on target messenger RNAs (mRNA). We identified profilin 2 (Pfn2) mRNA, which encodes an actin-binding protein involved in endocytosis and neurotransmitter release, as a novel IRP-interacting transcript, and studied its role in iron metabolism. A combination of electrophoretic mobility shift assay experiments and bioinformatic analyses led to the identification of an atypical and conserved IRE in the 3' untranslated region of Pfn2 mRNA. Pfn2 mRNA levels were significantly reduced in duodenal samples from mice with intestinal IRP ablation, suggesting that IRPs exert a positive effect on Pfn2 mRNA expression in vivo. Overexpression of Pfn2 in HeLa and Hepa1-6 cells reduced their metabolically active iron pool. Importantly, Pfn2-deficient mice showed iron accumulation in discrete areas of the brain (olfactory bulb, hippocampus, and midbrain) and reduction of the hepatic iron store without anemia. Despite low liver iron levels, hepatic hepcidin expression remained high, likely because of compensatory activation of hepcidin by mild inflammation. Splenic ferroportin was increased probably to sustain hematopoiesis. Overall, our results indicate that Pfn2 expression is controlled by the IRPs in vivo and that Pfn2 contributes to maintaining iron homeostasis in cell lines and mice.

Activation of the TGFß pathway impairs endothelial to haematopoietic transition.

The endothelial to haematopoietic transition (EHT) is a key developmental process where a drastic change of endothelial cell morphology leads to the formation of blood stem and progenitor cells during embryogenesis. As TGFß signalling triggers a similar event during embryonic development called epithelial to mesenchymal transition (EMT), we hypothesised that TGFß activity could play a similar role in EHT as well. We used the mouse embryonic stem cell differentiation system for in vitro recapitulation of EHT and performed gain and loss of function analyses of the TGFß pathway. Quantitative proteomics analysis showed that TGFß treatment during EHT increased the secretion of several proteins linked to the vascular lineage. Live cell imaging showed that TGFß blocked the formation of round blood cells. Using gene expression profiling we demonstrated that the TGFß signalling activation decreased haematopoietic genes expression and increased the transcription of endothelial and extracellular matrix genes as well as EMT markers. Finally we found that the expression of the transcription factor Sox17 was up-regulated upon TGFß signalling activation and showed that its overexpression was enough to block blood cell formation. In conclusion we showed that triggering the TGFß pathway does not enhance EHT as we hypothesised but instead impairs it.

Functional consequences of transferrin receptor-2 mutations causing hereditary hemochromatosis type 3.

Hereditary hemochromatosis (HH) type 3 is an autosomal recessive disorder of iron metabolism characterized by excessive iron deposition in the liver and caused by mutations in the transferrin receptor 2 (TFR2) gene. Here, we describe three new HH type 3 Spanish families with four TFR2 mutations (p.Gly792Arg, c.1606-8A>G, Gln306*, and Gln672*). The missense variation p.Gly792Arg was found in homozygosity in two adult patients of the same family, and in compound heterozygosity in an adult proband that also carries a novel intronic change (c.1606-8A>G). Two new nonsense TFR2 mutations (Gln306* and Gln672*) were detected in a pediatric case. We examine the functional consequences of two TFR2 variants (p.Gly792Arg and c.1606-8A>G) using molecular and computational methods. Cellular protein localization studies using immunofluorescence demonstrated that the plasma membrane localization of p.Gly792Arg TFR2 is impaired. Splicing studies in vitro and in vivo reveal that the c.1606-8A>G mutation leads to the creation of a new acceptor splice site and an aberrant TFR2 mRNA. The reported mutations caused HH type 3 by protein truncation, altering TFR2 membrane localization or by mRNA splicing defect, producing a nonfunctional TFR2 protein and a defective signaling transduction for hepcidin regulation. TFR2 genotyping should be considered in adult but also in pediatric cases with early-onset of iron overload.

Intracellular iron trafficking: role of cytosolic ligands.

Iron acquired by cells is delivered to mitochondria for metabolic processing via pathways comprising undefined chemical forms. In order to assess cytosolic factors that affect those iron delivery pathways, we relied on microscopy and flow-cytometry for monitoring iron traffic in: (a) K562 erythroleukemia cells labeled with fluorescent metal-sensors targeted to either cytosol or mitochondria and responsive to changes in labile iron and (b) permeabilized cells that retained metabolically active mitochondria accessible to test substrates. Iron supplied to intact cells as transferrin-Fe(III) or Fe(II)-salts evoked concurrent metal ingress to cytosol and mitochondria. With either supplementation modality, iron ingress into cytosol was mostly absorbed by preloaded chelators, but ingress into mitochondria was fully inhibited only by some chelators, indicating different cytosol-to-mitochondria delivery mechanisms. Iron ingress into cytosol or mitochondria were essentially unaffected by depletion of cytosolic iron ligands like glutathione or the hypothesized 2,5 dihydroxybenzoate (2,5-DHBA) siderophore/chaperone. These ligands also failed to affect mitochondrial iron ingress in permeabilized K562 cells suspended in cytosol-simulating medium. In such medium, mitochondrial iron uptake was >6-eightfold higher for Fe(II) versus Fe(III), showed saturable properties and submicromolar K(1/2) corresponding to cytosolic labile iron levels. When measured in iron(II)-containing media, ligands like AMP, ADP or ATP, did not affect mitochondrial iron uptake whereas in iron(III)-containing media ADP and ATP reduced it and AMP stimulated it. Thus, cytosolic iron forms demonstrably contribute to mitochondrial iron delivery, are apparently not associated with DHBA analogs or glutathione but rather with resident components of the cytosolic labile iron pool.

Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein.

MitoNEET (mNT) is an outer mitochondrial membrane target of the thiazolidinedione diabetes drugs with a unique fold and a labile [2Fe-2S] cluster. The rare 1-His and 3-Cys coordination of mNT's [2Fe-2S] leads to cluster lability that is strongly dependent on the presence of the single histidine ligand (His87). These properties of mNT are similar to known [2Fe-2S] shuttle proteins. Here we investigated whether mNT is capable of cluster transfer to acceptor protein(s). Facile [2Fe-2S] cluster transfer is observed between oxidized mNT and apo-ferredoxin (a-Fd) using UV-VIS spectroscopy and native-PAGE, as well as with a mitochondrial iron detection assay in cells. The transfer is unidirectional, proceeds to completion, and occurs with a second-order-reaction rate that is comparable to known iron-sulfur transfer proteins. Mutagenesis of His87 with Cys (H87C) inhibits transfer of the [2Fe-2S] clusters to a-Fd. This inhibition is beyond that expected from increased cluster kinetic stability, as the equivalently stable Lys55 to Glu (K55E) mutation did not inhibit transfer. The H87C mutant also failed to transfer its iron to mitochondria in HEK293 cells. The diabetes drug pioglitazone inhibits iron transfer from WT mNT to mitochondria, indicating that pioglitazone affects a specific property, [2Fe-2S] cluster transfer, in the cellular environment. This finding is interesting in light of the role of iron overload in diabetes. Our findings suggest a likely role for mNT in [2Fe-2S] and/or iron transfer to acceptor proteins and support the idea that pioglitazone's antidiabetic mode of action may, in part, be to inhibit transfer of mNT's [2Fe-2S] cluster.

Transferrin-iron routing to the cytosol and mitochondria as studied by live and real-time fluorescence.

In the present study we analysed the mechanism of intracellular routing of iron acquired by erythroid cells via receptor-mediated endocytosis of Tf-Fe [Tf (transferrin)-iron]. Using real-time fluorimetry and flow cytometry, in conjunction with targeted fluorescent metal sensors, we monitored concurrently the cytosolic and mitochondrial changes in labile iron evoked by endocytosed Tf-Fe. In K562 human erythroleukaemia cells, most of the Tf-Fe was found to be delivered to the cytosolic labile iron pool by a saturable mechanism [60-120 nM Km (app)] that was quantitatively dependent on: Tf receptor levels, endosomal acidification/reduction for dislodging iron from Tf and ensuing translocation of labile iron into the cytosolic compartment. The parallel ingress of iron to mitochondria was also saturable, but with a relatively lower Km (app) (26-42 nM) and a lower maximal ingress per cell than into the cytosol. The ingress of iron into the mitochondrial labile iron pool was blocked by cytosol-targeted iron chelators, implying that a substantial fraction of Tf-Fe delivered to these organelles passes through the cytosol in non-occluded forms that remain accessible to high-affinity ligands. The present paper is the first report describing intracellular iron routing measured in intact cells in real-time and in quantitative terms, opening the road for also exploring the process in mixed-cell populations of erythroid origin.

Intracellular labile iron.

Cells maintain organellar pools of "labile iron" (LI), despite its propensity for catalyzing the formation of reactive oxygen species. These pools are identifiable by iron-chelating probes and accessible to pharmacological agents. Cytosolic LI has been assumed to have a dual function: providing a rapidly adjustable source of iron for immediate metabolic utilization, and for sensing by iron-regulatory proteins (IRPs) that regulate iron uptake and compartmentalization via transferrin receptors and ferritin. However, it now appears that IRPs may respond both to fluctuations in LI per se and to secondary signals associated with redox-active species. Recent information also indicates that iron can be delivered to mitochondria via pathways that circumvent cytosolic LI, suggesting possible alternative mechanisms of cell iron mobilization and trafficking. We discuss the changing views of intracellular LI pools in relation to iron homeostasis and cellular distribution in physiological and pathological states.

Non-transferrin-bound iron reaches mitochondria by a chelator-inaccessible mechanism: biological and clinical implications.

Non-transferrin-bound iron, commonly found in the plasma of iron-overloaded individuals, permeates into cells via pathways independent of the transferrin receptor. This may lead to excessive cellular accumulation of labile iron followed by oxidative damage and eventually organ failure. Mitochondria are the principal destination of iron in cells and a primary site of prooxidant generation, yet their mode of acquisition of iron is poorly understood. Using fluorescent probes sensitive to iron or to reactive oxygen species, targeted to cytosol and/or to mitochondria, we traced the ingress of labile iron into these compartments by fluorescence microscopy and quantitative fluorimetry. We observed that 1) penetration of non-transferrin-bound iron into the cytosol and subsequently into mitochondria occurs with barely detectable delay and 2) loading of the cytosol with high-affinity iron-binding chelators does not abrogate iron uptake into mitochondria. Therefore, a fraction of non-transferrin-bound iron acquired by cells reaches the mitochondria in a nonlabile form. The physiological role of occluded iron transfer might be to confer cells with a "safe and efficient cytosolic iron corridor" to mitochondria. However, such a mechanism might be deleterious in iron-overload conditions, because it could lead to surplus accumulation of iron in these critical organelles.

Intracellular labile iron pools as direct targets of iron chelators: a fluorescence study of chelator action in living cells.

The primary targets of iron chelators used for treating transfusional iron overload are prevention of iron ingress into tissues and its intracellular scavenging. The present study was aimed at elucidating the capacity of clinically important iron chelators such as deferiprone (DFP), desferrioxamine, and ICL670 to (a) gain direct access to intracellular iron pools of key cells of iron accumulation (macrophages, hepatocytes, and cardiomyocyte cell lines); (b) chelate the labile iron present in discrete cell compartments/organelles; and (c) prevent labile iron involvement in the generation of reactive oxidant species. Chelation of cytosolic and organellar cell iron was visualized dynamically and quantitatively in living cells by fluorescence microscopic imaging of fluorescent metallosensors (used as iron-quenched complexes of calceins) targeted to either cytosol, endosome-lysosomes, or mitochondria. The rate and extent of fluorescence recovery provided an in situ measure of the accessibility of chelators to particular cell sites/organelles. Complementary, fluorogenic redox probes associated with cell compartments enabled identification of chelator-sensitive, localized reactive oxidant production. Our studies indicate that chelation by desferrioxamine is slow and is enhanced in cells with relatively high endocytic activities, while ICL670 and DFP readily enter most cells and efficiently reach the major intracellular sites of iron accumulation.