Iron Mining for Erythropoiesis.

Iron is necessary for essential processes in every cell of the body, but the erythropoietic compartment is a privileged iron consumer. In fact, as a necessary component of hemoglobin and myoglobin, iron assures oxygen distribution; therefore, a considerable amount of iron is required daily for hemoglobin synthesis and erythroid cell proliferation. Therefore, a tight link exists between iron metabolism and erythropoiesis. The liver-derived hormone hepcidin, which controls iron homeostasis via its interaction with the iron exporter ferroportin, coordinates erythropoietic activity and iron homeostasis. When erythropoiesis is enhanced, iron availability to the erythron is mainly ensured by inhibiting hepcidin expression, thereby increasing ferroportin-mediated iron export from both duodenal absorptive cells and reticuloendothelial cells that process old and/or damaged red blood cells. Erythroferrone, a factor produced and secreted by erythroid precursors in response to erythropoietin, has been identified and characterized as a suppressor of hepcidin synthesis to allow iron mobilization and facilitate erythropoiesis.

Induction of erythroferrone in healthy humans by micro-dose recombinant erythropoietin or high-altitude exposure.

The erythropoietin (Epo)-erythroferrone (ERFE)-hepcidin axis coordinates erythropoiesis and iron homeostasis. While mouse studies have established that Epo-induced ERFE production represses hepcidin synthesis by inhibiting hepatic BMP/SMAD signaling, evidence for the role of ERFE in humans is limited. To investigate the role of ERFE as a physiological erythroid regulator in humans, we conducted two studies: first, 24 males received six injections of saline (placebo), recombinant Epo (rhEpo) 20 UI kg-1 (micro-dose) or 50 UI kg-1 (low-dose). Second, we quantified ERFE in 22 subjects exposed to high altitude (3800 m) for 15 hours. In the first study, total hemoglobin mass (Hbmass) increased after low- but not after micro-dose injections, when compared to placebo. Serum ERFE levels were enhanced by rhEpo, remaining higher than after placebo for 48 (micro-dose) or 72 hours (low-dose) post-injections. Conversely, hepcidin levels decreased when Epo and ERFE arose, before any changes in serum iron parameters occurred. In the second study, serum Epo and ERFE increased at high altitude. The present results demonstrate that in healthy humans ERFE responds to slightly increased Epo levels not associated with Hbmass expansion and down-regulates hepcidin in an apparently iron-independent way. Notably, ERFE flags micro-dose Epo, thus holding promise as novel anti-doping biomarker.

Iron Availability in Tissue Microenvironment: The Key Role of Ferroportin.

Body iron levels are regulated by hepcidin, a liver-derived peptide that exerts its function by controlling the presence of ferroportin (FPN), the sole cellular iron exporter, on the cell surface. Hepcidin binding leads to FPN internalization and degradation, thereby inhibiting iron release, in particular from iron-absorbing duodenal cells and macrophages involved in iron recycling. Disruption in this regulatory mechanism results in a variety of disorders associated with iron-deficiency or overload. In recent years, increasing evidence has emerged to indicate that, in addition to its role in systemic iron metabolism, FPN may play an important function in local iron control, such that its dysregulation may lead to tissue damage despite unaltered systemic iron homeostasis. In this review, we focus on recent discoveries to discuss the role of FPN-mediated iron export in the microenvironment under both physiological and pathological conditions.

Macrophage ferroportin is essential for stromal cell proliferation in wound healing.

Iron recycling by macrophages is essential for erythropoiesis, but may also be relevant for iron redistribution to neighboring cells at the local tissue level. Using mice with iron retention in macrophages due to targeted inactivation of the iron exporter ferroportin, we investigated the role of macrophage iron release in hair follicle cycling and wound healing, a complex process leading to major clinical problems, if impaired. Genetic deletion of ferroportin in macrophages resulted in iron deficiency and decreased proliferation in epithelial cells, which consequently impaired hair follicle growth and caused transient alopecia. Hair loss was not related to systemic iron deficiency or anemia, thus indicating the necessity of local iron release from macrophages. Inactivation of macrophage ferroportin also led to delayed skin wound healing with defective granulation tissue formation and diminished fibroplasia. Iron retention in macrophages had no impact on the inflammatory processes accompanying wound healing, but affected stromal cell proliferation, blood and lymphatic vessel formation, and fibrogenesis. Our findings reveal that iron/ferroportin plays a largely underestimated role in macrophage trophic function in skin homeostasis and repair.

Molecular regulation of cellular iron balance.

Handling a life-supporting yet redox-active metal like iron represents a significant challenge to cells and organisms that must not only tightly balance intra- and extracellular iron concentrations but also chaperone it during its journey from its point of entry to final destinations, to prevent inappropriate generation of damaging reactive oxygen species. Accordingly, regulatory mechanisms have been developed to maintain appropriate cellular and body iron levels. In intracellular compartments, about 95% of iron is protein-bound and the expression of the major proteins of iron metabolism is controlled by an integrated and dynamic system involving multilayered levels of regulation. However, dysregulation of iron homeostasis, which could result from both iron-related and unrelated effectors, may occur and have important pathological consequences in a number of human disorders. In this review, we describe the current understanding of the mechanisms that keep cellular iron balance and outline recent advances that increased our knowledge of the molecular physiology of iron metabolism. © 2017 IUBMB Life, 69(6):389-398, 2017.

Erythropoietin's inhibiting impact on hepcidin expression occurs indirectly.

Under conditions of accelerated erythropoiesis, elevated erythropoietin (Epo) levels are associated with inhibition of hepcidin synthesis, a response that ultimately increases iron availability to meet the enhanced iron needs of erythropoietic cells. In the search for erythroid regulators of hepcidin, many candidates have been proposed, including Epo itself. We aimed to test whether direct interaction between Epo and the liver is required to regulate hepcidin. We found that prolonged administration of high doses of Epo in mice leads to great inhibition of liver hepcidin mRNA levels, and concomitant induction of the hepcidin inhibitor erythroferrone (ERFE). Epo treatment also resulted in liver iron mobilization, mediated by increased ferroportin activity and accompanied by reduced ferritin levels and increased TfR1 expression. The same inhibitory effect was observed in mice that do not express the homodimeric Epo receptor (EpoR) in liver cells because EpoR expression is restricted to erythroid cells. Similarly, liver signaling pathways involved in hepcidin regulation were not influenced by the presence or absence of hepatic EpoR. Moreover, Epo analogs, possibly interacting with the postulated heterodimeric ß common EpoR, did not affect hepcidin expression. These findings were supported by the lack of inhibition on hepcidin found in hepatoma cells exposed to various concentrations of Epo for different periods of times. Our results demonstrate that hepcidin suppression does not require the direct binding of Epo to its liver receptors and rather suggest that the role of Epo is to stimulate the synthesis of the erythroid regulator ERFE in erythroblasts, which ultimately downregulates hepcidin.

Liver iron modulates hepcidin expression during chronically elevated erythropoiesis in mice.

UNLABELLED: The liver-derived peptide hepcidin controls the balance between iron demand and iron supply. By inhibiting the iron export activity of ferroportin, hepcidin modulates iron absorption and delivery from the body's stores. The regulation of hepcidin, however, is not completely understood and includes a variety of different signals. We studied iron metabolism and hepcidin expression in mice constitutively overexpressing erythropoietin (Epo) (Tg6 mice), which leads to excessive erythropoiesis. We observed a very strong down-regulation of hepcidin in Tg6 mice that was accompanied by a strong increase in duodenal expression of ferroportin and divalent metal tranporter-1, as well as enhanced duodenal iron absorption. Despite these compensatory mechanisms, Tg6 mice displayed marked circulating iron deficiency and low levels of iron in liver, spleen, and muscle. To elucidate the primary signal affecting hepcidin expression during chronically elevated erythropoiesis, we increased iron availability by either providing iron (thus further increasing the hematocrit) or reducing erythropoiesis-dependent iron consumption by means of splenectomy. Both treatments increased liver iron and up-regulated hepcidin expression and the BMP6/SMAD pathway despite continuously high plasma Epo levels and sustained erythropoiesis. This suggests that hepcidin expression is not controlled by erythropoietic signals directly in this setting. Rather, these results indicate that iron consumption for erythropoiesis modulates liver iron content, and ultimately BMP6 and hepcidin. Analysis of the BMP6/SMAD pathway targets showed that inhibitor of DNA binding 1 (ID1) and SMAD7, but not transmembrane serine protease 6 (TMPRSS6), were up-regulated by increased iron availability and thus may be involved in setting the upper limit of hepcidin. CONCLUSION: We provide evidence that under conditions of excessive and effective erythropoiesis, liver iron regulates hepcidin expression through the BMP6/SMAD pathway.

Iron Absorption: Molecular and Pathophysiological Aspects.

Iron is an essential nutrient for growth among all branches of life, but while iron is among the most common elements, bioavailable iron is a relatively scarce nutrient. Since iron is fundamental for several biological processes, iron deficiency can be deleterious. On the other hand, excess iron may lead to cell and tissue damage. Consequently, iron balance is strictly regulated. As iron excretion is not physiologically controlled, systemic iron homeostasis is maintained at the level of absorption, which is mainly influenced by the amount of iron stores and the level of erythropoietic activity, the major iron consumer. Here, we outline recent advances that increased our understanding of the molecular aspects of iron absorption. Moreover, we examine the impact of these recent insights on dietary strategies for maintaining iron balance.

Evidence of synergistic/additive effects of sildenafil and erythropoietin in enhancing survival and migration of hypoxic endothelial cells.

Endothelial cell dysfunction is a common event to several pathologies including pulmonary hypertension, which is often associated with hypoxia. As the endothelium plays an essential role in regulating the dynamic interaction between pulmonary vasodilatation and vasoconstriction, this cell type is fundamental in the development of vascular remodeling and increased vascular resistance. We investigated the protective effects of sildenafil, a phosphodiesterase type 5 inhibitor, given in combination with erythropoietin (Epo), as it has been demonstrated that both drugs have antiapoptotic effects on several cell types. Specifically, we examined the viability and angiogenic properties of rat pulmonary artery endothelial cells upon exposure to either 21% or 1% oxygen, in presence of sildenafil (1 and 100 nM) and Epo (5 and 20 U/ml) alone or in combination (1 nM and 20 U/ml). Cell proliferation and viability were analyzed by Trypan blue staining, MTT assay, and Annexin V/propidium iodide stainings. In all assays, the ability of the combination treatment in improving cell viability was superior to that of either drug alone. The angiogenic properties were studied using a migration and a 3D collagen assay, and the results revealed increases in the migration potential of endothelial cells as well as the ability to form tube-like structures in response to sildenafil and the combination treatment. We therefore conclude that both drugs exert protective effects on endothelial cells on hypoxia and that sildenafil enhances the migratory and angiogenic properties, especially in hypoxic conditions. Furthermore, we present evidence of possible additive or synergistic effects of both drugs.

The Emerging Role of Tumor Microenvironmental Stimuli in Regulating Metabolic Rewiring of Liver Cancer Stem Cells.

Primary liver cancer (PLC) is one of the most devastating cancers worldwide. Extensive phenotypical and functional heterogeneity is a cardinal hallmark of cancer, including PLC, and is related to the cancer stem cell (CSC) concept. CSCs are responsible for tumor growth, progression, relapse and resistance to conventional therapies. Metabolic reprogramming represents an emerging hallmark of cancer. Cancer cells, including CSCs, are very plastic and possess the dynamic ability to constantly shift between different metabolic states depending on various intrinsic and extrinsic stimuli, therefore amplifying the complexity of understanding tumor heterogeneity. Besides the well-known Warburg effect, several other metabolic pathways including lipids and iron metabolism are altered in PLC. An increasing number of studies supports the role of the surrounding tumor microenvironment (TME) in the metabolic control of liver CSCs. In this review, we discuss the complex metabolic rewiring affecting liver cancer cells and, in particular, liver CSCs. Moreover, we highlight the role of TME cellular and noncellular components in regulating liver CSC metabolic plasticity. Deciphering the specific mechanisms regulating liver CSC-TME metabolic interplay could be very helpful with respect to the development of more effective and innovative combinatorial therapies for PLC treatment.

Unconventional endocytosis and trafficking of transferrin receptor induced by iron.

The posttranslational regulation of transferrin receptor (TfR1) is largely unknown. We investigated whether iron availability affects TfR1 endocytic cycle and protein stability in HepG2 hepatoma cells exposed to ferric ammonium citrate (FAC). NH4Cl and bafilomycin A1, but not the proteasomal inhibitor MG132, prevented the FAC-mediated decrease in TfR1 protein levels, thus indicating lysosomal involvement. Knockdown experiments showed that TfR1 lysosomal degradation is independent of 1) endocytosis mediated by the clathrin adaptor AP2; 2) Tf, which was suggested to facilitate TfR1 internalization; 3) H-ferritin; and 4) MARCH8, previously implicated in TfR1 degradation. Notably, FAC decreased the number of TfR1 molecules at the cell surface and increased the Tf endocytic rate. Colocalization experiments confirmed that, upon FAC treatment, TfR1 was endocytosed in an AP2- and Tf-independent pathway and trafficked to the lysosome for degradation. This unconventional endocytic regulatory mechanism aimed at reducing surface TfR1 may represent an additional posttranslational control to prevent iron overload. Our results show that iron is a key regulator of the trafficking of TfR1, which has been widely used to study endocytosis, often not considering its function in iron homeostasis.

Adenosine A(2)A receptor but not HIF-1 mediates Tyrosine hydroxylase induction in hypoxic PC12 cells.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamines released by oxygen-sensitive cells in response to hypoxic conditions. Adenosine is released in response to hypoxia in the central nervous system and CGS21680, an adenosine A(2)A receptor agonist, induces TH transcription. As we have previously demonstrated the A(2)A receptor-mediated induction of HIF-1 in macrophages and hepatocytes, we investigated the involvement of HIF-1 in the adenosine-mediated activation of TH expression. Exposure to adenosine or CGS21680 increased TH mRNA and protein levels in PC12 cells. Transcription of a reporter gene under the control of the wild type rat TH promoter was induced 3.5-fold in CGS21680-treated cells, but neither the mutation of the hypoxia responsive element in the TH promoter nor the co-transfection of a dominant negative of the HIF-1 beta subunit prevented the increase in transcription; furthermore, CGS21680 increased CREB binding activity but did not induce HIF-1 DNA binding activity or protein levels. To investigate whether HIF-1 was involved in the hypoxia-mediated induction of TH, PC12 cells were exposed to hypoxia in the presence of the A(2)A receptor antagonist ZM241385, which prevented hypoxia-dependent TH induction despite HIF-1 activation; in line with this finding, the inhibition of HIF-1 did not abolish TH induction in hypoxic PC12 cells. These results indicate that, under hypoxic conditions, TH (a key factor in systemic adaptation to reduced oxygen availability) is not regulated by HIF-1, the primary modulator of the response to hypoxia, but by the adenosine A(2)A receptor-mediated signalling pathway.

Adenosine-dependent activation of hypoxia-inducible factor-1 induces late preconditioning in liver cells.

UNLABELLED: The cellular mechanisms by which ischemic preconditioning increases liver tolerance to ischemia/reperfusion injury are still poorly understood. This study investigated the role of the hypoxia-inducible factor-1 (HIF-1) in the protection associated with the late phase of liver preconditioning. Late preconditioning was induced in primary cultured rat hepatocytes by a transient (10 minute) hypoxic stress or by 15 minutes incubation with the adenosine A(2A) receptors agonist CGS21680 24 hours before exposure to 90 minutes of hypoxia in a serum-free medium. Late preconditioning induced the nuclear translocation of HIF-1 and the expression of carbonic anhydrase IX (CAIX), a HIF-1-regulated transmembrane enzyme that catalyzes bicarbonate production. Such effects were associated with prevention of hepatocyte killing by hypoxia and the amelioration of intracellular acidosis and Na+ accumulation. The inhibition of PKC-mediated and PI3-kinase-mediated signals with, respectively, chelerythrine and wortmannin abolished HIF-1 activation and blocked both CAIX expression and the protective action of late preconditioning. CAIX expression was also prevented by interfering with the transcriptional activity of HIF-1 using a dominant negative HIF-1beta subunit. The inhibition of CAIX with acetazolamide or the block of bicarbonate influx with disodium-4-acetamido-4'-isothiocyanato-stilben-2,2'-disulfonate also reverted the protective effects of late preconditioning on intracellular acidosis and Na+ accumulation. CONCLUSION: The stimulation of adenosine A(2A) receptors induced late preconditioning in liver cells through the activation of HIF-1. HIF-1-induced expression of CAIX increases hepatocyte tolerance to ischemia by maintaining intracellular Na+ homeostasis. These observations along with the importance of HIF-1 in regulating cell survival indicates HIF-1 activation as a possible key event in liver protection by late preconditioning.

Dysregulation of iron metabolism in cancer stem cells.

Cancer stem cells (CSCs) are a distinct subpopulation of tumor cells endowed with stem-like properties. Importantly, CSCs can survive current standard therapies, resulting in metastatic disease and tumor recurrence. Here we describe the alterations of iron homeostasis occurring in CSCs, which in general are characterized by high intracellular iron content. Importantly, abnormalities of iron metabolism correlate with faster tumor growth and adverse prognosis in cancer patients. In line with the dependence of cancer on iron, we also discuss iron-dependent mechanisms as druggable pathways, as iron chelators have been considered for tumor therapy and new molecules currently proposed and studied as antineoplastic drugs may impinge on iron and its capacity to promote oxidative stress to have therapeutic value in cancer.

Ironing out Macrophage Immunometabolism.

Over the last decade, increasing evidence has reinforced the key role of metabolic reprogramming in macrophage activation. In addition to supporting the specific immune response of different subsets of macrophages, intracellular metabolic pathways also directly control the specialized effector functions of immune cells. In this context, iron metabolism has been recognized as an important component of macrophage plasticity. Since macrophages control the availability of this essential metal, changes in the expression of genes coding for the major proteins of iron metabolism may result in different iron availability for the macrophage itself and for other cells in the microenvironment. In this review, we discuss how macrophage iron can also play a role in immunometabolism.

Iron Metabolism in Liver Cancer Stem Cells.

Cancer stem cells (CSC) which have been identified in several tumors, including liver cancer, represent a particular subpopulation of tumor cells characterized by properties similar to those of adult stem cells. Importantly, CSC are resistant to standard therapies, thereby leading to metastatic dissemination and tumor relapse. Given the increasing evidence that iron homeostasis is deregulated in cancer, here we describe the iron homeostasis alterations in cancer cells, particularly in liver CSC. We also discuss two paradoxically opposite iron manipulation-strategies for tumor therapy based either on iron chelation or iron overload-mediated oxidant production leading to ferroptosis. A better understanding of iron metabolism modifications occurring in hepatic tumors and particularly in liver CSC cells may offer new therapeutic options for this cancer, which is characterized by increasing incidence and unfavorable prognosis.

Indirect down-regulation of nuclear NF-kappaB levels by cobalamin in the spinal cord and liver of the rat.

We used electrophoretic mobility shift assays to investigate the effects of cobalamin (Cbl) deficiency on the levels of activated nuclear factor-kappa B (NF-kappaB) in the spinal cords (SCs) and livers of rats made Cbl-deficient (Cbl-D) by total gastrectomy or a Cbl-D diet. We chose the SC and liver because they are severely or scarcely affected, respectively, by Cbl deficiency in terms of histological damage. We found permanently increased NF-kappaB levels (particularly the p50 and p65 subunits) in the SCs and livers of both types of Cbl-D rats, and Western blot analysis demonstrated increased p65 levels. NF-kappaB and p65 protein levels normalized when the totally gastrectomized (TGX) rats were treated with Cbl replacement. As we have previously demonstrated that Cbl deficiency increases tumor necrosis factor (TNF)-alpha and nerve growth factor (NGF) levels in the SC (each of which is a known NF-kappaB activator), we redetermined NF-kappaB levels in the SCs and livers of TGX rats treated with anti-TNF-alpha or anti-NGF antibodies and found that NF-kappaB levels normalized in both tissues after either treatment. These results demonstrate that: (1) Cbl physiologically and indirectly down-regulates NF-kappaB levels in rat SC and liver, and (2) NF-kappaB is an important signaling molecule after Cbl deficiency injury.

Cobalamin deficiency-induced down-regulation of p75-immunoreactive cell levels in rat central nervous system.

We investigated immunoreactivity for p75 neurotrophin receptor (NTR) in the spinal cord white matter and septum of rats made cobalamin-deficient (Cbl-D) by means of total gastrectomy or a Cbl-D diet. Cbl deficiency down-regulates p75NTR-immunoreactive cell levels in spinal cord white matter and septum with different time courses. On the whole, the spinal cord white matter seems to be more affected in terms of p75NTR-immunoreactive cells, most of which are astrocytes. The p75NTR-immunoreactive cell levels in the spinal cord white matter and septum normalized in rats treated with Cbl (scheme b) and killed 4 months after total gastrectomy. However, Western blot analysis of p75NTR in the spinal cords of Cbl-D rats shows increased p75NTR protein levels, which are resistant to Cbl replacement. These findings demonstrate that a neurotrophic vitamin (Cbl) positively regulates the levels of a neurotrophic receptor (p75NTR) (at least in terms of immunohistochemistry) in rat central nervous system, although the underlying mechanism(s) are still unknown.