Induction Of Ferritin Research Articles

Iron cellular uptake from almond and coconut beverages using an in vitro intestinal cell model

Plant-based beverages could contain iron but also phenolic compounds (PC) and ascorbic acid, which are considered modulators of iron uptake. We investigated whether iron from almond and coconut beverages can be taken up by Caco-2 cells. The iron content in almond and coconut beverages was 19.20 and 19.34 mg kg−1 dry weight (dw), respectively; PC were 727.71 and 673.11 mg gallic acid equivalents kg−1, respectively; and ascorbic acid 10.96 mg kg−1 and 24.44 mg∙kg−1, respectively. We observed an increase in iron uptake by induction of cellular ferritin at concentrations of 15 and 30 g L−1 (almond) and 30 g L−1 (coconut). Both beverages significantly increased ferritin induction when iron sulfate was added. Thus, these beverages represent iron sources which can either contribute directly to iron supply or indirectly by enhancing absorption of exogenous iron and contribute to decrease the impact of diseases considered public health problems.

Airway Epithelial Cells Differentially Adapt Their Iron Metabolism to Infection With Klebsiella pneumoniae and Escherichia coli In Vitro.

BackgroundPneumonia is often elicited by bacteria and can be associated with a severe clinical course, respiratory failure and the need for mechanical ventilation. In the alveolus, type-2-alveolar-epithelial-cells (AECII) contribute to innate immune functions. We hypothesized that AECII actively adapt cellular iron homeostasis to restrict this essential nutrient from invading pathogens – a defense strategy termed ‘nutritional immunity’, hitherto mainly demonstrated for myeloid cells.MethodsWe established an in-vitro infection model using the human AECII-like cell line A549. We infected cells with Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli), two gram-negative bacteria with different modes of infection and frequent causes of hospital-acquired pneumonia. We followed the entry and intracellular growth of these gram-negative bacteria and analyzed differential gene expression and protein levels of key inflammatory and iron metabolism molecules.ResultsBoth, K. pneumoniae and E. coli are able to invade A549 cells, whereas only K. pneumoniae is capable of proliferating intracellularly. After peak bacterial burden, the number of intracellular pathogens declines, suggesting that epithelial cells initiate antimicrobial immune effector pathways to combat bacterial proliferation. The extracellular pathogen E. coli induces an iron retention phenotype in A549 cells, mainly characterized by the downregulation of the pivotal iron exporter ferroportin, the upregulation of the iron importer transferrin-receptor-1 and corresponding induction of the iron storage protein ferritin. In contrast, cells infected with the facultative intracellular bacterium K. pneumoniae exhibit an iron export phenotype indicated by ferroportin upregulation. This differential regulation of iron homeostasis and the pathogen-specific inflammatory reaction is likely mediated by oxidative stress.ConclusionAECII-derived A549 cells show pathogen-specific innate immune functions and adapt their iron handling in response to infection. The differential regulation of iron transporters depends on the preferential intra- or extracellular localization of the pathogen and likely aims at limiting bacterial iron availability.

Heme Burden and Ensuing Mechanisms That Protect the Kidney: Insights from Bench and Bedside.

With iron at its core, the tetrapyrrole heme ring is a cardinal prosthetic group made up of many proteins that participate in a wide array of cellular functions and metabolism. Once released, due to its pro-oxidant properties, free heme in sufficient amounts can result in injurious effects to the kidney and other organs. Heme oxygenase-1 (HO-1) has evolved to promptly attend to such injurious potential by facilitating degradation of heme into equimolar amounts of carbon monoxide, iron, and biliverdin. HO-1 induction is a beneficial response to tissue injury in diverse animal models of diseases, including those that affect the kidney. These protective attributes are mainly due to: (i) prompt degradation of heme leading to restraining potential hazardous effects of free heme, and (ii) generation of byproducts that along with induction of ferritin have proven beneficial in a number of pathological conditions. This review will focus on describing clinical aspects of some of the conditions with the unifying end-result of increased heme burden and will discuss the molecular mechanisms that ensue to protect the kidneys.

Bioavailability of iron from novel hydrogen reduced iron powders: Studies in Caco-2 cells and rat model.

The bioavailability of iron from elemental iron powders, including hydrogen reduced iron powder (HRIP), is influenced by particle size and surface area. In the present study, we investigated the solubility, bioaccessibility, and bioavailability of iron from novel HRIPs (particle size≤25 and 38µmgenerated at low [LT] and high [HT] temperature), with porous morphology and high surface area, in intestinal Caco-2 cells and in rat models. The acceptability of fortified wheat flour was tested in human volunteers. The iron solubility and ferritin induction in Caco-2 cells were significantly higher from wheat flour fortified with HRIPs compared to electrolytic iron powder (EIP,≤45µmsize) either in the absence or presence of ascorbic acid. Nevertheless, ferritin induction in Caco-2 cells was significantly higher with FeSO4 compared to HRIP or EIP. The relative biological value of HRIPs was significantly higher (≤38HT) or similar compared to EIP in rats. However, serum ferritin was significantly higher in rats fed HRIPs than EIP. Further, wheat flour fortified with HRIP was found to be acceptable for consumption. These findings demonstrate higher iron bioavailability from novel HRIPs compared to the reference EIP (≤45 µm) and merits further studies on toxicity and efficacy. PRACTICAL APPLICATION: The use of elemental iron powders for food fortification to alleviate iron deficiency is limited due to its poor bioavailability. The novel hydrogen-reduced elemental iron powders used in this study had higher bioaccessibility and bioavailability compared to reference EIP (≤45µm) in in vitro and in vivo models, respectively. Further, there were no sensory differences between roti prepared with fortified or unfortified wheat flour. These results suggest that the novel hydrogen reduced elemental iron powders used in the present study are suitable for wheat flour fortification.

Heme cytotoxicity is the consequence of endoplasmic reticulum stress in atherosclerotic plaque progression

Hemorrhage and hemolysis with subsequent heme release are implicated in many pathologies. Endothelial cells (ECs) encounter large amount of free heme after hemolysis and are at risk of damage from exogenous heme. Here we show that hemorrhage aggravates endoplasmic reticulum (ER) stress in human carotid artery plaques compared to healthy controls or atheromas without hemorrhage as demonstrated by RNA sequencing and immunohistochemistry. In EC cultures, heme also induces ER stress. In contrast, if cultured ECs are pulsed with heme arginate, cells become resistant to heme-induced ER (HIER) stress that is associated with heme oxygenase-1 (HO-1) and ferritin induction. Knocking down HO-1, HO-2, biliverdin reductase, and ferritin show that HO-1 is the ultimate cytoprotectant in acute HIER stress. Carbon monoxide-releasing molecules (CORMs) but not bilirubin protects cultured ECs from HIER stress via HO-1 induction, at least in part. Knocking down HO-1 aggravates heme-induced cell death that cannot be counterbalanced with any known cell death inhibitors. We conclude that endothelium and perhaps other cell types can be protected from HIER stress by induction of HO-1, and heme-induced cell death occurs via HIER stress that is potentially involved in the pathogenesis of diverse pathologies with hemolysis and hemorrhage including atherosclerosis.

Iron loss triggers mitophagy through induction of mitochondrial ferritin.

Mitochondrial quality is controlled by the selective removal of damaged mitochondria through mitophagy. Mitophagy impairment is associated with aging and many pathological conditions. An iron loss induced by iron chelator triggers mitophagy by a yet unknown mechanism. This type of mitophagy may have therapeutic potential, since iron chelators are clinically used. Here, we aimed to clarify the mechanisms by which iron loss induces mitophagy. Deferiprone, an iron chelator, treatment resulted in the increased expression of mitochondrial ferritin (FTMT) and the localization of FTMT precursor on the mitochondrial outer membrane. Specific protein 1 and its regulator hypoxia-inducible factor 1α were necessary for deferiprone-induced increase in FTMT. FTMT specifically interacted with nuclear receptor coactivator 4, an autophagic cargo receptor. Deferiprone-induced mitophagy occurred selectively for depolarized mitochondria. Additionally, deferiprone suppressed the development of hepatocellular carcinoma (HCC) in mice by inducing mitophagy. Silencing FTMT abrogated deferiprone-induced mitophagy and suppression of HCC. These results demonstrate the mechanisms by which iron loss induces mitophagy and provide a rationale for targeting mitophagic activation as a therapeutic strategy.

The development of a novel ferric phytate compound for iron fortification of bouillons (part I)

In a series of two studies, we report the development (this study) and evaluation (part II) of a novel ferric phytate compound designed as a condiment iron fortificant. Condiments are used as iron fortification vehicles to reduce the prevalence of iron deficiency. The challenge for iron fortificants in e.g. a bouillon matrix is to avoid undesired sensory effects and to ensure a reasonable cost. We added phytic acid to chelate iron, and hydrolysed protein to counteract the inhibiting effect of phytic acid on iron bioaccessibility. We characterised four novel ferric phytate compounds, destabilised by hydrolysed plant protein or amino acids. Colour stability of fortified bouillons with ferric phytate compounds was superior to bouillons fortified with ferrous sulfate. The iron-phytate-hydrolysed corn protein compound (Fe-PA-HCP) resulted in highest cellular ferritin induction in Caco-2 cells, in both vegetable (36.1 ± 13.40 ng/mg protein) and chicken (73.9 ± 19.93 ng/mg protein) bouillon matrices as observed in the human Caco-2/HepG2 cell model. Iron uptake (as estimated by ferritin production) from the Fe-PA-HCP compound was about 55% (chicken bouillon) and 66% (vegetable bouillon) of the iron uptake from ferrous sulfate. Based on this study, the Fe-PA-HCP compound was chosen for further evaluation (part II).

Deferoxamine deconditioning increases neuronal vulnerability to hemoglobin

Concomitant treatment with deferoxamine (DFO) protects neural cells from iron and heme-mediated oxidative injury, but also disrupts cell responses to iron loading that may be protective. We hypothesized that DFO treatment and withdrawal would subsequently increase neuronal vulnerability to hemoglobin. Pretreatment with DFO followed by its washout increased neuronal loss after subsequent hemoglobin exposure by 3-4-fold compared with control vehicle-pretreated cultures. This was associated with reduced ferritin induction by hemoglobin; expression of heme oxygenase-1, which catalyzes iron release from heme, was not altered. Increased neuronal loss was prevented by exogenous apoferritin or by continuing DFO or antioxidants throughout the experimental course. Cell nonheme iron levels after hemoglobin treatment were similar in DFO-pretreated and control cultures. These results indicate that DFO deconditions neurons and subsequently increases their vulnerability to heme-mediated injury. Its net effect after CNS hemorrhage may be highly dependent on the timing and duration of its administration. Withdrawal of DFO while heme or iron levels remain elevated may be deleterious, and may negate any benefit of prior concomitant therapy.

Time-dependent hemeoxygenase-1, lipocalin-2 and ferritin induction after non-contusion traumatic brain injury

Traumatic brain injury (TBI) often presents with focal contusion and parenchymal bleeds, activating heme oxygenase (HO) to degrade released hemoglobin. Here we show that diffuse, midline fluid percussion injury causes time-dependent induction of HO-1 and iron binding proteins within both hemorrhagic neocortex and non-hemorrhagic hippocampus. Rats subjected to midline fluid percussion injury (FPI) survived 1-15d postinjury and tissue was collected for Western blot and immunohistochemical assays. HO-1 was elevated 1d after FPI, peaked at 3d, and returned to control baseline 7-15d. Iron management proteins lipocalin 2 (LCN2) and ferritin (FTL) exhibited distinct postinjury time courses, where peak LCN2 response preceded, and FTL followed that of HO-1. LCN2 elevation supported not only its role in iron transport, but also mediation of matrix metalloproteinase 9 (MMP9) activity. Upregulation of FTL for intracellular iron sequestration was delayed relative to both HO-1 and LCN2 induction. In the neocortex IBA-1+ microglia around the injury core expressed HO-1, but astrocytes co-localized with HO-1 in perilesional parenchyma. Non-hemorrhagic dentate gyrus showed predominant HO-1 labeling in hilar microglia and in molecular layer astrocytes. At 1d postinjury, LCN2 and HO-1 co-localized in a subpopulation of reactive glia within both brain regions. Notably, FTL was distributed within cells around injured vessels, damaged subcortical white matter, and along vessels of the hippocampal fissure. Together these results confirm that even the moderate, non-contusional insult of diffuse midline FPI can significantly activate postinjury HO-1 heme processing pathways and iron management proteins. Moreover, this activation is time-dependent and occurs in the absence of overt hemorrhage.

Crosstalk Between Oxidative Stress and Endoplasmic Reticulum (ER) Stress in Endothelial Dysfunction and Aberrant Angiogenesis Associated With Diabetes: A Focus on the Protective Roles of Heme Oxygenase (HO)-1.

Type-2 diabetes prevalence is continuing to rise worldwide due to physical inactivity and obesity epidemic. Diabetes and fluctuations of blood sugar are related to multiple micro- and macrovascular complications, that are attributed to oxidative stress, endoplasmic reticulum (ER) activation and inflammatory processes, which lead to endothelial dysfunction characterized, among other features, by reduced availability of nitric oxide (NO) and aberrant angiogenic capacity. Several enzymatic anti-oxidant and anti-inflammatory agents have been found to play protective roles against oxidative stress and its downstream signaling pathways. Of particular interest, heme oxygenase (HO) isoforms, specifically HO-1, have attracted much attention as major cytoprotective players in conditions associated with inflammation and oxidative stress. HO operates as a key rate-limiting enzyme in the process of degradation of the iron-containing molecule, heme, yielding the following byproducts: carbon monoxide (CO), iron, and biliverdin. Because HO-1 induction was linked to pro-oxidant states, it has been regarded as a marker of oxidative stress; however, accumulating evidence has established multiple cytoprotective roles of the enzyme in metabolic and cardiovascular disorders. The cytoprotective effects of HO-1 depend on several cellular mechanisms including the generation of bilirubin, an anti-oxidant molecule, from the degradation of heme; the induction of ferritin, a strong chelator of free iron; and the release of CO, that displays multiple anti-inflammatory and anti-apoptotic actions. The current review article describes the major molecular mechanisms contributing to endothelial dysfunction and altered angiogenesis in diabetes with a special focus on the interplay between oxidative stress and ER stress response. The review summarizes the key cytoprotective roles of HO-1 against hyperglycemia-induced endothelial dysfunction and aberrant angiogenesis and discusses the major underlying cellular mechanisms associated with its protective effects.

Transferrin receptor 1 controls systemic iron homeostasis by fine-tuning hepcidin expression to hepatocellular iron load

AbstractTransferrin receptor 1 (Tfr1) mediates uptake of circulating transferrin-bound iron to developing erythroid cells and other cell types. Its critical physiological function is highlighted by the embryonic lethal phenotype of Tfr1-knockout (Tfrc−/−) mice and the pathologies of several tissue-specific knockouts. We generated TfrcAlb-Cre mice bearing hepatocyte-specific ablation of Tfr1 to explore implications in hepatocellular and systemic iron homeostasis. TfrcAlb-Cre mice are viable and do not display any apparent liver pathology. Nevertheless, their liver iron content (LIC) is lower compared with that of control Tfrcfl/fl littermates as a result of the reduced capacity of Tfr1-deficient hepatocytes to internalize iron from transferrin. Even though liver Hamp messenger RNA (mRNA) and serum hepcidin levels do not differ between TfrcAlb-Cre and Tfrcfl/fl mice, Hamp/LIC and hepcidin/LIC ratios are significantly higher in the former. Importantly, this is accompanied by modest hypoferremia and microcytosis, and it predisposes TfrcAlb-Cre mice to iron-deficiency anemia. TfrcAlb-Cre mice appropriately regulate Hamp expression following dietary iron manipulations or holo-transferrin injection. Holo-transferrin also triggers proper induction of Hamp mRNA, ferritin, and Tfr2 in primary TfrcAlb-Cre hepatocytes. We further show that these cells can acquire 59Fe from 59Fe-transferrin, presumably via Tfr2. We conclude that Tfr1 is redundant for basal hepatocellular iron supply but essential for fine-tuning hepcidin responses according to the iron load of hepatocytes. Our data are consistent with an inhibitory function of Tfr1 on iron signaling to hepcidin via its interaction with Hfe. Moreover, they highlight hepatocellular Tfr1 as a link between cellular and systemic iron-regulatory pathways.

Metabolic Adaptation Establishes Disease Tolerance to Sepsis

SummarySepsis is an often lethal syndrome resulting from maladaptive immune and metabolic responses to infection, compromising host homeostasis. Disease tolerance is a defense strategy against infection that preserves host homeostasis without exerting a direct negative impact on pathogens. Here, we demonstrate that induction of the iron-sequestering ferritin H chain (FTH) in response to polymicrobial infections is critical to establish disease tolerance to sepsis. The protective effect of FTH is exerted via a mechanism that counters iron-driven oxidative inhibition of the liver glucose-6-phosphatase (G6Pase), and in doing so, sustains endogenous glucose production via liver gluconeogenesis. This is required to prevent the development of hypoglycemia that otherwise compromises disease tolerance to sepsis. FTH overexpression or ferritin administration establish disease tolerance therapeutically. In conclusion, disease tolerance to sepsis relies on a crosstalk between adaptive responses controlling iron and glucose metabolism, required to maintain blood glucose within a physiologic range compatible with host survival.

Hyperferritinemia and inflammation.

Understanding of ferritin biology has traditionally centered on its role in iron storage and homeostasis, with low ferritin levels indicative of deficiency and high levels indicative of primary or secondary hemochromatosis. However, further work has shown that iron, redox biology and inflammation are inexorably linked. During infection, increased ferritin levels represent an important host defense mechanism that deprives bacterial growth of iron and protects immune cell function. It may also be protective, limiting the production of free radicals and mediating immunomodulation. Additionally, hyperferritinemia is a key acute-phase reactants, used by clinicians as an indication for therapeutic intervention, aimed at controlling inflammation in high-risk patients. One school of thought maintains that hyperferritinemia is an 'innocent bystander' biomarker of uncontrolled inflammation that can be used to gauge effectiveness of intervention. Other schools of thought maintain that ferritin induction could be a protective negative regulatory loop. Others maintain that ferritin is a key mediator of immune dysregulation, especially in extreme hyperferritinemia, via direct immune-suppressive and pro-inflammatory effects. There is a clear need for further investigation of the role of ferritin in uncontrolled inflammatory conditions both as a biomarker and mediator of disease because its occurrence identifies patients with high mortality risk and its resolution predicts their improved survival.

Nutrigenomic effects of glucosinolates on liver, muscle and distal kidney in parasite-free and salmon louse infected Atlantic salmon.

BackgroundReduction of Lepeophtheirus salmonis infection in Atlantic salmon achieved by glucosinolates (GLs) from Brassica plants was recently reported. However, wider application of functional feeds based on GLs requires better knowledge of their positive and adverse effects.MethodsLiver, distal kidney and muscle transcriptomes of salmon exposed to the extreme dose of GLs were profiled by microarray, while qPCR analysis followed up selected hepatic and renal responses under the extreme and moderate GLs dose during the L. salmonis challenge. Transcriptional analysis were complemented with measurements of organ indices, liver steatosis and plasma profiling, including indicators of cytolysis and bilirubin. Finally, the third trial was performed to quantify the effect of lower GLs doses on growth.ResultsThe extreme GLs dose caused a decrease in hepatic fat deposition and growth, in line with microarray findings, which suggested tissue remodeling and reduction of cellular proliferation in the skeletal muscle and liver. Lower GLs inclusion levels in a follow-up trial did not show negative effects on growth. Microarray analysis of the distal kidney pointed to activation of anti-fibrotic responses under the overexposure. However, analyses of ALT, CK and AST enzymes in plasma provided no evidence of increased cytolysis and organ damage. Prevalent activation of phase-2 detoxification genes that occurred in all three tissues could be considered part of beneficial effects caused by the extreme dose of GLs. In addition, transcriptomic evidence suggested GLs-mediated iron and heme withdrawal response, including increased heme degradation in muscle (upregulation of heme oxygenase-1), decrease of its synthesis in liver (downregulation of porphobilinogen deaminase) and increased iron sequestration from blood (hepatic induction of hepcidin-1 and renal induction of intracellular storage protein ferritin). This response could be advantageous for salmon upon encountering lice, which depend on the host for the provision of iron carrying heme. Most of the hepatic genes studied by qPCR showed similar expression levels in fish exposed to GLs, lice and their combination, while renal induction of leptin suggested heightened stress by the combination of extreme dose of GLs and lice. High expression of interferon γ (cytokine considered organ-protective in mammalian kidney) was detected at the moderate GLs level. This fish also showed highest plasma bilirubin levels (degradation product of heme), and had lowest number of attached lice, further supporting hypothesis that making heme unavailable to lice could be part of an effective anti-parasitic strategy.ConclusionsModulation of detoxification and iron metabolism in Atlantic salmon tissues could be beneficial prior and during lice infestations. Investigation of anti-lice functional feeds based on low and moderate GLs inclusion levels thus deserves further attention.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1921-7) contains supplementary material, which is available to authorized users.

Haptoglobin increases the vulnerability of CD163-expressing neurons to hemoglobin.

Haptoglobin (Hp) binds hemoglobin (Hb) with high affinity and provides the primary defense against its toxicity after intravascular hemolysis. Neurons are exposed to extracellular Hb after CNS hemorrhage, and a therapeutic effect of Hp via Hb sequestration has been hypothesized. In this study, we tested the hypothesis that Hp protects neurons from Hb in primary mixed cortical cell cultures. Treatment with low micromolar concentrations of human Hb for 24h resulted in loss of 10-20% of neurons without injuring glia. Concomitant treatment with Hp surprisingly increased neuronal loss five-sevenfold, with similar results produced by Hp 1-1 and 2-2 phenotypes. Consistent with a recent invivo observation, neurons expressed the CD163 receptor for Hb and the Hb-Hp complex in these cultures. Hp reduced overall Hb uptake, directed it away from the astrocyte-rich CD163-negative glial monolayer, and decreased induction of the iron-binding protein ferritin. Hb-Hp complex neuronal toxicity, like that of Hb per se, was iron-dependent and reduced by deferoxamine and 2,2' bipyridyl. These results suggest that Hp increases the vulnerability of CD163+ neurons to Hb by permitting Hb uptake while attenuating the protective response of ferritin induction by glial cells. Cover Image for this issue: doi: 10.1111/jnc.13342.

The chelation of colonic luminal iron by a unique sodium alginate for the improvement of gastrointestinal health.

Iron is an essential nutrient. However, in animal models, excess unabsorbed dietary iron residing within the colonic lumen has been shown to exacerbate inflammatory bowel disease and intestinal cancer. Therefore, the aims of this study were to screen a panel of alginates to identify a therapeutic that can chelate this pool of iron and thus be beneficial for intestinal health. Using several in vitro intestinal models, it is evident that only one alginate (Manucol LD) of the panel tested was able to inhibit intracellular iron accumulation as assessed by iron-mediated ferritin induction, transferrin receptor expression, intracellular (59) Fe concentrations, and iron flux across a Caco-2 monolayer. Additionally, Manucol LD suppressed iron absorption in mice, which was associated with increased fecal iron levels indicating iron chelation within the gastrointestinal tract. Furthermore, the bioactivity of Manucol LD was found to be highly dependent on both its molecular weight and its unique compositional sequence. Manucol LD could be useful for the chelation of this detrimental pool of unabsorbed iron and it could be fortified in foods to enhance intestinal health.

Iron overload inhibits osteogenic commitment and differentiation of mesenchymal stem cells via the induction of ferritin

Osteogenic differentiation of multipotent mesenchymal stem cells (MSCs) plays a crucial role in bone remodeling. Numerous studies have described the deleterious effect of iron overload on bone density and microarchitecture. Excess iron decreases osteoblast activity, leading to impaired extracellular matrix (ECM) mineralization. Additionally, iron overload facilitates osteoclast differentiation and bone resorption. These processes contribute to iron overload-associated bone loss. In this study we investigated the effect of iron on osteogenic differentiation of human bone marrow MSCs (BMSCs), the third player in bone remodeling.We induced osteogenic differentiation of BMSCs in the presence or absence of iron (0–50μmol/L) and examined ECM mineralization, Ca content of the ECM, mRNA and protein expressions of the osteogenic transcription factor runt-related transcription factor 2 (Runx2), and its targets osteocalcin (OCN) and alkaline phosphatase (ALP). Iron dose-dependently attenuated ECM mineralization and decreased the expressions of Runx2 and OCN. Iron accomplished complete inhibition of osteogenic differentiation of BMSCs at 50μmol/L concentration. We demonstrated that in response to iron BMSCs upregulated the expression of ferritin. Administration of exogenous ferritin mimicked the anti-osteogenic effect of iron, and blocked the upregulation of Runx2, OCN and ALP. Iron overload in mice was associated with elevated ferritin and decreased Runx2 mRNA levels in compact bone osteoprogenitor cells. The inhibitory effect of iron is specific toward osteogenic differentiation of MSCs as neither chondrogenesis nor adipogenesis were influenced by excess iron. We concluded that iron and ferritin specifically inhibit osteogenic commitment and differentiation of BMSCs both in vitro and in vivo.

Iron Overload Exacerbates Busulfan-Melphalan Toxicity Through a Pharmacodynamic Interaction in Mice.

Busulfan-melphalan high-dose chemotherapy followed by autologous stem cell transplantation is an essential consolidation treatment of high-risk neuroblastoma in children. Main treatment limitation is hepatic veno-occlusive disease, the most severe and frequent extra-hematological toxicity. This life threatening toxicity has been related to a drug interaction between busulfan and melphalan which might be increased by prior disturbance of iron homeostasis, i.e. an increased plasma ferritin level. We performed an experimental study of busulfan and melphalan pharmacodynamic and pharmacokinetics in iron overloaded mice. Iron excess dramatically increased the toxicity of melphalan or busulfan melphalan combination in mice but it did not modify the clearance of either busulfan or melphalan. We show that prior busulfan treatment impairs the clearance of melphalan. This clearance alteration was exacerbated in iron overloaded mice demonstrating a pharmacokinetic interaction. Additionally, iron overload increased melphalan toxicity without altering its pharmacokinetics, suggesting a pharmacodynamic interaction between iron and melphalan. Based on iron homeostasis disturbance, we postulated that prior induction of ferritin, through Nrf2 activation after oxidative stress, may be associated with the alteration of melphalan metabolism. Iron overload increases melphalan and busulfan-melphalan toxicity through a pharmacodynamic interaction and reveals a pharmacokinetic drug interaction between busulfan and melphalan.

Ferritin Assembly in Enterocytes of Drosophila melanogaster.

Ferritins are protein nanocages that accumulate inside their cavity thousands of oxidized iron atoms bound to oxygen and phosphates. Both characteristic types of eukaryotic ferritin subunits are present in secreted ferritins from insects, but here dimers between Ferritin 1 Heavy Chain Homolog (Fer1HCH) and Ferritin 2 Light Chain Homolog (Fer2LCH) are further stabilized by disulfide-bridge in the 24-subunit complex. We addressed ferritin assembly and iron loading in vivo using novel transgenic strains of Drosophila melanogaster. We concentrated on the intestine, where the ferritin induction process can be controlled experimentally by dietary iron manipulation. We showed that the expression pattern of Fer2LCH-Gal4 lines recapitulated iron-dependent endogenous expression of the ferritin subunits and used these lines to drive expression from UAS-mCherry-Fer2LCH transgenes. We found that the Gal4-mediated induction of mCherry-Fer2LCH subunits was too slow to effectively introduce them into newly formed ferritin complexes. Endogenous Fer2LCH and Fer1HCH assembled and stored excess dietary iron, instead. In contrast, when flies were genetically manipulated to co-express Fer2LCH and mCherry-Fer2LCH simultaneously, both subunits were incorporated with Fer1HCH in iron-loaded ferritin complexes. Our study provides fresh evidence that, in insects, ferritin assembly and iron loading in vivo are tightly regulated.

Ferritin-Mediated Iron Sequestration Stabilizes Hypoxia-Inducible Factor-1α upon LPS Activation in the Presence of Ample Oxygen.

Both hypoxic and inflammatory conditions activate transcription factors such as hypoxia-inducible factor (HIF)-1α and nuclear factor (NF)-κB, which play a crucial role in adaptive responses to these challenges. In dendritic cells (DC), lipopolysaccharide (LPS)-induced HIF1α accumulation requires NF-κB signaling and promotes inflammatory DC function. The mechanisms that drive LPS-induced HIF1α accumulation under normoxia are unclear. Here, we demonstrate that LPS inhibits prolyl hydroxylase domain enzyme (PHD) activity and thereby blocks HIF1α degradation. Of note, LPS-induced PHD inhibition was neither due to cosubstrate depletion (oxygen or α-ketoglutarate) nor due to increased levels of reactive oxygen species, fumarate, and succinate. Instead, LPS inhibited PHD activity through NF-κB-mediated induction of the iron storage protein ferritin and subsequent decrease of intracellular available iron, a critical cofactor of PHD. Thus, hypoxia and LPS both induce HIF1α accumulation via PHD inhibition but deploy distinct molecular mechanisms (lack of cosubstrate oxygen versus deprivation of co-factor iron).