Induction Of Ferritin Synthesis Research Articles

Induction of ferritin synthesis by water deficit and iron excess in common bean (Phaseolus vulgaris L.)

Ferritins are molecules for iron storage present in most living beings. In plants, ferritin is an essential iron homeostasis regulator and therefore plays a fundamental role in control of iron induced by oxidative stress or by excess of iron ions. Ferritin gene expression is modulated by various environmental factors, including the intensity of drought, cold, light and pathogenic attack. Common bean, one of the most important species in the Brazilian diet, is also affected by insufficiency or lack of water. Thus, the present study was conducted for the purpose of determining the levels of expression of ferritins transcripts in leaf tissues of three common bean cultivars (BAT 477, Carioca Comum and IAC-Diplomata) under osmotic shock caused by polyethylene glycol 6000 and by iron excess. The expression of three ferritins genes (PvFer1, PvFer2 and PvFer3), determined by quantitative PCR, indicated a difference in the expression kinetics among the cultivars. All the ferritin genes were actively transcribed under iron excess and water deficit conditions. The cultivars most responsive to treatments were BAT 477 and IAC-Diplomata. All the cultivars responded to treatments. Nevertheless, the ferritin genes were differentially regulated according to the cultivars. Analysis of variance indicated differences among cultivars in expression of the genes PvFer1 and PvFer3. Both genes were most responsive to treatments. This result suggests that ferritin genes may be functionally important in acclimatization of common bean under iron excess or water deficit conditions.

Heme oxygenase-1: A cellular Hercules

Heme oxygenase-1: A cellular Hercules

Endogenous ferritin protects cells with iron-laden lysosomes against oxidative stress

Previous studies have shown that a variety of mammalian cell types, including macrophages, contain small amounts of redox-active iron in their lysosomes. Increases in the level of this iron pool predispose the cell to oxidative stress. Limiting the availability of intralysosomal redox-active iron could therefore represent potential cytoprotection for cells under oxidative stress.In the present study we have shown that an initial 6 h exposure of J774 macrophages to 30 μM iron, added to the culture medium as FeCl3, increased the lysosomal iron content and their sensitivity to H2O2-induced (0.25 mM for 30 min) oxidative stress. Over time (24–72 h), however, the cells were desensitized to the cytotoxic effects of H2O2; most likely as a consequence of both lysosomal iron exocytosis and of ferritin synthesis (demonstrated by atomic absorption spectrophotometry, autometallography, and immunohistochemistry). When the cells were exposed to a second dose of iron, their lysosomal content of iron increased again but the cells became no further sensitized to the cytotoxic effects of H2O2. Using the lysosomotropic weak base, acridine orange, we demonstrated that after the second exposure to iron and H2O2, lysosomes remained intact and were no different from control cells which were exposed to H2O2 but not iron.These data suggest that the initial induction of ferritin synthesis leads to enrichment of lysosomes with ferritin via autophagocytosis. This limits the redox-availability of intralysosomal iron and, in turn, decreases the cells' sensitivity to oxidative stress. These in vitro observations could also explain why cells under pathological conditions, such as haemochromatosis, are apparently able to withstand high iron concentrations for some time in vivo.

Induction of ferritin synthesis in ischemic-reperfused rat liver: Analysis of the molecular mechanisms

BACKGROUND & AIMS: Iron may catalyze the production of reactive oxygen species (ROS) during postischemic reoxygenation. Ferritin, a cellular iron storage protein, can either represent a source of iron or perform a cytoprotective action against ROS. The aim of this study was to address the role of ferritin in postischemic reperfusion. METHODS: Transcriptional and posttranscriptional mechanisms controlling ferritin gene expression were studied in reperfused rat livers. RESULTS: Proteolysis reduced ferritin levels 2 hours after reperfusion, but a concomitant increase of synthesis, accompanied by enhanced transcription and accumulation of H and L ferritin subunit messenger RNAs (mRNAs), almost re-established normal ferritin content at 4 hours. Pretreatment with interleukin 1 receptor antagonist (IL-1RA) did not prevent the rise of ferritin mRNAs. RNA bandshift assays showed that the activity of the iron regulatory proteins (IRPs), which control ferritin mRNA translation, declined early after reperfusion and recovered progressively thereafter. Pretreatment with either the antioxidant N-acetyl cysteine or IL-1RA was sufficient to prevent almost completely down-regulation of IRP activity. CONCLUSIONS: Postischemic reperfusion causes degradation of ferritin, possibly increasing iron levels. However, induction of ferritin gene transcription, possibly mediated by ferritin-derived iron and ROS- mediated inactivation of IRP, which allows translation of ferritin mRNAs, counteracts this effect and concurs to reestablish the amount of ferritin, which may thus act to limit reperfusion damage. (Gastroenterology 1997 Sep;113(3):946-53)

Mobilization of iron from urban particulates leads to generation of reactive oxygen species in vitro and induction of ferritin synthesis in human lung epithelial cells.

Many of the biochemical effects of asbestos in cultured cells have been shown to be due to iron, which can be as high as 27% by weight. Urban air particulates also contain iron, and some of the pathological effects after inhalation may be due to reactive oxygen species produced by iron-catalyzed reactions. Two standard reference material (SRM) urban air particulate samples were used for the studies described here. SRM 1648 (3.9% iron by weight) was collected in the St. Louis, MO, area, and SRM 1649 (3% iron by weight) was collected in the Washington, DC, area. To determine if iron associated with urban particulates could be mobilized, as it is from asbestos, SRMs 1648 and 1649 were incubated with 1 mM citrate or EDTA, in the presence or absence of ascorbate. Iron was mobilized from both particulates by either chelator, especially in the presence of ascorbate. Citrate, in the presence of ascorbate, mobilized 30.9 nmol of Fe/mg of SRM 1648 and 65.1 nmol of Fe/mg of SRM 1649 in 24 h. EDTA, in the presence of ascorbate, mobilized 53.8 nmol of Fe/mg of SRM 1648 and 98.8 nmol of Fe/mg of SRM 1649 in 24 h. To determine whether reactive oxygen species were being produced by the particulate iron, each particulate was incubated with phi X174 RFI DNA in the presence or absence of ascorbate. Single-strand breaks (SSBs) were produced by either particulate, but only in the presence of ascorbate. Incubation of SRM 1648 or 1649 (0.5 mg/mL) with DNA in the presence of ascorbate and citrate resulted in 20% or 34% DNA with SSBs, respectively. Incubation of SRM 1648 or 1649 (0.1 mg/mL) with DNA in the presence of ascorbate and EDTA resulted in 26% or 45% DNA with SSBs, respectively. To determine if iron associated with urban particulates could be mobilized by human lung epithelial cells (A549), cells were treated with particulates and the amount of the iron storage protein ferritin was determined at the end of treatment. The 6.4- or 8.4-fold increase in ferritin observed in cells treated with SRM 1648 or 1649, respectively, over that of control (untreated) cells strongly suggested that iron was mobilized in the cultured cells. If similar mobilization and reactivity of the iron occurs in the lung, this may explain some of the pathological effects of urban particulates.

Induction of Ferritin Synthesis in Human Lung Epithelial Cells Treated with Crocidolite Asbestos

Crocidolite asbestos is a known human carcinogen containing 27% iron by weight. It has previously been shown that iron was mobilized intracellularly from crocidolite after treatment of human lung epithelial cells (A549) and that the toxicity of the fibers was directly related to how much mobilized iron was in the <10,000 MW (low-molecular-weight, LMW) fraction [C. C. Chao, L. G. Lund, K. R. Zinn, and A. E. Aust (1994)Arch. Biochem. Biophys.314, 384–391]. The data here show that iron mobilization from crocidolite began immediately after treatment of the A549 cells and increased linearly with time. However, the synthesis of ferritin, an iron storage protein, did not begin until after 4 h of treatment, reaching a sustained maximum after 12 h. Mobilized iron was preferentially incorporated into the nonferritin-protein fraction up to 7 h after treatment, when the amount of iron mobilized was low and before significant accumulation of newly synthesized ferritin had occurred. This suggested that these cultured cells needed additional iron for synthesis of iron-requiring proteins and that iron mobilized from crocidolite could be utilized directly for this purpose. Subsequent to this, additional mobilized iron was incorporated into newly synthesized ferritin. Even though iron from crocidolite was incorporated into newly synthesized ferritin or into other proteins, the amount of iron from crocidolite in the LMW fraction remained constant during the 24 h. Thus, it appeared that synthesis of ferritin may not have fully protected the cells from the toxic effects of iron mobilized from crocidolite.

On the Cytoprotective Role of Ferritin in Macrophages and its Ability to Enhance Lysosomal Stability

Macrophages have a great capacity to take up (eg. by endocytosis and phagocytosis) exogenous sources of iron which could potentially become cytotoxic, particularly following the intralysosomal formation of low-molecular weight, redox active iron, and under conditions of oxidative stress. Following autophago-cytosis of endogenous ferritin/apoferritin, these compounds may serve as chelators of such lysosomal iron and counteract the occurrence of iron-mediated intralysosomal oxidative reactions. Such redox-reactions have been shown to lead to destabilisation of lysosomal membranes and result in leakage of damaging lysosomal contents to the cytosol. In this study we have shown: (i) human monocyte-derived macrophages to accumulate ferritin in response to iron exposure; (ii) iron to destabilise macrophage secondary lysosomes when the cells are exposed to H2O2; and (iii) endocytosed apoferritin to act as a stabiliser of the acidic vacuolar compartment of iron-loaded macrophages. While the endogenous ferritin accumulation which was induced by iron exposure was not sufficient to protect cells from the damaging effects of H2O2, exogenously added apoferritin, as well as the potent iron chelator desferrioxamine, afforded significant protection. It is suggested that intralysosomal formation of haemosiderin, from partially degraded ferritin, is a protective strategy to suppress intralysosomal iron-catalysed redox reactions. However, under conditions of severe macrophage lysosomal iron-overload, induction of ferritin synthesis is not enough to completely prevent the enhanced cytotoxic effects of H2O2.

Induction of Ferritin Synthesis in Cells Infected with Mengo Virus

We have recently identified ferritin as a cellular protein particle whose synthesis is stimulated in mouse or human cells infected by the picornavirus Mengo. Immunoprecipitation of the particle from infected murine L929 cells showed a 4- and 6-fold increase in the intracellular concentrations of H and L apoferritin subunits, respectively. This differential expression altered the H/L subunit ratio from 3.0 in uninfected cells to 2.2 in Mengo virus-infected cells. The induction is not due to an increase in transcription of the apoferritin L and H genes, nor is it due to an increase in stability of the apoferritin mRNAs. At the level of translation, the iron regulatory protein (IRP) remained intact, with similar amounts being detected in uninfected and infected cells. The Mengo virus RNA genome does not compete with the iron regulatory element (IRE) for the binding of IRP, and sequence analysis confirmed that there are no IREs in the virus RNA. The IRE binding activity of IRP in infected cells decreased approximately 30% compared with uninfected cells. The decrease in binding activity could be overcome by the addition of Desferal (deferoxamine mesylate; CIBA) an intracellular iron chelator, which suggests that virus infection causes an increase in intracellular free iron. Electron paramagnetic resonance (EPR) studies have confirmed the increase in free iron in Mengo virus infected cells. The permeability of cells for iron does not change in virus infected cells, suggesting that the induction of ferritin by Mengo virus is due to a change in the form of intracellular iron from a bound to a free state.

Induction of ferritin synthesis in maize leaves by an iron‐mediated oxidative stress

Oxygen is essential for aerobic life, although reactive oxygen intermediates can be highly toxic to cells. Superoxide radicals and hydrogen peroxide become particularly harmful in vivo due to their reactivity with transition metals like iron, producing hydroxyl radicals, which are involved in DNA mutagenesis and lipid peroxidation. Control of the intracellular concentration of transition metals can prevent hydroxyl radical formation, protecting cells against oxidative damage. The iron storage protein ferritin plays an important role in these processes, and it has been shown that its synthesis is inducible by iron‐overload in plants. In this paper, the induction of ferritin synthesis in response to iron‐overload is investigated using de‐rooted maize plantlets. In this system, a rapid increase of iron concentration in the leaves is observed, leading to ferritin mRNA and subunit accumulation. Using the vp2 maize mutant, it is demonstrated that abscisic acid is not involved in this response, in contrast to previous results obtained using plants induced under hydroponic culture. In de‐rooted plantlets, ferritin transcript accumulation is inhibited when plants are co‐treated by iron and anti‐oxidant reagents like N‐acetyl cysteine or reduced glutathione. Furthermore, hydrogen peroxide treatment induced a dose‐dependent ferritin mRNA accumulation at a low concentration of iron. It can be concluded from these results that reactive oxygen intermediates are involved in the pathway leading to ferritin synthesis in response to iron in de‐rooted maize plantlets. Ferritin emerges, therefore, as an important component of the oxidative stress response in plants.

Cellular and molecular aspects of iron metabolism in plants

Iron is an essential element for plant metabolism because of its redox properties. Its long distance and intracellular trafficking require specialized proteins and low molecular mass chelates because of its insolubility and toxicity in presence of oxygen. Iron deficiency induces various morphological and biochemical changes. They include root hair morphogenesis, differentiation of rhizodermal cells into transfer cells, yellowing of leaves and ultrastructural disorganisation of chloroplasts and mitochondria, as well as increased synthesis of organic acids and phenolics, and activation of root systems responsible for an enhanced iron uptake capacity. Upon iron resupply, these alterations disappeared within few days and a transient accumulation of the iron storage protein ferritin in the plastids is one of the early events in this process. Iron excess can also occur in plants where it elicits an oxidative stress leading to necrotic spots in the leaves. Induction of ferritin synthesis is again an early event of the plant response to this iron toxicity. Plant hormones such as auxin, abscisic acid and ethylene, as well as reactive oxygen intermediates play an important role in the transduction pathways, allowing plants to respond to these iron-deficiency and excess stresses. Similarities and differences among the various mechanisms responsible for iron uptake and storage in mammals, higher plants and yeast are outlined. Relationships between iron and copper metabolism are also indicated.

Regulation of Ferritin Levels in Cultured Lens Epithelial Cells

In most eukaryotic cells, synthesis of the iron storage protein, ferritin is regulated by iron levels and redox conditions. Proper iron storage is important to protect against damaging iron-catalysed free radical reactions. Although iron-catalysed reactions are believed to contribute to oxidative damage and cataractogenesis, little is known about iron storage in the lens. In this study, ferritin concentration was measured in cultured canine lens epithelial cells. Baseline ferritin concentration ranged from 76-163 ng (mg protein)-1; cells cultured in low-iron media had significantly lower ferritin levels than cells cultured in iron-supplemented media. Addition of a large excess of iron as hemin resulted in an eight-fold increase in ferritin concentration. The iron chelator, Desferal, significantly decreased ferritin concentration. The reducing agent dithiothreitol decreased the hemin-induced increase in ferritin levels, but not baseline levels. In contrast, ascorbic acid induced a large increase in ferritin content. Other studies have shown that induction of ferritin synthesis can protect against oxidative damage. Regulation of ferritin levels may represent a mechanism by which the lens epithelium is protected from oxidative damage. In vivo, epithelial cells are normally exposed to much lower iron concentrations than the cultured lens epithelial cells in this study. However, in pathological circumstances, the iron content and redox state of the aqueous humor is dramatically altered and may affect the steady state levels of ferritin within the lens. This remains to be determined.

Irreversible steps in the ferritin synthesis induction pathway.

The ability of cells to re-repress ferritin synthesis after removal of an inducing agent (iron or heme) was investigated. Re-repression was found to be a slow process, requiring approximately 4 (after iron removal) to 10 h (after heme removal) for completion. Desferrioxamine mesylate (Desferal) had only a slight effect on the rate of re-repression, whereas cycloheximide was strongly inhibitory, indicating that new protein synthesis is required for re-repression. Re-repression occurred at a slow but significant rate in the presence of both Desferal and cycloheximide. These results indicate that, in the absence of an iron chelator, the induction of ferritin synthesis is essentially irreversible. The kinetics of the previously reported covalent modification of IRE-binding protein (IRE-BP) were then examined, to see whether this phenomenon might account (at least in part) for the irreversibility of induction. It was found that the heme- or iron-dependent disappearance of 98-kDa IRE-BP occurred rapidly (within 1 h), and was equally rapidly reversed upon removal of heme after a 1-h exposure. By contrast, after a 4-h exposure to heme, little 98-kDa IRE-BP could be regenerated after heme removal. These results suggest that the slow, irreversible covalent modification of IRE-BP correlates closely over time with the induction of ferritin synthesis. The covalent modification of IRE-BP depends on cell growth rate, and is most readily detected in rapidly growing cells.

Enhanced degradation of the ferritin repressor protein during induction of ferritin messenger RNA translation.

Induction of ferritin synthesis in cultured cells by heme or iron is accompanied by degradation of the ferritin repressor protein (FRP). Intermediates in the degradative pathway apparently include FRP covalently linked in larger aggregates. The effect of iron on FRP degradation is enhanced by porphyrin precursors but is decreased by inhibitors of porphyrin synthesis, which implies that heme is an active agent. These results suggest that translational induction in this system may be caused by enhanced repressor degradation. While unique among translational regulatory systems, this process is common to a variety of other biosynthetic control mechanisms.

Induction of Ferritin Synthesis in vitro: Problems of Specificity Inherent in the Use of Iron Compounds

The potential importance of heme as a translational regulator has been recognized for nearly 30 years. However, the ability to distinguish the specific regulatory effects of heme from the nonspecific effects has been hampered by its high reactivity and its tendency to generate reactive by-products in most systems. In this article we discuss some of the technical difficulties in studying the effects of iron salts and compounds, notably heme, in biochemical systems in vitro. Data are presented which show that the nonspecific inhibitory effects of heme on two restriction endonucleases can be eliminated by (a) including a redox buffer in the reaction mixture, and (b) maintaining a sufficiently high total protein concentration. Under these conditions, the specific effects of hemin on the ferritin repressor protein are still observed. A possible relationship between these observations and the status of 'free' heme in vivo is considered.

Thyrotropin stimulates transcription from the ferritin heavy chain promoter

Thyrotropin (TSH) is the primary hormone regulating the activity of the thyroid gland. We have recently shown that TSH stimulates H-ferritin mRNA levels in rat thyroid. Ferritin plays a key role in determining the intracellular fate of iron. The induction of ferritin synthesis by iron in liver is regulated both at transcriptional and translational levels. Here we present evidence that the mechanisms by which TSH regulates the mRNA levels are mediated by a diffusible product acting in trans on its own promoter. In fact, the H-ferritin promoter mediates increased CAT activity in response to hormone induction. Our results identify transcription as an important regulatory step of TSH action. They suggest that TSH induces expression of the ferritin gene, and that continuous protein synthesis is required to maintain basal ferritin gene expression in the absence of hormone.

Iron induction of ferritin synthesis and secretion in human hepatoma cell (Hep G2) cultures

In order to determine if iron was able to stimulate specifically ferritin synthesis and secretion in transformed human hepatocytes in culture, human hepatoma cell (HepG2) cultures were submitted to increasing doses of ferric nitrilotriacetate. Iron uptake by the cells was demonstrated by incorporation of 59 Fe and the staining method of Perls. The following results were obtained: 1. iron incorporation within the hepatocytes increased as a function of culture time; 2. during the first 24 h of treatment, ferritin synthesis increased progressively, in parallel to the iron uptake; 3. a dose-dependent significant stimulation of ferritin synthesis and secretion were observed when the medium iron concentration increased from 5 to 20 mumol/l; 4. albumin, transthyretin and transferrin secretions were unaffected. These data demonstrated that, in our hepatocyte culture model, iron load increased the expression of ferritin in a highly specific manner.

Iron induction of ferritin synthesis and secretion in a human hepatoblastoma cellline (HepG2)

Iron induction of ferritin synthesis and secretion in a human hepatoblastoma cellline (HepG2)

Regulation of ferritin mRNA: a possible gene-sparing phenomenon. Induction of ferritin synthesis by iron in liver as well as red cells combines high translational efficiency with increased utilization of preformed ferritin mRNA.

Control of ferritin synthesis by iron at the level of transcription is potentially hazardous to DNA because of the iron-catalyzed degradation of DNA. The induction of ferritin synthesis in reticulocytes of embryos (bullfrog tadpoles) occurs by two types of translational control i.e. increased availability of stored ferritin mRNA, in response to iron, coupled with a high translational efficiency. Since erythroid cell nuclei have large amounts of heterochromatin and may be relatively inactive genetically, the translational control of ferritin by iron observed in red cells was studied in other tissue by isolating poly (A+) RNA from tadpole liver and analyzing protein synthesis in vitro. Liver ferritin mRNA directed the synthesis of 7.0% of the protein in a wheat germ system, compared to 1.2% in vivo, suggesting that tadpole liver contained a large amount of stored ferritin mRNA. At levels of poly (A+) RNA which were saturating for total protein synthesis, ferritin synthesis was still linearly dependent upon RNA concentration, indicating a high efficiency of translation of ferritin mRNA. The results are analogous to those previously observed in red cells and confirm the storage of ferritin mRNA deduced from studies of the polysomal and nonpolysomal distribution of the mRNA in rat liver. The results indicate that the increased availability for translation of stored ferritin mRNA, in response to iron, and the high translational efficiency of ferritin mRNA are a general characteristic of ferritin synthesis rather than a specific feature of red cell maturation. This novel form of regulation of ferritin gene expression can be attributed to a need to protect DNA from degradation by iron and oxygen. The normal barrier between DNA and iron is apparently breached by the iron-oxygen complex of the drug bleomycin, an antitumor agent thought to act in vivo by iron-catalyzed cleavage of DNA.

The induction of ferritin synthesis in circulating larval red blood cells.

The circulating red blood cells formed in bullfrog larvae, chicken embryos, and mouse embryos contain large amounts of ferritin and storage iron in excess of the need for hemoglobin. In contrast, the circulating red cells of adult animals contain little ferritin. Ferritin synthesis and iron storage are coordinated with differentiation and hemoglobin synthesis in the red cells of adults. In order to test the hypothesis that ferritin synthesis could be controlled independently of hemoglobin synthesis and differentiation in the red cells formed early in life, bullfrog larvae were injected with iron to determine if ferritin synthesis was increased in the circulating red cells. Within 17 h after the injection of iron, the synthesis of ferritin, assayed as the incorporation of [14C]leucine by cell suspensions prepared from circulating red cells, was increased from 2.9 to 10.2% of the total protein, and the specific activity of the ferritin synthesized increased from 1100 to 3000 cpm/A280. There was no change in the hematocrit of the animals nor in the specific activity of hemoglobin synthesized by suspensions of red cells (average, 720 cpm/A280). The results suggest that in mature, larval red cells, ferritin synthesis can be controlled by changes in the extracellular environment. The results also indicate that ferritin synthesis can be controlled independently of hemoglobin synthesis with which it is coordinated during erythroid differentiation in adult animals.

FERRITIN SYNTHESIS IN HUMAN EMBRYONIC AND FETAL TISSUES

Ferritin synthesis by various tissues of the human conceptus was examined in the present study using tissue cultures. Normal embryonic and fetal tissues were obtained from 21 spontaneously or therapeutically aborted conceptuses of 29 days to 21 weeks of gestation. The tissues were cultured in the presence of 14C-amino acids and the culture fluids were examined for synthesized radioactive ferritin by means of immunodiffusion and autoradiography. Ferritin synthesis was detected in the earliest embryo studied, 29 days of gestation; this embryo was too small to permit culture of individual tissues and all tissues were cultured together. Ferritin synthesis by the liver and yolk sac was clearly established at the earliest stages in which these organs were cultured separately, 4.5 and 5.5 weeks of gestation, respectively. Synthesis of ferritin by bone marrow was detectable in some embryos at 8.5 weeks and was established in this tissue and in the spleen by 11 weeks. The chronological development of ferritin synthesis from liver and yolk sac to bone marrow and then spleen approximates the ontogenetic development of hematopoiesis. Ferritin synthesis was noted in the stomach and duodenum at 8.5 weeks, but was not well established at these sites until 21 weeks, suggesting possible induction of ferritin synthesis in the latter organs through fetal swallowing of amniotic fluid containing transferrin iron.