Ferritin synthesis in rat L-6 cells: response to iron challenge

The short term response of the L-6 cell line of rat skeletal myoblasts to elevated extracellular iron concentrations was studied. It was found in all cases that iron as the nitrilotriacetate (NTA) chelate was effective at donating iron to the cells and at stimulating ferritin synthesis. After 48 h in 50 microM ferric NTA, the cellular ferritin levels rose from an undetectable level to 1.11 (+/- 0.07) ng ferritin/microgram cell protein, or 0.1% of total cell protein. Similarly, the total iron in the cells rose under the same conditions from an unmeasurable level to plateau at over 10 fmol iron/cell. In addition, it was found that these cells synthesize ferritin in response to iron in a dose-dependent manner over a range of iron concentrations from 5-1000 microM. A sensitive and specific immunoradiometric assay for rat ferritin was used in these studies to quantitate ferritin in cell lysates.     

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.     

Correlation between levels of ferritin and the iron-containing component of ribonucleotide reductase in hydroxyurea-sensitive, -resistant, and -revertant cell lines.

The reduction of ribonucleotides to deoxyribonucleotides, a rate-limiting step in DNA synthesis, is catalyzed by ribonucleotide reductase. This enzyme is composed of two components, M1 and M2. Recent work has shown that inhibition of ribonucleotide reductase by the antitumor drug hydroxyurea leads to a destabilized iron centre in protein M2. We have examined the relationship between the levels of ferritin, the iron storage protein, and the iron-containing M2 component of ribonucleotide reductase. These studies were carried out with hydroxyurea-sensitive, -resistant, and -revertant cell lines. Hydroxyurea-resistant mouse L cells contained M2 gene amplification and elevated levels of enzyme activity, M2 message, and total cellular M2 protein concentration. Hydroxyurea-revertant cells exhibited a wild-type M2 gene copy number, and approximately wild-type levels of enzyme activity, M2 message, and M2 protein concentration. In addition, we observed that the hydroxyurea-resistant cells possessed elevated levels of L-chain ferritin message and total cellular H-chain ferritin protein when compared to wild-type cells. In contrast, the revertant cell population contained approximately wild-type levels of ferritin mRNA and protein. In keeping with these observations, obtained with mouse L cells, was the finding that hydroxyurea-resistant Chinese hamster ovary cells with increased ribonucleotide reductase activity exhibited elevated expression of both ferritin and M2 genes, which declined in drug-sensitive revertant hamster cell lines with decreased levels of ribonucleotide reductase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of extracellular hemin on hemoglobin and ferritin content of erythroleukemia cells

Mouse (MEL) and human (K-562) erythroleukemia cell lines can be induced to undergo erythroid differentiation, including hemoglobin (Hb) synthesis, by extra cellular hemin. In order to study the effect of extracellular hemin on intracellular ferritin and Hb content, we have used Mossabauer spectroscopy to measure the amount of 57Fe incorporated into ferritin or Hb and a fluorescent enzyme-linked immunosorbent assay (ELISA) to measure the ferritin protein content. When K-562 cells were cultured in the presence of a 57Fe source either as transferrin or citrate, in the absence of a differentiation inducer, all the intracellular 57Fe was detected in ferritin. When the cells were cultured in the presence of 57Fe-hemin, 57Fe was found in both ferritin and Hb. 57Fe in ferritin increased rapidly, and after 2 days it reached a plateau at 5 X 10(-14) g/cell. 57Fe in Hb increased linearly with time and reached the same value after 12 days. Addition of other iron sources such as iron-saturated transferrin, iron citrate, or iron ammonium citrate caused a much lower increase in ferritin protein content as compared to hemin. When K-562 cells were induced by 57Fe-hemin in the presence of 56Fe-transferrin, 57Fe was found to be incorporated in equal amounts into both ferritin and Hb. However, when the cells were induced by 56Fe-hemin in the presence of 57Fe-transferrin, 57Fe was incorporated only into ferritin, but not into Hb, which contained 56Fe iron. These results indicate that in K-562 cells, when hemin is present in the culture medium it is preferentially incorporated into Hb, regardless of the availability of other extra- or intracellular iron sources such as transferrin or ferritin. In MEL cells induced to differentiate by dimethylsulfoxide (DMSO) a different pattern of iron incorporation was observed; 57Fe from both transferrin and hemin was found to incorporate in ferritin as well as in Hb.     

Ferritin levels in microglia depend upon activation: modulation by reactive oxygen species

Iron is one of the trace elements playing a key role in the normal cellular metabolism. Since an excess of free iron is catalyzing the Fenton reaction, most of the intracellular iron is sequestered in the iron storage protein ferritin. The binding of iron into ferritin is well described for physiological conditions, however, under certain pathophysiological situations, the efficiency of this process is unknown. In the brain, microglial cells are among others the cell population most importantly responsible for the maintenance of the extracellular environment. These cells might undergo activation, and little is known about the expression of ferritin during activation of microglial cells. Therefore, we tested the microglial model cell line RAW264.7 for the expression of ferritin after LPS activation. A significant decrease in the levels of the ferritin H-chain during activation and a significant increase in the early recovery phase were found. We were able to demonstrate that reactive oxygen species are responsible for a suppression of the H-chain of ferritin, whereas iNOS expression and NO synthesis are counteracting the reactive oxygen species effect. The balance of reactive oxygen species and NO production are, therefore, determining expression levels of the ferritin H-chain during activation of microglial cells.     

Effects of interferon-gamma and lipopolysaccharide on macrophage iron metabolism are mediated by nitric oxide-induced degradation of iron regulatory protein 2

Iron regulatory proteins (IRP-1 and IRP-2) control the synthesis of transferrin receptors (TfR) and ferritin by binding to iron-responsive elements, which are located in the 3'-untranslated region and the 5'-untranslated region of their respective mRNAs. Cellular iron levels affect binding of IRPs to iron-responsive elements and consequently expression of TfR and ferritin. Moreover, NO(*), a redox species of nitric oxide that interacts primarily with iron, can activate IRP-1 RNA binding activity resulting in an increase in TfR mRNA levels. Recently we found that treatment of RAW 264.7 cells (a murine macrophage cell line) with NO(+) (nitrosonium ion, which causes S-nitrosylation of thiol groups) resulted in a rapid decrease in RNA binding of IRP-2 followed by IRP-2 degradation, and these changes were associated with a decrease in TfR mRNA levels (Kim, S., and Ponka, P. (1999) J. Biol. Chem. 274, 33035-33042). In this study, we demonstrated that stimulation of RAW 264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) increased IRP-1 binding activity, whereas RNA binding of IRP-2 decreased and was followed by a degradation of this protein. Moreover, the decrease of IRP-2 binding/protein levels was associated with a decrease in TfR mRNA levels in LPS/IFN-gamma-treated cells, and these changes were prevented by inhibitors of inducible nitric oxide synthase. Furthermore, LPS/IFN-gamma-stimulated RAW 264.7 cells showed increased rates of ferritin synthesis. These results suggest that NO(+)-mediated degradation of IRP-2 plays a major role in iron metabolism during inflammation.     

Increased ferritin gene expression is associated with increased ribonucleotide reductase gene expression and the establishment of hydroxyurea resistance in mammalian cells

In the present study, we show that hydroxyurea-inactivated ribonucleotide reductase protein M2 has a destabilized iron center, which readily releases iron. In addition, evidence is presented which indicates that single or multistep selection for hydroxyurea resistance, in a variety of mammalian cell lines, leads to alterations in the expression of the gene for the iron storage protein, ferritin. In all hydroxyurea-resistant cell lines examined, including human, hamster, rat, and mouse, there was an elevation in ferritin heavy (H)- and/or light (L)-mRNA levels, but no change in the corresponding gene copy number. A detailed analysis of ferritin expression in a hydroxyurea-resistant mouse L cell line showed that when compared to its wild type counterpart, there was an increase in H subunit concentration but no significant change in L subunit levels. The increased H/L subunit ratio was not brought about by specific changes in the rates of ferritin subunit biosynthesis, but rather resulted from changes in the post-translational stability of H subunits relative to L subunits in the resistant cell line compared to its parental wild type. Also, we show that treatment of cells with hydroxyurea results in an increased rate of ferritin biosynthesis in the absence of changes in H- or L-mRNA levels. These results indicate that the development of even low level hydroxyurea resistance in mammalian cells may require alterations in ferritin gene expression, and they show an interesting relationship between the expressions of two highly regulated activities, ribonucleotide reductase and ferritin.     

Ferritin is not a required intermediate for iron utilization in heme synthesis

Pulse-chase analysis of newt (Triturus cristatus) erythroblasts has shown that ferritin is not a primary source of iron for heme synthesis. During chase incubation with and without non-radioactive plasma iron in the medium, no transfer of 59Fe from ferritin to hemoglobin was detected although the integrity of heme synthesis was maintained. In puromycin-inhibited cells where iron uptake was drastically curtailed, heme synthesis continued to occur, though at reduced levels; incorporation of 59Fe from the plasma appeared initially in heme and hemoglobin without any prior labelling of ferritin. These results indicate that ferritin is neither an obligatory iron intermediate in heme synthesis nor a cytosolic transport molecule involved in mobilization of iron from the transferrin-receptor complex. The most likely role for erythroid ferritin is storage of excess iron.     

Decoupling ferritin synthesis from free cytosolic iron results in ferritin secretion

Ferritin is a multisubunit protein that is responsible for storing and detoxifying cytosolic iron. Ferritin can be found in serum but is relatively iron poor. Serum ferritin occurs in iron overload disorders, in inflammation, and in the genetic disorder hyperferritinemia with cataracts. We show that ferritin secretion results when cellular ferritin synthesis occurs in the relative absence of free cytosolic iron. In yeast and mammalian cells, newly synthesized ferritin monomers can be translocated into the endoplasmic reticulum and transits through the secretory apparatus. Ferritin chains can be translocated into the endoplasmic reticulum in an in?vitro translation and membrane insertion system. The insertion of ferritin monomers into the ER occurs under low-free-iron conditions, as iron will induce the assembly of ferritin. Secretion of ferritin chains provides a mechanism that limits ferritin nanocage assembly and ferritin-mediated iron sequestration in the absence of the translational inhibition of ferritin synthesis.     

Effect of actinomycin D, cycloheximide, and acute blood loss on ferritin synthesis in rat liver

1. The mechanism of the stimulation of ferritin synthesis by iron in vivo has been studied in rat liver. Ferritin synthesis and turnover was measured by [(14)C]leucine incorporation. 2. Actinomycin D had no inhibitory effect, after administration of iron, on [(14)C]leucine incorporation into ferritin but appeared to augment the effect of iron on ferritin synthesis. 3. Cycloheximide completely abolished the stimulation by iron of [(14)C]leucine into ferritin and was subsequently utilized to show that iron acts in vivo by translational induction of apoferritin synthesis, rather than by stabilization of apoferritin or its precursors. 4. This conclusion was confirmed by showing that 2 days after acute bleeding, when iron was in the process of being removed from hepatic ferritin stores, ferritin synthesis was decreased whereas breakdown rates were unchanged.     

Tumor necrosis factor-alpha and interleukin 1-alpha regulate transferrin receptor in human diploid fibroblasts. Relationship to the induction of ferritin heavy chain

We have studied transferrin receptor expression in MRC5 human fibroblasts in response to tumor necrosis factor-alpha (TNF, cachectin) or interleukin 1-alpha (IL-1). Treatment of exponentially growing MRC5 cells with these cytokines led to a 3-4-fold increase in transferrin receptor mRNA and a coordinate increase in transferrin receptor protein by 24 h. Under these conditions, stimulation of [3H]thymidine incorporation was minimal, suggesting that the induction of transferrin receptor by TNF and IL-1 is mediated by a growth-independent regulatory mechanism. A study of the time course of this response showed that cytokine-mediated increases in transferrin receptor mRNA and protein proceeded after a lag of 12-24 h. A simultaneous analysis of the effects of TNF and IL-1 on ferritin in MRC5 cells was also performed. Ferritin L mRNA levels were unchanged. However, induction of ferritin H mRNA was seen within 4 h, preceding the induction of the transferrin receptor. The synthesis of ferritin H (but not ferritin L) protein peaked at 8 h after TNF or IL-1 treatment, followed by a rapid decrease in both ferritin H and L protein synthesis. As ferritin H synthesis declined, levels of transferrin receptor protein increased, reaching a maximum by 24 h. These results suggest that the cytokine-dependent induction of ferritin H and subsequent increase in the transferrin receptor are related and possibly interdependent events. This study demonstrates that the complex role of TNF and IL-1 in iron homeostasis includes modulation of the transferrin receptor.     

Induction of programmed cell death in a dorsal root ganglia × neuroblastoma cell line

Growth factor-dependent neurons die when they are deproved of their specific growth factor. This “programmed” cell death (PCD) requires macromolecular synthesis and is distinct from necrotic cell death. To investigate the mechanisms involved in neuronal PCD, we have studied the sequence of events that occur when a neuronal cell line (F-11: Mouse neuroblastoma X rat dorsal root ganglia) is deprived of serum in a manner analogous to growth factor deprivation from neurons. Protein synthesis was inhibited within the first 8 h of serum deprivation, while DNA cleavage into nucleosome ladders was prominent by 24 h. The DNA cleavage could be inhibited by cycloheximide, consistent with a requirement for protein synthesis. In contrast, mitochondrial function was not compromised by serum deprivation. Rather, the cells appeared to be metabolically activated after serum removal as shown by an increased reduction of MTT by mitochondrial dehydrogenases and an increase in cellular autofluorescence, which is thought to be due to elevated levels of NADH and flavoproteins. Assessment of cell viability by propidium iodide staining showed no indication of cell death within 24 h. After 48 h of serum deprivation, cells decreased in size and increased propidium iodide uptake. Thus, serum deprivation activates PCD in F-11 cells and may be a useful model to study the intracellular events responsible for PCD. © 1993 John Wiley & Sons, Inc.     

Oxidation-induced ferritin turnover in microglial cells: role of proteasome

Highly oxidized protein aggregates accumulating in the brain during neurodegenerative diseases are often surrounded by microglia. Most of the microglial cells surrounding these plaques are activated and release a high amount of oxidizing species. In order to develop their toxic effects numerous oxidizing species need iron. To prevent this iron-dependent oxidation an iron-sequestering apparatus exists, including the major iron storage protein ferritin. Microglial cells damage their own protein pool during activation and it is still unknown whether microglial cells are able to maintain their iron-sequestering function during oxidative stress. Therefore, we explored the microglial cell line RAW to test the maintenance of ferritin under oxidizing conditions. Our investigations revealed a half-life of both ferritin chains of 3-3.5 h and a reduced half-life due to oxidation. This was due to the removal of oxidized ferritin by the proteasomal system. Ferritin de novo synthesis was also severely affected by oxidation. This results in a decreased ferritin pool due to acute oxidative stress. These data let us conclude that microglial cells do not increase their ferritin amount after oxidative stress and an increase in the iron storage capacity in these cells after treatment might be achieved only by a high iron saturation of the existing ferritin molecules.     

Hypoxia Alters Iron Homeostasis and Induces Ferritin Synthesis in Oligodendrocytes

Abstract: Both iron and the major iron-binding protein ferritin are enriched in oligodendrocytes compared with astrocytes and neurons, but their functional role remains to be determined. Progressive hypoxia dramatically induces the synthesis of ferritin in both neonatal rat oligodendrocytes and a human oligodendroglioma cell line. We now report that the release of iron from either transferrin or ferritin-bound iron, after a decrease in intracellular pH, also leads to the induction of ferritin synthesis. The hypoxic induction of ferritin synthesis can be blocked either with iron chelators (deferoxamine or phenanthroline) or by preventing intracellular acidification (which is required for the release of transferrin-bound iron) with weak base treatment (ammonium chloride and amantadine). Two sources of exogenous iron (hemin and ferric ammonium citrate) were able to stimulate ferritin synthesis in both oligodendrocytes and HOG in the absence of hypoxia. This was not additive to the hypoxic stimulation, suggesting a common mechanism. We also show that ferritin induction may require intracellular free radical formation because hypoxia-mediated ferritin synthesis can be further enhanced by cotreatment with hydrogen peroxide. This in turn was blocked by the addition of exogenous catalase to the culture medium. Our data suggest that disruption of intracellular free iron homeostasis is an early event in hypoxic oligodendrocytes and that ferritin may serve as an iron sequestrator and antioxidant to protect cells from subsequent iron-catalyzed lipid peroxidation injury.