Ferritin Gene Research Articles

Hemodialysis reduces inhibitory effect of plasma ultrafiltrate on LDL oxidation and subsequent endothelial reactions

Oxidative modification of low-density lipoprotein (LDL) and its deleterious effect on endothelium is implicated in the pathogenesis of atherosclerosis. Endothelium responds to such an insult by upregulating the synthesis of heme oxygenase-1 (HO-1) and ferritin. Endothelial cell damage and dysfunction have been observed in patients with chronic kidney disease (CKD) on maintenance hemodialysis (HD). We studied the effect of low-molecular-weight components of uremic plasma on LDL oxidation and LDL-oxidation-provoked endothelial cell reactions, such as the induction of cytotoxicity and the upregulation of cell-protective HO-1 and ferritin. Plasma ultrafiltrate (molecular weight<5000 Da) from CKD patients on HD or when treated conservatively exhibited a pronounced inhibition on heme-mediated oxidative modification of LDL. Endothelial cell cytotoxicity provoked by LDL oxidation was also attenuated by plasma ultrafiltrate from CKD patients. During HD treatment, a dramatic drop occurred in the retardation of oxidative reactions, and a loss of endothelial cytoprotection exerted by plasma ultrafiltrate was noted. The upregulation of HO-1 and ferritin in response to oxidative stress of LDL was blunted by uremic plasma ultrafiltrate that was released by the end of HD. The decreased antioxidant capacity of ultrafiltrate after HD occurred as a consequence of the intradialytic removal of L-ascorbic acid, uric acid, bilirubin, 3-indoxyl sulfate, indoxyl-beta-D-glucuronide, p-cresol, and phenol. Intradialytic removal of L-ascorbic acid, uric acid, bilirubin, 3-indoxyl sulfate, indoxyl-beta-D-glucuronide, p-cresol, and phenol increases the risk of LDL oxidation and subsequent endothelial cell damage, which underlines the importance of activation of cytoprotective HO-1 and ferritin in endothelium.

Genetic Background of Primary Iron Overload Syndromes in Japan

The different prevalences of iron overload syndromes between Caucasians and Asians may be accounted for by the differences in genetic background. The major mutation of hemochromatosis in Celtic ancestry, C282Y of HFE, was reported in a Japanese patient. Five patients of 3 families with the hepatic transferrin receptor gene (TFR2)-linked hemochromatosis were found in different areas of Japan, suggesting that TFR2 is a major gene in Japanese people. Three patients with mutations in the hemojuvelin gene, HJV, showed also middle-age-onset hemochromatosis. A heterozygous mutation in the H ferritin gene, FTH1, was found in a family of 3 affected patients. Another autosomal dominant SLC40A1-linked hyperferritinemia (ferroportin disease) was found in 3 patients of 2 families. Two patients with hemochromatosis were free from any mutations in the genes investigated. In conclusion, the genetic backgrounds of Japanese patients with primary iron overload syndromes were partially clarified, showing some phenotype-genotype correlations.

Expression of transferrin receptor and ferritin following ferumoxides–protamine sulfate labeling of cells: implications for cellular magnetic resonance imaging

Ferumoxides-protamine sulfate (FE-Pro) complexes are used for intracellular magnetic labeling of cells to non-invasively monitor cell trafficking by in vivo MRI. FE-Pro labeling is non-toxic to cells; however, the effects of FE-Pro labeling on cellular expression of transferrin receptor (TfR-1) and ferritin, proteins involved in iron transport and storage, has not been reported. FE-Pro-labeled human mesenchymal stem cells (MSCs), HeLa cells and primary macrophages were cultured from 1 week to 2 months and evaluated for TfR-1 and ferritin gene expression by RT-PCR and protein levels were determined using Western blots. MTT (proliferation assay) and reactive oxygen species (ROS) analysis were performed. FE-Pro labeling of HeLa and MSCs resulted in a transient decrease in TfR-1 mRNA and protein levels. In contrast, Fe-Pro labeling of primary macrophages resulted in an increase in TfR-1 mRNA but not in TfR-1 protein levels. Ferritin mRNA and protein levels increased transiently in labeled HeLa and macrophages but were sustained in MSCs. No changes in MTT and ROS analysis were noted. In conclusion, FE-Pro labeling elicited physiological changes of iron metabolism or storage, validating the safety of this procedure for cellular tracking by MRI.

Iron at the center of ferritin, metal/oxygen homeostasis and novel dietary strategies

Bioiron - central to respiration, photosynthesis and DNA synthesis and complicated by radical chemistry with oxygen - depends on ferritin, the super family of protein nanocages (maxi-ferritins in humans, animals, plant, and bacteria, and mini-ferritins, also called DPS proteins, in bacteria) for iron and oxygen control. Regulation of ferritin synthesis, best studied in animals, uses DNA transcription and mRNA translation check points. Ferritin is a member of both the "oxidant stress response" gene family that includes thioredoxin reductase and quinine reductase, and a member of the iron responsive gene family that includes ferroportin and mt-aconitase ferritin DNA regulation responds preferentially to oxidant response inducers and ferritin mRNA to iron inducers: heme confers regulator synergy. Ferritin proteins manage iron and oxygen, with ferroxidase sites and iron + oxygen substrates to form mineral of both Fe and O atoms; maxi-ferritins contribute more to cellular iron metabolism and mini-ferritins to stress responses. Iron recovery from ferritin is controlled by gated protein pores, possibly contributing to iron absorption from ferritin, a significant dietary iron source. Ferritin gene regulation is a model for integrating DNA/mRNA controls, while ferritin protein function is central to molecular nutrition cellular metabolism at the crossroads of iron and oxygen in biology.

Cellular Iron Metabolism Studies Demonstate Safety of Magnetic Tracking of Mesenchymal Stem Cells.

Background: Ferumoxides-protamine sulfate (Fe-Pro) complexes are used for intracellular magnetic labeling of mesenchymal stem cells (MSCs) to non invasively monitor cell trafficking and tissue distribution by in vivo magnetic resonance imaging (MRI). It has been previously shown that there was no short or long- term toxic effects on cell's viability, proliferation, reactive oxygen species and biological functions including differentiation capacity of MSCs, however the effects of dextran coated superparamagnetic iron oxide (SPIO) nanoparticles labeling on cellular iron metabolism and storage is unknown.Objective: Evaluate the effect of using Fe-Pro complexes for magnetic cell labeling in MSCs on the expression of transferrin receptor (TfR-1) and ferritin, proteins involved in intracellular iron metabolism and storage.Methods: Confluent MSCs were labeled with Fe-Pro complexes at the ratio of 100 ug/mL to 6 ug/mL. After labeling, cells were washed and cultured for 2 months. TfR-1 and ferritin expression were evaluated at the gene and protein levels in total cell lysates, on day 1, 3, 7, 14, 28 and 35 days post-labeling, by real-time polymerase chain reaction (RT-PCR) and Western blotting. MSCs were evaluated for the presence of iron by Prussian blue stain and light microscopy. In addition, TfR-1 and ferritin levels were determined for Fe-Pro labeled and unlabeled MSCs, allowed to slowly multiply, as a result of dividing the cells in half when they reached confluence.Results: All cells were Prussian blue positive on light microscopy following labeling with Fe-Pro. Labeling of MSCs grown to confluence resulted in short-term increase of TfR-1 mRNA (day 1 and day 3, p= 0.0056) without changes in TfR-1 protein levels. Fe- Pro labeled MSC demonstrated an increase in ferritin gene expression on day 7 and 14 (p=0.0003) compared to unlabeled cells, while protein levels were higher compared to unlabeled MSC at each time point (p=0.005). However, ferritin protein levels from Fe-Pro labeled MSCs were not significantly increased after 7 passages.Conclusions: Magnetic labeling of cells with FDA approved ferumoxides complexed to protamine sulfate elicited short-term changes in cellular iron metabolism and storage that was dependent on ability to divide. Fe-Pro labeling did not have any long-term implications validating the safety of Fe-Pro labeling technique for MSCs trafficking studies by MRI.

Regulation of the ferritin heavy‐chain homologue gene in the yellow fever mosquito, Aedes aegypti

In the yellow fever mosquito Aedes aegypti, the ferritin heavy-chain homologue (HCH) gene is induced by blood feeding. This suggests that ferritin may serve as a cytotoxic protector against the oxidative challenge of the blood meal and may be essential for the survival of the insect. In this study, various cis-acting elements for the gene were identified and mapped. Transfection assays showed that the strength and activity of a subset of these elements are orientation-dependent. The shift observed for the ferritin HCH cis-acting elements is unique among known ferritin genes. DNase I footprinting data together with Transfac analyses identified a number of putative sites known for their involvement in developmental and cell proliferation processes.

Isolation and Expression Pattern Analysis of Two Ferritin Genes in Tobacco

Abstract: For understanding of the ferritin gene expression pattern and the mechanism of iron homeostasis in tobacco (Nicotiana tabaccum, L.) plants, two full‐length ferritin cDNAs, NtFer1, and NtFer2, were isolated from tobacco seedlings and characterized. These cDNAs are 1 214 and 1 125 bp nucleotides and encode 251 and 259 amino acid residues, respectively. The deduced amino acid sequences showed that two tobacco ferritins share the same characteristics as the plant ferritins from Arabidopsis, soybean, and maize. Southern blotting analysis indicated that both NtFer1, and NtFer2, were probably multicopy genes in the tobacco genome. Northern blotting analysis indicated that iron loading of tobacco plantlets increased the ferritin mRNA abundance and that NtFer1, expression was higher and more sensitive to iron than NtFer2 expression. Furthermore, NtFer1, was expressed in both leaves and roots, whereas NtFer2, was expressed mainly in leaves.(Managing editor: Li‐Hui ZHAO)

Yugoslav HD Phenocopies Analyzed on the Presence of Mutations in Prp, Ferritin, and Jp-3 Genes

Huntington disease (HD) is a well-defined autosomal dominant neurodegenerative disease caused by CAG repeat expansions in HD gene. There are a significant number of HD cases where this mutation was not found and such cases are named HD-like phenotype (HDL). This article reports 48 patients with HDL phenotype. Patients were analyzed on the presence of mutations in prion (PrP), ferritin and junctophilin-3 (JP-3) genes. None of the patients showed the presence of the mutation in analyzed genes. This could suggest that there is some other gene/genes where the mutation can cause the disease with clinical features of HD.

Different cytoprotective effect of antioxidants and change in the iron regulatory system in rodent cells exposed to paraquat or formaldehyde

To study the mechanism of toxicity of paraquat and formaldehyde, the response of oxidant-exposed cultured NIH3T3 cells to antioxidants or an iron chelator was investigated. Paraquat-induced cell death was reduced by treatment with 10 microM pyrrolidine dithiocarbamate (PDTC) and 10 microM desferrioxamine (DFO), but not with N-acetyl-L-cysteine (NAC). Cells were protected from formaldehyde-induced cytotoxicity by 1 mM NAC, but not by PDTC or DFO. Moreover, paraquat modulated the cellular iron regulatory system. Paraquat induced a time-dependent increase in the binding of iron regulatory protein 1 (IRP1) to iron-responsive element (IRE), and the enhanced IRP1 activity continued over 24 h. On the other hand, no induction of increased IRP1 binding to IRE was observed in rodent cells exposed to formaldehyde. Previously, we observed stimulation of EpRE-mediated ferritin mRNA expression in the cells exposed to hydrogen peroxide. However, paraquat did not induce any transcriptional activation of ferritin genes. These results suggest that intracellular iron may be involved in paraquat-mediated cytotoxicity and the influence of paraquat on iron metabolism differs from that of hydrogen peroxide.

Identification of Fur regulated genes in the bacterial fish pathogen Photobacterium damselae ssp. piscicida using the Fur titration assay

Bacteria have developed a series of iron-scavenging and transport systems. The expression of many of the iron utilization genes is tightly regulated by the Fe2+ loaded Fur repressor protein. In this study, the Fur titration assay (FURTA) was used to screen for DNA fragments from a genomic DNA library of Photobacterium damselae ssp. piscicida containing potential Fe2+ Fur binding sites or iron binding-proteins which withdraw iron from Fur. One of the clones encoded a tonB gene and adjacent a functionally related exbB gene. An additional and complete tonB exbB exbD gene cluster was identified and sequenced. A gene homologous to the ferritin gene was found whose FURTA-positive phenotype may be explained by its iron-binding ability. Genes encoding a putative complete iron-regulated outer membrane transport protein and a pseudogene of a transport protein were found. The FURTA assay also revealed iron regulation of the AraC type transcriptional regulation.

Transcriptional Regulation and Life-span Modulation of Cytosolic Aconitase and Ferritin Genes in C.elegans

Ferritin is the major iron storage protein regulating cytosolic concentration of iron by storing excess iron. Vertebrate ferritins are heteropolymeric proteins composed of heavy chain and light chain subunits. We have characterized two Caenorhabditis elegans genes (ftn-1 and ftn-2), which encode ferritin homologs showing high degree of similarity to mammalian ferritin heavy chains. Even though these two ferritins are more than 78% identical in amino acid sequence, our data show that expression patterns and responses to iron are quite different. Cytosolic aconitase (aco-1), iron regulatory protein, is known to regulate cellular iron concentration by modulating translation of the ferritin mRNA in addition to its enzymatic activity that converts citrate into iso-citrate. We have shown that the expression levels of aco-1 and ftn-1 genes are both regulated by iron treatment but in opposite ways. Interestingly, mutant animals lacking ACO-1 and FTN-1 show significantly reduced life-span upon iron stress, while N2 and ftn-2 animals show no difference. Our results suggest that ftn-1 and aco-1 are transcriptionally regulated by iron and are important for iron homeostasis affecting life-span upon iron stress conditions in C.elegans.

Transcriptional regulation of the Bacteroides fragilis ferritin gene (ftnA) by redox stress.

This study shows that the iron-storage protein ferritin is a component of the redox-stress response in the obligate anaerobe Bacteroides fragilis. It is up-regulated at transcriptional level under aerobic conditions but constitutively expressed at low levels under anaerobic conditions. Northern hybridization and primer extension analysis revealed that ftnA is transcribed as a monocistronic mRNA of approximately 600 nt. Under reduced anaerobic conditions, ftnA mRNA levels were not dependent on the iron content of the culture medium. Following oxygen exposure ftnA message increased about 10-fold in iron-replete medium compared to a fourfold increase under low-iron conditions. Addition of the oxidant potassium ferricyanide induced expression of ftnA mRNA anaerobically, suggesting that the oxidation of the medium affected expression of ftnA. Two transcription initiation start sites were identified. Both transcripts were expressed constitutively under anaerobic conditions but one promoter was induced by oxidative stress or the addition of the oxidant potassium ferricyanide. The effect of redox stress on ftnA expression was further investigated by addition of diamide, a thiol-oxidizing agent, which induced ftnA mRNA levels anaerobically, suggesting that an unbalanced cellular redox state also affects ftnA expression. Induction by hydrogen peroxide and oxygen was decreased in an oxyR deletion mutant but some oxygen induction still occurred. This strongly suggests that ftnA is regulated by both the peroxide response transcriptional activator, OxyR, and another unidentified oxygen-dependent regulator. Taken together, these data show that ftnA mRNA levels are controlled by both iron and oxidative stress; this coordinated regulation may be important for survival in an adverse aerobic environment.

&lt;I&gt;Ferritin&lt;/I&gt; Gene Coding Sequences Are Conserved Among Eight Hard Tick Species (Ixodida: Ixodidae)

Eight complete cDNAs encoding for ferritins from the hard tick species Amblyomma americanum (L.), Amblyomma maculatum Koch, Boophilus microplus (Canestrini), Dermacentor albipictus (Packard), Dermacentor variabilis (Say), Hemaphysalis longicornis Neumann, Ixodes scapularis Say, and Rhipicephalus sanguineus (Latreille) were obtained using the techniques of rapid amplification of cDNA ends. The deduced amino acid sequences from these tick ferritin genes share high identities (84–98%) with those of two published tick ferritins. The sequences and positions of the iron-responsive element in the 5′-untranslated region of the cDNAs are highly conserved in ticks. Similar to other known animal ferritins, a ferroxidase center, consisting of seven conserved amino acid residues, was identified in the deduced tick ferritins. Sequence comparison indicates that these tick ferritin genes belong to the type of invertebrate cytosolic heavy chain homologue. Two separate phylogenetic analyses of ferritin amino acid sequences, one with 52 arthropod ferritin sequences and another one using 10 tick ferritins, suggested that ferritin is a suitable nuclear protein-encoding gene for tick phylogenetic reconstruction.

Upregulation of the genes for ferritin, RNase, and DnaJ in leaves of rice plants in response to sulfur deficiency

Microarray analysis was carried out with mRNAs isolated from aerial portions of rice plants subjected to sulfur deficiency. Among the candidate clones regulated by sulfur deficiency, three genes were identified by northern analysis that showed altered levels of mRNA accumulation. Genes similar to those for ferritin, S-like ribonuclease, and DnaJ-like protein were down-, down-, and upregulated by sulfate deficiency, respectively. These genes were found for the first time to be responsive to sulfur nutrition in the present study.

Iron-regulated expression of a cytosolic ascorbate peroxidase encoded by the APX1 gene in Arabidopsis seedlings.

Iron (Fe) is an essential element for living organisms. However, under aerobic conditions, its use is complicated because of its high insolubility and its potential toxicity through reactivity with reduced forms of oxygen. In plants, Fe overload can lead to intracellular concentrations beyond the storage and detoxification capacities of cells. Such a displacement toward a pro-oxidant state can activate antioxidant defenses, including Fe-mediated expression of ascorbate peroxidase genes. In this work, we demonstrate that Fe overload specifically induces the AtAPX1 gene encoding a cytosolic ascorbate peroxidase in Arabidopsis leaves. The strong constitutive expression of the AtAPX1 gene in roots is unaffected by Fe and depends on the first 5'-untranslated region intron. Presence of an AtAPX1 expressed sequence tag in the Arabidopsis database, longer in its 5' region than what could be predicted from the published AtAPX1transcription initiation site, leads to define a new transcription initiation region for this gene. A minimal promoter sequence enabling Fe-induced expression of the AtAPX1 gene is defined by following expression of various AtAPX1::beta-glucuronidase constructs in transformed Arabidopsis plantlets. This 118-bp minimal promoter sequence contains an Fe-dependent regulatory sequence-like cis-element known to be necessary for maize (Zea mays) and Arabidopsis ferritin gene derepression in response to Fe overload. Site-directed mutagenesis of this element within the AtAPX1 promoter sequence does not abolish the Fe-dependent activation of a reporter gene, indicating that it is likely not involved in the Fe-regulated expression of the AtAPX1 gene.

Screening for mutations of the ferritin light and heavy genes in Parkinson's disease patients with hyperechogenicity of the substantia nigra

Recently, an insertional mutation in the ferritin-L gene was reported in some patients with familial basal ganglia degeneration, which, however, could not be detected in another Parkinson's disease (PD) population. We investigated 186 PD patients, in whom an increased amount of iron of the substantia nigra (SN) was priorly identified by transcranial ultrasound, for mutations of the whole coding region of ferritin-L and ferritin-H by denaturing high-pressure liquid chromatography and subsequent sequencing. In the ferritin-L gene two silent mutations were detected. In the ferritin-H gene the sequence variation 161A→G was found in one patient but none of the 186 controls. Although functional analysis will show, whether this sequence variation might be causative for single cases of PD, the results indicate that mutations in the ferritin genes are not a common cause for PD with increased levels of iron of the SN.

Screening for mutations of the ferritin light and heavy genes in Parkinson's disease patients with hyperechogenicity of the substantia nigra

Recently, an insertional mutation in the ferritin-L gene was reported in some patients with familial basal ganglia degeneration, which, however, could not be detected in another Parkinson's disease (PD) population. We investigated 186 PD patients, in whom an increased amount of iron of the substantia nigra (SN) was priorly identified by transcranial ultrasound, for mutations of the whole coding region of ferritin-L and ferritin-H by denaturing high-pressure liquid chromatography and subsequent sequencing. In the ferritin-L gene two silent mutations were detected. In the ferritin-H gene the sequence variation 161A→G was found in one patient but none of the 186 controls. Although functional analysis will show, whether this sequence variation might be causative for single cases of PD, the results indicate that mutations in the ferritin genes are not a common cause for PD with increased levels of iron of the SN.

Molecular analyses of patients with hyperferritinemia and normal serum iron values reveal both L ferritin IRE and 3 new ferroportin (slc11A3) mutations

AbstractUnexplained hyperferritinemia is a common clinical finding, even in asymptomatic persons. When early onset bilateral cataracts are also present, the hereditary hyperferritinemia-cataract syndrome (HHCS), because of heterozygous point mutation in the L ferritin iron-responsive element (IRE) sequence, can be suspected. We sequenced the L ferritin exon 1 in 52 DNA samples from patients referred to us for molecular diagnosis of HHCS. We identified 24 samples with a point mutation/deletion in the IRE. For the 28 samples in which no IRE mutation was present, we also genotyped HFE mutations and sequenced both H ferritin and ferroportin genes. We found an increased frequency of His63Asp heterozygotes (12 of 28) but no H ferritin mutations. We identified 3 new ferroportin mutations, producing, respectively, Asp157Gly, Gln182His, and Gly323Val amino acid replacements, suggesting that these patients have dominant type 4 hemochromatosis. This study demonstrates that both L ferritin IRE and ferroportin mutations can account for isolated hyperferritinemia. The presence of cataract does not permit the unambiguous identification of patients with HHCS, although the existence of a family history of cataract was only encountered in these patients. This raises the intriguing possibility that lens ferritin accumulation might be a factor contributing to age-related cataract in the general population. Additional causes of isolated hyperferritinemia remain to be identified.

In situ Western hybridization: a new, highly sensitive technique to detect foreign and endogenous protein distribution in rice seeds.

We have developed a highly sensitive in situ Western hybridization technique to study tissue-specific expression of foreign and endogenous genes in transgenic and non-transformed rice seed. The expression pattern of the soybean ferritin gene directed by a rice glutelin gene promoter, GluB-1, in transgenic rice seed revealed by this method was exactly the same as that revealed by immunological tissue printing but much clearer than the latter, and corresponded well to the results of GluB-1 promoter characterization studies. This method provides an alternative choice for studying the tissue-specific expression of a promoter, omitting the complicated transgenic procedure. The method can also be used to study the expression and accumulation pattern of endogenous genes, such as glutelin and prolamine genes, in non-transformed plants.

Differential expression and evolutionary analysis of the three ferritin genes in the legume plant Lupinus luteus

We identified three genes coding for ferritins in the legume plant – yellow lupine (LlFer1, LlFer2, LlFer3). Proteins coded by these genes have a typical ‘plant’ structure, and precursors of their functional subunits are probably targeted to plastids. Despite very high amino acid sequence identity of mature polypeptides (86–90%), their genes are differentially induced by iron and the ‘stress‐related’ hormone abscisic acid (ABA). In tissues of lupine plants grown in hydroponics, the transcription of all three classes was induced by both iron and ABA with different kinetics. However, in homogenous lupine cell suspension, the accumulation of all classes of ferritin messages was also induced with iron, whereas only one gene (LlFer2 class) was transcribed in response to ABA. Moreover, the activity of LlFer3 gene was repressed by ABA, but up‐regulated by light. During the development of symbiotic interactions of lupine plants with the bacteria Bradyrhizobium lupini, only two out of three ferritin genes were active –LlFer2 and LlFer3. Increased accumulation of ferritin RNAs and polypeptide(s) correlated with late developmental stages of root nodules, suggesting the involvement of two types of ferritin subunits in the protection against senescence‐related iron release. The phylogenetic analysis of ferritin amino acid sequences is in agreement with classical systematics. However, because of clear grouping of some legume ferritins outside all other legume sequences, a possibility of a more diverse character of ferritins in this family should also be considered.