Iron prevents ferritin turnover in hepatic cells

It has long been assumed that iron regulates the turnover of ferritin, but evidence for or against this idea has been lacking. This issue was addressed using rat hepatoma cells with characteristics of hepatocytes subjected to a continuous influx of iron. Iron-pretreated cells were pulsed with [(35)S]Met for 60 min or with (59)Fe overnight and harvested up to 30 h thereafter, during which they were/were not cultured with ferric ammonium citrate (FAC; 180 microm). Radioactivity in ferritin/ferritin subunits of cell heat supernatants was determined by autoradiography of rockets obtained by immunoelectrophoresis or after precipitation with ferritin antibody and SDS-PAGE. Both methods gave similar results. During the +FAC chase, the concentration of ferritin in the cells increased linearly with time. Without FAC, the half-life of (35)S-ferritin was 19-20 h; with FAC there was no turnover. Without FAC, the iron in ferritin had an apparent half-life of 20 h; in the presence of FAC there was no loss of (59)Fe. Without FAC, concentrations of ferritin iron and protein also decreased in parallel. We conclude that a continuous influx of excess iron can completely inhibit the degradation of ferritin protein and that the iron and protein portions of ferritin molecules may be coordinately degraded.     

Early effects in chemical-induced rat liver carcinogenesis: an immunohistochemical study following exposure to 0.04% AAF

To investigate effects that distinguish AAF from incomplete carcinogens, the rate of cell death (apoptosis) and cell proliferation was studied at early stages of AAF induced rat liver carcinogenesis. Male Wistar rats were fed 0.04% AAF in the diet for 2, 6 and 16 weeks and immunohistochemical markers were measured in the liver. The formation of initiated cells and preneoplastic foci was followed by staining for GST-P (glutathione-S-transferase). GST-P-positive foci were present from 6 weeks on. Apoptosis was increased in the periportal area and in preneoplastic foci at all time points. Cell proliferation was enhanced in the periportal area in oval cells and in bile duct-like cells particularly at 2 and 6 weeks and mainly in GST-P positive foci at 16 weeks. Notably, more cells always proliferated than were eliminated. Other apoptosis-related markers like p53 and FAS/Apo-1 could not be demonstrated in either normal hepatocytes, preneoplastic foci or in hepatocytes from treated animals. Scattered bcl-2 positive cells were present in livers at 16 weeks of treatment. The two cell growth and differentiation related proto-oncogenes c-FOS and c-JUN were increased in all treated animals at early stages. If feeding was stopped after 6 weeks, livers did not recover significantly within the following 10 weeks. The results support the complex effects of AAF in rat liver carcinogenesis. Chronic toxicity locally impairs the balance between cell proliferation and cell death and induces morphological alterations that promote the growth of initiated cells.     

Multiple ferritin subunit genes of the Pacific oyster Crassostrea gigas and their distinct expression patterns during early development

Multiple ferritin subunit genes are reported in mollusks, but they have not been systematically classified. Based on the recently published whole genome sequence, we screened out the four ferritin subunit genes (cgi-fer1–cgi-fer4) from the Pacific oyster Crassostrea gigas. The four genes were predicted to encode two non-secretory and two secretory peptides. Further phylogenetic analyses revealed two groups of non-secretory and secretory ferritin subunits in mollusks. This differs dramatically from the situation in mammals or insects, which contain only non-secretory or secretory ferritin subunits. These results emphasize the evolution of molluscan ferritin subunit genes. The expression patterns of the four genes during early development exhibited dramatic differences, indicating the functional diversity of these genes. Among them, cgi-fer2 was the only gene expressed prevalently and is thus suggested to be the major house-keeping ferritin subunit gene. The expression of the other three genes was tissue-specific beginning in the D-veliger stage. Based on their expression patterns, we inferred important functions of cgi-fer2 in ciliated tissues and of the other three genes in the digestive system. Moreover, our results indicated potentially different roles of ferritin subunit genes during larval shell formation in gastropods and bivalves, which may be helpful in understanding the molecular mechanisms that cause the different shells of gastropods and bivalves. In addition, we conducted a further semi-quantitative analysis of the four genes in four major developmental stages and five adult tissues. The results also revealed dramatically different expression patterns of the genes, which brought additional functional indications. This work may promote studies on molluscan ferritins and shed light on the evolution of ferritin subunit genes among different animal groups.     

Ferritin accumulation and uroporphyrin crystal formation in hepatocytes of C57BL/10 mice: a time-course study

To establish the time-sequence relationship between ferritin accumulation and uroporphyrin crystal formation in livers of C57BL/10 mice, a biochemical, morphological and morphometrical study was performed. Uroporphyria was induced by the intraperitoneal administration of hexachlorobenzene plus iron dextran and of iron dextran alone. Uroporphyrin crystal formation started in hepatocytes of mice treated with hexachlorobenzene plus iron dextran at 2 weeks and in mice treated with iron dextran alone at 9 weeks. In the course of time, uroporphyrin crystals gradually increased in size. Uroporphyrin crystals were initially formed in hepatocytes in the periportal areas of the liver, in which also ferric iron staining was first detected. The amount and the distribution of the main storage form of iron in hepatocytes, ferritin, did not differ between the two treatment groups. Ferritin accumulation preceded the formation of uroporphyrin crystals in hepatocytes in both treatment groups. Moreover, uroporphyrin crystals were nearly always found close to ferritin iron. We conclude that uroporphyrin crystals are only formed in hepatocytes in which also iron (ferritin) accumulates. Hexachlorobenzene accelerates the effects of iron in porphyrin metabolism, but does not influence the accumulation of iron into the liver.     

Molecular size heterogeneity of ferritin in mouse liver

As much as 4% of the total protein in pure liver ferritin from mice with short-term parenteral iron overload produces a minor band migrating anodally to the major (alpha) band of holoferritin with non-denaturing polyacrylamide gel electrophoresis. The components in this minor band and the alpha band have been isolated to purity by preparative electrophoretic fractionation. The protein in the minor band is ferritin, since it contains ferric iron and fulfills defining criteria at the level of biochemistry, immunology and ultrastructure. Native polyacrylamide electrophoresis with pore-size-gradient gels shows that the ferritin molecules in the minor band have a slightly smaller diameter than the holoferritin in the alpha band. Isoelectric focusing reveals that the smaller ferritin has an identical number and range of charge isomers (pI 4.9-5.3) as the larger ferritin, but the relative amount of each size class within some isoferritin bands differs. The smaller ferritin molecules are structurally intact and are made from polypeptide subunits with Mr 18 000; the larger ferritin molecules have subunits with Mr 22 000. The minor species of hepatic ferritin thus has a smaller molecular size because it is made mainly from smaller subunits. No minor electrophoretic band can be detected in liver ferritin obtained from mice with normal iron levels. These results demonstrate that siderosis induces the formation of molecular size polymorphism (macroheterogeneity) in mouse liver ferritin. The new smaller hepatic ferritin could serve to redistribute excess iron into the main storage organs during the early response to iron overload, since it appears to be identical to one of the two types of serum ferritin molecules present in these siderotic mice.     

Localization of cAMP-dependent signal transducers in early rat liver carcinogenesis

 Cyclic AMP (cAMP) is an important regulator of liver growth and differentiation. The main intracellular cAMP receptor, cAMP-dependent protein kinase (PKA), consists of two regulatory (R) and two catalytic (C) subunits. There are two classes, RI and RII, of the regulatory subunit, giving rise to type I (RI₂C₂) and type II (RII₂C₂) PKA. The RI/RII ratio generally decreases during organ development, and increases during carcinogenesis. Alterations in this ratio have been implicated as an important factor in experimental and clinical carcinogenesis. We have studied the expression of RIα, RIIα, Cα, and an important substrate of PKA, the cAMP-response element binding protein, during rat liver carcinogenesis. Two-color immunofluorescence and confocal laser scan microscopy were used to characterize localization of the cAMP-dependent signal transducers in hepatocytes, bile ducts, oval cells, and preneoplastic lesions. We found that bile ducts and oval cells (putative liver stem cells) contained a higher RI/RII ratio than hepatocytes and preneoplastic lesions. Thus, an altered RI/RII ratio was not detected during early rat liver carcinogenesis, but may contribute to differentiation of putative liver stem cells to hepatocytes. Accepted: 19 August 1997     

Ferroportin-mediated mobilization of ferritin iron precedes ferritin degradation by the proteasome

Ferritin is a cytosolic molecule comprised of subunits that self-assemble into a nanocage capable of containing up to 4500 iron atoms. Iron stored within ferritin can be mobilized for use within cells or exported from cells. Expression of ferroportin (Fpn) results in export of cytosolic iron and ferritin degradation. Fpn-mediated iron loss from ferritin occurs in the cytosol and precedes ferritin degradation by the proteasome. Depletion of ferritin iron induces the monoubiquitination of ferritin subunits. Ubiquitination is not required for iron release but is required for disassembly of ferritin nanocages, which is followed by degradation of ferritin by the proteasome. Specific mammalian machinery is not required to extract iron from ferritin. Iron can be removed from ferritin when ferritin is expressed in Saccharomyces cerevisiae, which does not have endogenous ferritin. Expressed ferritin is monoubiquitinated and degraded by the proteasome. Exposure of ubiquitination defective mammalian cells to the iron chelator desferrioxamine leads to degradation of ferritin in the lysosome, which can be prevented by inhibitors of autophagy. Thus, ferritin degradation can occur through two different mechanisms.     

Immune proteasomes in the development of the rat immune system

The dynamics of the expression of LMP7 and LMP2 proteasome subunits during embryonic and early postnatal development of rat spleen and liver was studied in comparison with the dynamics of chymotrypsin-like and caspase-like proteasome activities and expression of MHC (major histocompatibility complex) class I molecules. The distribution of LMP7 and LMP2 immune subunits in spleen and liver cells was also evaluated throughout development. The common tendency of both organs to increase the expression of both LMP7 and LMP2 subunits on the 21st postnatal day (P21) was found. However, the total proteasome level was shown to be constant. At certain developmental stages, the dynamics of immune subunits expression in the spleen and liver was different. While the gradual enhancement of both immune subunits was observed on P1, P18 and P21 in the spleen, the periods of gradual increase observed on E16 (the 16th embryonic day) and E18 gave way to a period of decrease in immune subunits on P5 in the liver. This level did not reliably change until P18 and increased on P21. The revealed changes were accompanied by an increase in chymotrypsin-like activity and a decrease in caspase-like activity in the spleen at P21 compared to the embryonic period. This indicates the increase in proteasome ability to form antigenic epitopes for MHC class I molecules. In the liver, both activities increased compared to the embryonic period by P21. The dynamics of caspase-like activity can be explained not only by the change of proteolytic constitutive and immune subunits, but also by additional regulatory mechanisms. Moreover, it was discovered that the increase in the expression of immune subunits during early spleen development is associated with the process of formation of white pulp by B- and T-lymphocytes enriched with immune subunits. In the liver, the increase in the level of immune subunits by P21 was also accompanied by an increase of their expression in hepatocytes. While the decrease of their level by P5 may be associated with the fact that the liver has lost its function as the primary lymphoid organ in the immune system by this time, as well as with the disappearance of B-lymphocytes enriched with immune proteasomes. In the spleen and the liver, MHC class I molecules were found during the periods of increased levels of proteasome immune subunits. On E21, the liver was enriched with neuronal nitric oxide synthase (nNOS); the level of nNOS decreased after birth and then increased by P18. This fact indicates the possibility of the induction of expression of the LMP7 and LMP2 immune subunits in hepatocytes via a signaling pathway involving nNOS. These results indicate that compared to the rat liver cells, splenic T cell immune response develops in rats starting around P19–P21. First, a T-area of white pulp is formed in the spleen during this period. Second, an increased level of immune proteasomes and MHC class I molecules in hepatocytes can ensure the formation of antigenic epitopes from foreign proteins and their delivery to the cell surface for subsequent presentation to cytotoxic T-lymphocytes.     

Uptake of ferritin and iron bound to ferritin by rat hepatocytes: modulation by apotransferrin, iron chelators and chloroquine

Rat liver ferritin is an effective donor of iron to rat hepatocytes. Uptake of iron from ferritin by the cells is partially inhibited by including apotransferrin in the culture medium, but not by inclusion of diferric transferrin. This inhibition is dependent on the concentration of apotransferrin, with a 30% depression in iron incorporation in the cells detected at apotransferrin concentrations above 40 micrograms/ml. However, apotransferrin does not interfere with uptake of 125I-labeled ferritin, suggesting that apotransferrin decreases retention of iron taken up from ferritin by hepatocytes by sequestering a portion of released iron before it has entered the metabolic pathway of the cells. The iron chelators desferrioxamine (100 microM), citrate (10 mM) and diethylenetriaminepentaacetate (100 microM) reduce iron uptake by the cells by 35, 25 and 8%, respectively. In contrast, 1 mM ascorbate increases iron accumulation by 20%. At a subtoxic concentration of 100 microM, chloroquine depresses ferritin and iron uptake by hepatocytes by more than 50% after 3 h incubation. Chloroquine presumably acts by retarding lysosomal degradation of ferritin and recycling of ferritin receptors.     

Release of iron from ferritin molecules and their iron-cores by 3-hydroxypyridinone chelators in vitro

Ferritin molecules contain 24 subunits forming a shell around an inorganic iron-core. Release of iron(III) from ferritin and its isolated iron-cores by a series of hydroxypyridinone chelators with high affinities for iron(III) has been compared. The results collectively suggest that the chelators act by penetrating the protein shell and interacting directly with the iron-core in ferritin. Iron(III) is probably removed bound to a single ligand, but once outside the protein shell, the trihydroxypyridinone iron(III) complex predominates. The order of effectiveness of a group of pyridinones found for iron removal from ferritin molecules in solution differs from that obtained with hepatocytes in culture or with whole animals, where membrane solubility and other factors may modulate the response.     

烟草内源铁蛋白NtFer1和NtFer2的相互作用

以2个烟草铁蛋白基因全长序列（NtFer1和Mnr2,GenBank登录号：AY083924和AY141105）为基础,利用细菌双杂交系统分析不同烟草铁蛋白亚基之间及相同铁蛋白亚基之间的互作关系,并利用Northern杂交分析2个铁蛋白基因的特异表达。结果表明,铁蛋白基因NtFer1和Mnr2在叶片中均有表达,同时2种亚基之间存在很强的互作关系,说明在叶片中组成铁蛋白的24个亚基可能有3种类型,或来自单一的NtFer1亚基,或来自单一的NtFer2亚基,也可能来源于不同的铁蛋白亚基。在烟草根部组织中只有铁蛋白NtFer1基因大量表达,而MFPr2基因的表达非常微弱,所以根部的铁蛋白大分子可能由单一的铁蛋白NtFer1亚基聚合而成的。     

Modulation of Fas-FasL related apoptosis by PBN in the early phases of choline deficient diet-mediated hepatocarcinogenesis in rats

This study focused on the detection of apoptosis related events in very early phases of choline-deficient (CD)-induced hepatocarcinogenesis (at 2-5 weeks). Flow cytometry of isolated intact primary hepatocytes from CD diet fed rats indicated increased expression of the apoptosis-associated protein Fas. Increased apoptosis in CD-treated livers was confirmed by Western blot analyses of caspases and cytochrome c. This study was also able to detect differences in apoptotic events following phenyl butyl nitrone (PBN) treatment. Fas expression was inhibited by PBN, indicating that PBN is anti-apoptotic. It is speculated that in the early stages of CD-induced hepatotoxicity, PBN is involved in inhibiting pro-inflammatory factor-driven apoptosis of normal hepatocytes, which protects against the initiation of carcinogenesis. The CD diet model is also considered as a model for non-alcoholic steatohepatitis (NASH) in humans and early expression of Fas could also be a good index of the progression of NASH.     

Modulation of Fas-FasL related apoptosis by PBN in the early phases of choline deficient diet-mediated hepatocarcinogenesis in rats

This study focused on the detection of apoptosis related events in very early phases of choline-deficient (CD)-induced hepatocarcinogenesis (at 2–5 weeks). Flow cytometry of isolated intact primary hepatocytes from CD diet fed rats indicated increased expression of the apoptosis-associated protein Fas. Increased apoptosis in CD-treated livers was confirmed by Western blot analyses of caspases and cytochrome c. This study was also able to detect differences in apoptotic events following phenyl butyl nitrone (PBN) treatment. Fas expression was inhibited by PBN, indicating that PBN is anti-apoptotic. It is speculated that in the early stages of CD-induced hepatotoxicity, PBN is involved in inhibiting pro-inflammatory factor-driven apoptosis of normal hepatocytes, which protects against the initiation of carcinogenesis. The CD diet model is also considered as a model for non-alcoholic steatohepatitis (NASH) in humans and early expression of Fas could also be a good index of the progression of NASH.     

Iron loading-induced aggregation and reduction of iron incorporation in heteropolymeric ferritin containing a mutant light chain that causes neurodegeneration

Hereditary ferritinopathy (HF) is a neurodegenerative disease characterized by intracellular ferritin inclusion bodies (IBs) and iron accumulation throughout the central nervous system. Ferritin IBs are composed of mutant ferritin light chain as well as wild-type light (Wt-FTL) and heavy chain (FTH1) polypeptides. In vitro studies have shown that the mutant light chain polypeptide p.Phe167SerfsX26 (Mt-FTL) forms soluble ferritin 24-mer homopolymers having a specific structural disruption that explains its functional problems of reduced ability to incorporate iron and aggregation during iron loading. However, because ferritins are usually 24-mer heteropolymers and all three polypeptides are found in IBs, we investigated the properties of Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymeric ferritins. We show here the facile assembly of Mt-FTL and FTH1 subunits into soluble ferritin heteropolymers, but their ability to incorporate iron was significantly reduced relative to Wt-FTL/FTH1 heteropolymers. In addition, Mt-FTL/FTH1 heteropolymers formed aggregates during iron loading, contrasting Wt-FTL/FTH1 heteropolymers and similar to what was seen for Mt-FTL homopolymers. The resulting precipitate contained both Mt-FTL and FTH1 polypeptides as do ferritin IBs in patients with HF. The presence of Mt-FTL subunits in Mt-FTL/Wt-FTL heteropolymers also caused iron loading-induced aggregation relative to Wt-FTL homopolymers, with the precipitate containing Mt- and Wt-FTL polypeptides again paralleling HF. Our data demonstrate that co-assembly with wild-type subunits does not circumvent the functional problems caused by mutant subunits. Furthermore, the functional problems characterized here in heteropolymers that contain mutant subunits parallel those problems previously reported in homopolymers composed exclusively of mutant subunits, which strongly suggests that the structural disruption characterized previously in Mt-FTL homopolymers occurs in a similar manner and to a significant extent in both Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymers.     

Characterization of ferritin from human placenta. Implications for analysis of tissue specificity and microheterogeneity of ferritins

Mammalian ferritins can be resolved into multiple components by isoelectric focusing, and each tissue contains a characteristic subset of isoferritins. Ferritin isolated from human liver was compared to acidic ferritin isolated from mid-gestational human placenta to define a structural basis for ferritin heterogeneity. Placenta ferritin contained several major bands with isoelectric points in the range of pI = 4.7-5.0 which were more acidic than the predominant isoferritins of human liver. Ferritin from each tissue was resistant to denaturation by 10 M urea and appeared to be identical by electron microscopy. Circular dichroism measurements revealed that placenta ferritin had substantially less ordered secondary structure than liver ferritin. Both types of ferritin contained only two subunits when analyzed by electrophoresis in sodium dodecyl sulfate gels, but isoelectric focusing of dissociated subunits in urea revealed 6-7 different components. In this system, placenta ferritin was enriched in the more acidic subunits and it completely lacked the most basic subunits noted in liver ferritin; placental ferritin had no unique components. Differences in isoelectric points among assembled ferritins from these two tissues appear to result from different proportions of these acidic and basic subunits.