Polymorphism in a ferritin H gene from chromosome 6p

Summary This paper addresses the question of whether abnormalities in ferritin expression in the iron storage disease hemochromatosis (HC) involve major deletions or alterations in regions containing the two ferritin H genes that lie near the disease locus on chromosome 6p. We present evidence from analyses of Southern blots that neither gene is deleted in hemochromatosis. We also describe a polymorphism in one of the genes that we have previously shown to be a processed pseudogene. This polymorphism does not correlate with the presence of HC. The PIC value for this polymorphism was calculated as 0.49.     

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.     

The p97 antigen is mapped to the q24-qter region of chromosome 3; the same region as the transferrin receptor.

Since the p97 antigen, a membrane-associated iron-binding protein, has extensive amino acid sequence with homology with transferrin, is functionally related to the transferrin receptor, and has been previously mapped to chromosome 3, we have performed additional studies for regional mapping of the gene expressing p97 antigen. In these experiments, Chinese hamster-human cell lines were chosen that contained a large spectrum of autosomal human chromosomes, but mainly consisted of clones expressing all or a part of chromosome 3. These cell lines included a clone that previously allowed for mapping of human transferrin receptor to q22-qter region. Human p97 expression was assessed by specific binding of [125I]monoclonal antibody 96.5, and human transferrin receptor expression was tested by specific [125I]human transferrin binding and [125I]monoclonal antibody OKT-9 specific for human transferrin receptor. Based on these analyses, both human p97 antigenic expression and human transferrin receptor are mapped concordantly to the q24-qter region. These data and previous reports, therefore, suggest that the related iron-transport proteins are closely linked and may be under coordinate regulation. However, studies of several cell lines that exhibit up-regulation of human transferrin receptor expression with cellular proliferation, and down-regulation of receptor with increased transferrin-iron in the media, showed no change in expression of p97 antigen. p97 antigenic expression increased when melanocyte-stimulating hormone was added to a human melanoma cell line in tissue culture. These latter studies suggest that in mammalian cells the two proteins do not show coordinate regulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of ferritin and transferrin receptor mRNAs

Iron regulates the synthesis of two proteins critical for iron metabolism, ferritin and the transferrin receptor, through novel mRNA/protein interactions. The mRNA regulatory sequence (iron-responsive element (IRE)) occurs in the 5'-untranslated region of all ferritin mRNAs and is repeated as five variations in the 3'-untranslated region of transferrin receptor mRNA. When iron is in excess, ferritin synthesis and iron storage increase. At the same time, transferrin receptor synthesis and iron uptake decrease. Location of the common IRE regulatory sequence in different noncoding regions of the two mRNAs may explain how iron can have opposite metabolic effects; when the IRE is in the 5'-untranslated region of ferritin mRNA, translation is enhanced by excess iron whereas the presence of the IREs in the 3'-untranslated region of the transferrin receptor mRNA leads to iron-dependent degradation. How and where iron actually acts is not yet known. A soluble 90-kDa regulatory protein which has been recently purified to homogeneity from liver and red cells specifically blocks translation of ferritin mRNA and binds IRE sequences but does not appear to be an iron-binding protein. The protein is the first specific eukaryotic mRNA regulator identified and confirms predictions 20 years old. Concerted regulation by iron of ferritin and transferrin receptor mRNAs may also define a more general strategy for using common mRNA sequences to coordinate the synthesis of metabolically related proteins.     

Chloramphenicol-induced mitochondrial dysfunction is associated with decreased transferrin receptor expression and ferritin synthesis in K562 cells and is unrelated to IRE-IRP interactions

Chloramphenicol is an antibiotic that consistently suppresses the bone marrow and induces sideroblastic anemia. It is also a rare cause of aplastic anemia. These toxicities are thought to be related to mitochondrial dysfunction, since chloramphenicol inhibits mitochondrial protein synthesis. We hypothesized that chloramphenicol-induced mitochondrial impairment alters the synthesis of ferritin and the transferrin receptor. After treating K562 erythroleukemia cells with a therapeutic dose of chloramphenicol (10 µg/ml) for 4 days, there was a marked decrease in cell surface transferrin receptor expression and de novo ferritin synthesis associated with significant decreases in cytochrome c oxidase activity, ATP levels, respiratory activity, and cell growth. Decreases in the transferrin receptor and ferritin were associated with reduced and unchanged message levels, respectively. The mechanism by which mitochondrial dysfunction alters these important proteins in iron homeostasis is not clear. A global decrease in synthetic processes seems unlikely, since the expression of the cellular adhesion proteins VLA4 and CD58 was not significantly decreased by chloramphenicol, nor were the message levels of β-actin or ferritin. The alterations were not accompanied by changes in binding of the iron response protein (IRP) to the iron-responsive element (IRE), although cytosolic aconitase activity was reduced by 27% in chloramphenicol-treated cells. A disturbance in iron homeostasis due to alterations in the transferrin receptor and ferritin may explain the hypochromic-microcytic anemia and the accumulation of nonferritin iron in the mitochondria in some individuals after chloramphenicol therapy. Also, these studies provide evidence of a link between mitochondrial impairment and iron metabolism in K562 cells. J. Cell. Physiol. 180:334–344, 1999. © 1999 Wiley-Liss, Inc.     

Characterization and Distribution of Ferritin Binding Sites in the Adult Mouse Brain

Abstract : Studies on iron uptake into the brain have traditionally focused on transport by transferrin. However, transferrin receptors are not found in all brain regions and are especially low in white matter tracts where high iron concentrations have been reported. Several lines of research suggest that a receptor for ferritin, the intracellular storage protein for iron, may exist. We present, herein, evidence for ferritin binding sites in the brains of adult mice. Autoradiographic studies using ¹²⁵I-recombinant human ferritin demonstrate that ferritin binding sites in brain are predominantly in white matter. Saturation binding analyses revealed a single class of binding sites with a dissociation constant ( K _D) of 4.65 × 10^-9 M and a binding site density ( B _max) of 17.9 fmol bound/μg of protein. Binding of radiolabeled ferritin can be competitively displaced by an excess of ferritin but not transferrin. Ferritin has previously been shown to affect cellular proliferation, protect cells from oxidative damage, and deliver iron. The significance of a cellular ferritin receptor is that ferritin is capable of delivering 2,000 times more iron per mole of protein than transferrin. The distribution of ferritin binding sites in brain vis-à-vis transferrin receptor distribution suggests distinct methods for iron delivery between gray and whi     

Recycling of RNA binding iron regulatory protein 1 into an aconitase after nitric oxide removal depends on mitochondrial ATP

Iron regulatory proteins (IRPs) control iron metabolism by specifically interacting with iron-responsive elements (IREs) on mRNAs. Nitric oxide (NO) converts IRP-1 from a [4Fe-4S] aconitase to a trans-regulatory protein through Fe-S cluster disassembly. Here, we have focused on the fate of IRE binding IRP1 from murine macrophages when NO flux stops. We show that virtually all IRP-1 molecules from NO-producing cells dissociated from IRE and recovered aconitase activity after re-assembling a [4Fe-4S] cluster in vitro. The reverse change in IRP-1 activities also occurred in intact cells no longer exposed to NO and did not require de novo protein synthesis. Likewise, inhibition of mitochondrial aconitase via NO-induced Fe-S cluster disassembly was also reversed independently of protein translation after NO removal. Our results provide the first evidence of Fe-S cluster repair of NO-modified aconitases in mammalian cells. Moreover, we show that reverse change in IRP-1 activities and repair of mitochondrial aconitase activity depended on energized mitochondria. Finally, we demonstrate that IRP-1 activation by NO was accompanied by both a drastic decrease in ferritin levels and an increase in transferrin receptor mRNA levels. However, although ferritin expression was recovered upon IRP-1-IRE dissociation, expression of transferrin receptor mRNA continued to rise for several hours after stopping NO flux.     

Effect of iron and retinoic acid on the control of transferrin receptor and ferritin in the human promonocytic cell line U937.

The effect of changes in iron availability and induction of differentiation on transferrin receptor expression and ferritin levels has been examined in the promonocytic cell line U937. Addition of iron (as 200 micrograms/ml saturated transferrin) or retinoic acid (1 microM) both caused approx. 70% reduction in the average number of surface transferrin receptors, while the iron chelator desferrioxamine caused an 84% increase. Comparable changes also occurred in the levels of transferrin receptor mRNA. Neither iron nor retinoic acid significantly altered the half-life of transferrin receptor mRNA in the presence of actinomycin D (approx. 75 min) but a 10-fold increase in stability occurred in the presence of desferrioxamine. Iron and retinoic acid both caused an increase in intracellular ferritin levels (approx. 4-and 3-fold, respectively), while desferrioxamine reduced ferritin levels by approx. two-thirds. The effect of iron and retinoic acid added together did not differ greatly from that of each agent alone. None of the treatments greatly affected levels of L-ferritin mRNA. Virtually no H-ferritin mRNA was detected in U937 cells. These results show that changes in ferritin and transferrin receptor caused by treatment with retinoic acid are similar to those induced by excess iron, and suggest that changes in these proteins during cell differentiation are due to redistribution of intracellular iron into the regulatory pool(s), rather than to iron-independent mechanisms.     

NK recognition of target structures: is the transferrin receptor the NK target structure?

That the transferrin receptor acts as a target antigen for human NK cells has previously been suggested. In this study we used two models to examine the hypothesis that the transferrin receptor is recognized by NK cells. In the first model, we employed mouse cloned NK cells in conjunction with the species-specific monoclonal antibody R17 217, which binds to the murine transferrin receptor. We show that there is no correlation between the amount of transferrin receptor expressed on targets and the susceptibility of these targets to NK lysis or NK binding in cold target competition assays. In the second model, we used human NK cells and transferrin receptor-positive transformants as targets. These transformants were derived from mouse L cells transfected with human DNA and selected for the presence of human transferrin receptor. Results show that, in contrast to the mouse system, there is a correlation between the expression of the human transferrin receptor on targets and the ability of these targets to competitively inhibit the lysis of K562 by NK cells. However, because inhibition is not complete, other cell surface antigens probably play a role in human NK-target interactions.     

Expression of transferrin receptors in phytohemagglutinin-stimulated human T-lymphocytes. Evidence for a three-step model

Resting human T-lymphocytes show an elevated intracellular concentration of ferritin, whereas transferrin receptors are not detectable. Stimulation by phytohemagglutinin markedly lowers their ferritin content, while inducing the synthesis of transferrin receptors. Addition of iron salts (ferric ammonium citrate) in activated T-lymphocyte cultures causes a marked enhancement of both [3H]uridine and [3H]thymidine incorporation. Nevertheless, it also induces a concentration-dependent decrease in transferrin receptor synthesis, associated with a marked rise of ferritin production. Hemin treatment exerts the same effects. Addition of picolinic acid in phytohemagglutinin-stimulated cultures causes a decrease of [3H]thymidine incorporation, whereas transferrin expression is markedly enhanced. The action of iron salts and chelators is specific for transferrin receptors, since the expression of other membrane markers of activated human T-lymphocytes (interleukin-2 receptor, insulin receptor, and HLA-DR antigen) is not modified by treatment with iron or picolinic acid. These observations suggest that expression of transferrin receptors in activated T-lymphocytes is specifically modulated by their intracellular iron level, rather than their proliferative rate. Addition of picolinic acid to resting T-lymphocytes in the absence of mitogen induces a marked decrease of their ferritin content, but not the appearance of transferrin receptors. On the basis of these results, we suggest a three-step model: (a) in resting T-lymphocytes, the gene for transferrin receptor is apparently "closed," in that it is not expressed under both normal conditions and following iron deprivation. (b) After mitogen stimulus, T-lymphocytes are reprogrammed into cell cycle progression, which necessarily entails synthesis of transferrin receptors (c) Expression of these receptors is modulated by the intracellular iron level, rather than the rate of proliferation per se.     

Tim‐2 is the receptor for H‐ferritin on oligodendrocytes

Oligodendrocytes stain more strongly for iron than any other cell in the CNS, and they require iron for the production of myelin. For most cell types transferrin is the major iron delivery protein, yet neither transferrin receptor protein nor mRNA are detectable in mature oligodendrocytes. Thus an alternative iron delivery mechanism must exist. Given the significant long term consequences of developmental iron deficiency and the iron requirements for normal myelination, identification of the iron delivery mechanism for oligodendrocytes is important. Previously we have reported that oligodendrocytes bind H‐ferritin and that H‐ferritin binds to white matter tracts in vivo. Recently, T cell immunoglobulin and mucin domain‐containing protein‐2 (Tim‐2) was shown to bind and internalize H‐ferritin. In the present study we show that Tim‐2 is expressed on oligodendrocytes both in vivo and in vitro. Further, the onset of saturable H‐ferritin binding in CG4 oligodendrocyte cell line is accompanied by Tim‐2 expression. Application of a blocking antibody to the extracellular domain of Tim‐2 significantly reduces H‐ferritin binding to the differentiated CG4 cells and primary oligodendrocytes. Tim‐2 expression on CG4 cells is responsive to iron; decreasing with iron loading and increasing with iron chelation. Taken together, these data provide compelling evidence that Tim‐2 is the H‐ferritin receptor on oligodendrocytes suggesting it is the primary mechanism for iron acquisition by these cells.     

Modulation of cellular iron metabolism by hydrogen peroxide. Effects of H2O2 on the expression and function of iron-responsive element-containing mRNAs in B6 fibroblasts

Cellular iron uptake and storage are coordinately controlled by binding of iron-regulatory proteins (IRP), IRP1 and IRP2, to iron-responsive elements (IREs) within the mRNAs encoding transferrin receptor (TfR) and ferritin. Under conditions of iron starvation, both IRP1 and IRP2 bind with high affinity to cognate IREs, thus stabilizing TfR and inhibiting translation of ferritin mRNAs. The IRE/IRP regulatory system receives additional input by oxidative stress in the form of H(2)O(2) that leads to rapid activation of IRP1. Here we show that treating murine B6 fibroblasts with a pulse of 100 microm H(2)O(2) for 1 h is sufficient to alter critical parameters of iron homeostasis in a time-dependent manner. First, this stimulus inhibits ferritin synthesis for at least 8 h, leading to a significant (50%) reduction of cellular ferritin content. Second, treatment with H(2)O(2) induces a approximately 4-fold increase in TfR mRNA levels within 2-6 h, and subsequent accumulation of newly synthesized protein after 4 h. This is associated with a profound increase in the cell surface expression of TfR, enhanced binding to fluorescein-tagged transferrin, and stimulation of transferrin-mediated iron uptake into cells. Under these conditions, no significant alterations are observed in the levels of mitochondrial aconitase and the Divalent Metal Transporter DMT1, although both are encoded by two as yet lesser characterized IRE-containing mRNAs. Finally, H(2)O(2)-treated cells display an increased capacity to sequester (59)Fe in ferritin, despite a reduction in the ferritin pool, which results in a rearrangement of (59)Fe intracellular distribution. Our data suggest that H(2)O(2) regulates cellular iron acquisition and intracellular iron distribution by both IRP1-dependent and -independent mechanisms.     

Binding to the transferrin receptor is required for endocytosis of HFE and regulation of iron homeostasis

HFE, the protein that is mutated in hereditary haemochromatosis, binds to the transferrin receptor (TfR). Here we show that wild-type HFE and TfR localize in endosomes and at the basolateral membrane of a polarized duodenal epithelial cell line, whereas the primary haemochromatosis HFE mutant, and another mutant with impaired TfR-binding ability accumulate in the ER/Golgi and at the basolateral membrane, respectively. Levels of the iron-storage protein ferritin are greatly reduced and those of TfR are slightly increased in cells expressing wild-type HFE, but not in cells expressing either mutant. Addition of an endosomal-targeting sequence derived from the human low-density lipoprotein receptor (LDLR) to the TfR-binding-impaired mutant restores its endosomal localization but not ferritin reduction or TfR elevation. Thus, binding to TfR is required for transport of HFE to endosomes and regulation of intracellular iron homeostasis, but not for basolateral surface expression of HFE.     

Identification of two human ferritin H genes on the short arm of chromosome 6

We have found by analyses of human-hamster hybrid cells that two human ferritin H genes lie near the locus of the iron storage disease idiopathic hemochromatosis on chromosome 6p. One of these genes was isolated and shown to be a processed pseudogene. Comparison of its sequence with those of other ferritin H pseudogenes indicates that they may be derived from a functional H gene other than that on chromosome 11.