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Iron is required for normal cell growth and proliferation. However, excess iron is potentially harmful, as it can catalyse the formation of toxic reactive oxygen species (ROS) via Fenton chemistry. For this reason, cells have evolved highly regulated mechanisms for controlling intracellular iron levels. Chief among these is the sequestration of iron in ferritin. Ferritin is a 24 subunit protein composed of two subunit types, termed H and L. The ferritin H subunit has a potent ferroxidase activity that catalyses the oxidation of ferrous iron, whereas ferritin L plays a role in iron nucleation and protein stability. In the present study we report that increased synthesis of both subunits of ferritin occurs in HeLa cells exposed to oxidative stress. An increase in the activity of iron responsive element binding proteins in response to oxidative stress was also observed. However, this activation was transient, allowing ferritin protein induction to subsequently proceed. To assess whether ferritin induction reduced the accumulation of ROS, and to test the relative contribution of ferritin H and L subunits in this process, we prepared stable transfectants that overexpressed either ferritin H or ferritin L cDNA under control of a tetracycline-responsive promoter. We observed that overexpression of either ferritin H or ferritin L reduced the accumulation of ROS in response to oxidant challenge.

The global increase in transcription of cytoprotective genes induced in response to oxidative challenge has been termed the antioxidant response. Ferritin serves as the major iron-binding protein in nonhematopoietic tissues, limiting the catalytic availability of iron for participation in oxygen radical generation. Here we demonstrate that ferritin is a participant in the antioxidant response through a genetically defined electrophile response element (EpRE). The EpRE of ferritin H identified in this report exhibits sequence similarity to EpRE motifs found in antioxidant response genes such as those encoding NAD(P)H:quinone reductase, glutathione S-transferase, and heme oxygenase. However, the EpRE of ferritin H is unusual in structure, comprising two bidirectional motifs arranged in opposing directions on complementary DNA strands. In addition to EpRE-mediated transcriptional activation, we demonstrate that ferritin is subject to time-dependent translational control through regulation of iron-regulatory proteins (IRP). Although IRP-1 is initially activated to its RNA binding (ferritin-repressing) state by oxidants, it rapidly returns to its basal state. This permits the translation of newly synthesized ferritin transcripts and ultimately leads to increased levels of ferritin protein synthesis following oxidant exposure. Taken together, these results clarify the complex transcriptional and translational regulatory mechanisms that contribute to ferritin regulation in response to prooxidant stress and establish a role for ferritin in the antioxidant response.

Ferritin is a protein that oxidizes and sequesters intracellular iron in a mineral core. We have reported that the E1A oncogene selectively represses ferritin H transcription, resulting in reduced levels of the ferritin H protein. Here we demonstrate that cells respond to pro-oxidant challenge by inducing ferritin mRNA and protein, and that this response is completely blocked by E1A. Concordantly, E1A sensitized cells to the cytotoxic effects of oxidative stress and enhanced the accumulation of reactive oxygen species in response to pro-oxidant challenge. These results demonstrate that expression of E1A impedes the cellular response to oxidative stress, including the induction of ferritin.

We previously identified a major enhancer of the mouse ferritin H gene (FER-1) that is central to repression of the ferritin H gene by the adenovirus E1A oncogene (Tsuji, Y., Akebi, N., Lam, T. K., Nakabeppu, Y., Torti, S. V., and Torti, F. M. (1995) Mol. Cell. Biol. 15, 5152-5164). To dissect the molecular mechanism of transcriptional regulation of ferritin H, E1A mutants were tested for their ability to repress FER-1 enhancer activity using cotransfection with ferritin H-chloramphenicol acetyltransferase (CAT) reporter constructs. Here we report that p300/CBP transcriptional adaptor proteins are involved in the regulation of ferritin H transcription through the FER-1 enhancer element. Thus, E1A mutants that failed to bind p300/CBP lost the ability to repress FER-1, whereas mutants of E1A that abrogated its interaction with Rb, p107, or p130 were fully functional in transcriptional repression. Transfection with E1A did not affect endogenous p300/CBP levels, suggesting that repression of FER-1 by E1A is not due to repression of p300/CBP synthesis, but to E1A and p300/CBP interaction. In addition, we have demonstrated that transfection of a p300 expression plasmid significantly activated ferritin H-CAT containing the FER-1 enhancer, but had a marginal effect on ferritin H-CAT with FER-1 deleted. Furthermore, both wild-type p300 and a p300 mutant that failed to bind E1A but retained an adaptor function restored FER-1 enhancer activity repressed by E1A. Sodium butyrate, an inhibitor of histone deacetylase, mimicked p300/CBP function in activation of ferritin H-CAT and elevation of endogenous ferritin H mRNA, suggesting that the histone acetyltransferase activity of p300/CBP or its associated proteins may contribute to the activation of ferritin H transcription. Recruitment of these broadly active transcriptional adaptor proteins for ferritin H synthesis may represent an important mechanism by which changes in iron metabolism are coordinated with other cellular responses mediated by p300/CBP.

H-kininogen is a multifunctional protein: it inhibits cysteine proteases, plays a role in contact activation of the coagulation cascade, and is the precursor of the potent proinflammatory peptide bradykinin. In the experiments described here, we identify H-kininogen as a ferritin-binding protein. Ferritin is a cellular and serum protein that is elevated in acute and chronic inflammation and many cancers. Despite numerous reports of ferritin-binding protein(s) in human serum, the nature and function of these proteins remain unclear. As a first step in characterizing the interaction between ferritin and its binding protein(s), we devised a ligand blot assay and used it to guide purification of a ferritin-binding protein from human serum. Edman degradation of the purified protein determined the sequence HNLGHGHK(H)ERDQGHG, a sequence with identity to residues 421-436 of human H-kininogen. These results were confirmed by demonstrating that commercially purified H-kininogen possessed ferritin binding activity and that ferritin binding could not be detected in plasma from kininogen-deficient individuals. Ligand blot assays mapped the ferritin binding domain to the light chain of H-kininogen chain, and revealed that both H and L recombinant ferritins possess H-kininogen binding activity. The unexpected identification of H-kininogen as a ferritin-binding protein may link ferritin in the complex chain of interactions by which H-kininogen mediates its multiple effects in contact activation and inflammation.

We have previously reported that the adenovirus E1A oncogene represses the transcription of the H subunit of the mouse ferritin gene. Subsequent analyses defined FER-1, a 37-nucleotide sequence located 4.1 kilobases proximal to the start site of transcription, as the target of E1A-mediated transcriptional repression and as an enhancer of the ferritin H gene. FER-1 is composed of an AP1-like sequence followed by an element with dyad symmetry. To achieve maximal enhancer activity and transcriptional repression by E1A, both elements were essential. Using gel retardation assays, we now demonstrate that the binding complex for the AP1-like sequence of FER-1 contains JunD, FosB, and ATF1. Furthermore, JunD and FosB were able to activate FER-1 enhancer activity by transient cotransfection with ferritin H-chloramphenicol acetyltransferase reporter constructs. This augmented enhancer activity was inhibited by E1A. In addition, we have defined the minimal sequence in the dyad element of FER-1 required for protein interaction. This was determined to be a C-rich sequence to which Sp1 and Sp3 bind. Experiments with recombinant proteins indicate that members of both transcription factor families simultaneously bind FER-1. Taken together, these results elucidate molecular mechanisms involved in the transcriptional regulation of a pivotal gene in iron metabolism and provide insights into the contribution of the Sp1 family to the activation of AP1-dependent enhancers.

We describe a strategy for the creation of recombinant ferritin heteropolymers which mimic the natural heterogeneity of this protein. This method entailed the co-expression of cDNA for both ferritin H and ferritin L subunits in a single bacterium using either a bicistronic vector, in which both cDNAs were expressed from the vector, or a dual vector expression strategy, in which each subunit was expressed from a separate compatible plasmid in a single bacterial host. Electron microscopy and sucrose density gradient centrifugation demonstrated that ferritin assembled spontaneously in such bacteria to form catalytically active proteins of the expected size and shape. Isoelectric focusing revealed that protein isolated from any of these bacteria exhibited a restricted heterogeneity in subunit composition. Such multi-subunit recombinant ferritins spontaneously assembled in bacteria may be useful in further studies of ferritin assembly and function. Our results further suggest that varying expression levels is a simple way to alter levels of individual components within a multi-subunit recombinant protein, and that this approach may be of general utility in assessing the contribution of individual components to the function of multi-subunit proteins or protein complexes.

Ferritin is an iron-binding protein composed of two subunits, H and L. Twenty-four of these subunits assemble to form apoferritins whose subunit composition varies in a characteristic way in different tissues. Using recombinant proteins, we have assessed the role of H and L subunits in mouse ferritin function and compared these to human ferritin subunits. We report that mouse ferritin subunits exhibit considerable functional similarity to their human counterparts, including a prominent role of the H subunit in the facilitation of rapid iron uptake, and a key role of amino acid residues Glu-62 and His-65 in this process. In addition, amino acid residues important to assembly of the protein are conserved between mouse and human, permitting the formation of fully functional hybrid proteins containing both mouse and human subunits. However, murine and human ferritin H subunits also evidenced substantial functional differences; murine ferritin H showed a consistent reduction in iron uptake activity relative to human ferritin H. Creation of chimeric human/mouse ferritin H subunits by "helix swapping" mapped the domain of the protein critical to this activity difference to the DE helix. These findings suggest a novel functional role for carboxyl-terminal domains of the ferritin H subunit.

Properties of the L- and H-type polypeptide subunits forming ferritin 24-mer molecules in mice were investigated, using the products of in vitro transcription and translation from the two cloned genes, and recombinant ferritin molecules (H24L0 or H0L24) produced by transformation in Escherichia coli. Several different conditions for analytical electrophoresis reproducibly show that the relative migration position of the two mouse ferritin subunits is reversed from that reported for ferritin H- and L-subunits in all other mammals; since mouse and human H-polypeptides almost co-migrate, this unusual relative mobility is due largely to novel properties of the murine L-subunit. This unusual electrophoretic property of the mouse L-subunit has led to conflicting reports about the subunit composition of natural mouse ferritin. Here, we show that the single major electrophoretic band given by liver ferritin purified from mice having a short-term iron overload matches that produced by the genetically defined L-polypeptide and that some bona fide H-subunits are also detected. In conclusion, it is reasonable to assume that, when mouse ferritin samples will be analyzed under the same conditions as those described here, the slower species will correspond to the L-type subunit. However, when dealing with ferritin from species other than human or mouse, it should be kept in mind that upon electrophoretic analysis of ferritin polypeptide, the designation of an electrophoretic band as being H- or L-type subunits will be very uncertain without corroboration from genetic, immunological, or amino acid sequencing data.

Ferritin, the major intracellular iron storage protein of eucaryotic cells, is regulated during inflammation and malignancy. We previously reported that transcription of the H subunit of ferritin (ferritin H) is negatively regulated by the adenovirus E1A oncogene in mouse NIH 3T3 fibroblasts (Y. Tsuji, E. Kwak, T. Saika, S. V. Torti, and F. M. Torti, J. Biol. Chem. 268:7270-7275, 1993). To elucidate the mechanism of transcriptional repression of the ferritin H gene by E1A, a series of deletions in the 5' flanking region of the mouse ferritin H gene were constructed, fused to the chloramphenicol acetyltransferase (CAT) gene, and transiently cotransfected into NIH 3T3 cells with an E1A expression plasmid. The results indicate that the E1A-responsive region is located approximately 4.1 kb 5' to the transcription initiation site of the ferritin H gene. Further analyses revealed that a 37-bp region, termed FER-1, is the target of E1A-mediated repression. This region also serves as an enhancer, augmenting ferritin H transcription independently of position and orientation. FER-1 was dissected into two component elements, i.e., a 22-bp dyad symmetry element and a 7-bp AP1-like sequence. Insertion of these DNA sequences into a ferritin H-CAT chimeric gene lacking an E1A-responsive region indicated that (i) the 22-bp dyad symmetry sequence by itself has no enhancer activity, (ii) the AP1-like sequence has moderate enhancer activity which is repressed by E1A, and (iii) the combination of the dyad symmetry element and the AP1-like sequence is required for maximal enhancer activity and repression by E1A. Gel retardation assays and cotransfection experiments with c-fos and c-jun expression vectors suggested that members of the Fos and Jun families bind to the AP1-like element of FER-1 and contribute to its regulation. In addition, gel retardation assays showed that E1A reduces the ability of nuclear proteins to bind to the AP1-like sequence without affecting the levels of nuclear factors that recognize the 22-bp dyad symmetry element. Taken together, these results demonstrate that FER-1 serves as both an enhancer of ferritin H transcription and a target for E1A-mediated repression.

Ferritin is a ubiquitously distributed iron-binding protein that plays a key role in cellular iron homeostasis. It is composed of two subunits, termed H (heavy or heart) and L (light or liver). In fibroblasts and other cells, the cytokine tumor necrosis factor-alpha (TNF) specifically induces synthesis of the ferritin H subunit. Using nuclear run-off assays, we demonstrate that this TNF-dependent increase in ferritin H is mediated by a selective increase in ferritin H transcription. Transfection of murine fibroblasts with chimeric genes containing the 5'-flanking region of murine ferritin H fused to the human growth hormone reporter gene reveals that the cis-acting element that mediates this response is located approximately 4.8 kilobases distal to the start site of transcription. Deletion analyses delimit the TNF-responsive region to a 40-nucleotide sequence located between nucleotides -4776 and -4736, which we term FER-2. Electrophoretic mobility shift assays and site-specific mutations indicate that this region contains two independent elements: one contains a sequence that binds a member of the NF-kappa B family of transcription factors, and a second contains a novel sequence that partially conforms to the NF-kappa B consensus sequence and may bind a different member of the NF-kappa B/Rel transcription factor family. Thus, effects of an inflammatory cytokine on ferritin are mediated by a family of transcription factors responsive to oxidative stress.

Ferritin is the major intracellular iron-storage protein in eucaryotic cells and plays a prominent role in maintaining intracellular iron homeostasis. We observed that transfection of NIH-3T3 mouse fibroblasts with the adenovirus E1A oncogene specifically repressed the mRNA for one of the subunits of ferritin, ferritin H. This occurred in the absence of any effect of E1A on the mRNA for the L subunit of ferritin. The repression of ferritin H was not a general feature of oncogene expression since transfection of NIH-3T3 cells with H-ras did not affect ferritin composition. Deletion of the conserved regions of E1A responsible for immortalization and transcriptional repression impaired the ability of E1A to repress ferritin H. Immunoprecipitation of ferritin in E1A transfectants demonstrated that the decrease in the ferritin H/L ratio observed at the mRNA level was also exhibited at the protein level. The E1A-dependent inhibition of ferritin H was also observed in a chimeric gene containing the ferritin H promoter ligated to the chloramphenicol acetyltransferase reporter gene, but was not observed in control genes in which chloramphenicol acetyltransferase activity was dependent on promoters derived from SV40 or the interleukin-3 gene. This suggests that E1A may repress ferritin H at the transcriptional level. These results demonstrate that the adenovirus E1A oncogene specifically modulates ferritin H expression. They also suggest that alterations in cellular iron metabolism may be among the diverse array of cellular responses induced by E1A.

Doxurubicin is an effective and widely used chemotherapeutic agent. However, use of this drug is often limited by its cardiotoxic side effects. We have observed that an early event accompanying doxorubicin cardiomyopathy is a selective decrease in levels of muscle gene transcripts in cardiac tissue (Ito et al., Proc. Natl. Acad. Sci. USA 87: 4275-4279). Since this decrease precedes ultrastructural evidence of cardiac damage, measurements of muscle transcripts might assist in the clinical evaluation of doxorubicin cardiotoxicity. We have therefore assessed the utility of the polymerase chain reaction in the measurement of mRNA in control and doxorubicin-treated animals. These measurements were performed on small tissue samples that simulate endomyocardial biopsies. We measured cardiac alpha-actin transcripts as a fraction of ferritin heavy chain transcripts using the method described by Chelly et al. (Nature 333: 858-860, 1988). 0.5 micrograms of total RNA, an amount equivalent to that obtainable from a typical endomyocardial biopsy, was efficiently co-amplified with cardiac alpha-actin and ferritin heavy chain specific primers. The cardiac alpha-actin/ferritin heavy chain ratio calculated from the PCR results correlated well (R = 0.981) with results obtained using Northern blot analysis of 10 micrograms RNA. The correlation was maintained over a wide range of cardiac alpha-actin transcript abundance. These results show that mRNA from cardiac tissue can be estimated by the polymerase chain reaction, even from a small, endomyocardial biopsy-sized sample.

Iron increases the synthesis of the iron-storage protein, ferritin, largely by promoting translation of preexisting mRNAs for both the H and L ferritin isoforms (H, heavy, heart, acidic; L, light, liver, basic). We have recently cloned and sequenced a full-length cDNA to murine ferritin H and identified ferritin H as a gene induced by tumor necrosis factor alpha (TNF-alpha, cachectin). Using primary human myoblasts, we have now examined the relationship between TNF-alpha and iron in regulating ferritin. Four lines of evidence suggest that TNF-alpha regulates ferritin independently of iron. First, evaluation of mRNA showed that TNF-alpha increased ferritin H chain specifically, provoking no change in steady-state levels of ferritin L mRNA; iron, in contrast, increased the mRNA of both isoforms. Second, the increase in ferritin H protein synthesis observed during TNF-alpha treatment was dependent on an increase in ferritin H mRNA: actinomycin D blocked the TNF-alpha-induced changes in ferritin H but did not inhibit the translational induction of ferritin seen with iron treatment. Third, equal ferritin mRNA induction was observed in iron-loaded cells and in cells depleted of iron by a permeant chelator, 2,2'-dipyridyl. Fourth, ferritin H induction by TNF-alpha and iron was additive over the entire range of iron concentrations, even at TNF-alpha doses known to maximally stimulate ferritin H mRNA levels. Nonetheless, the role of iron in translational regulation of ferritin was retained in TNF-alpha-treated cells; effective biosynthesis of TNF-alpha-induced, H-subunit-predominant ferritin protein required iron and could be enhanced by treatment of the cells with additional iron or blocked by 2,2'-dipyridyl. Finally, we observed that the TNF-alpha-mediated increase in ferritin synthesis peaked at 8 hr and was followed by a decrease in both H and L isoferritin synthesis; the addition of iron, however, reversed the late-occurring depression in ferritin synthesis. This suggests that TNF-alpha-induced synthesis of H-rich ferritin may reduce the regulatory pool of intracellular iron, secondarily inhibiting iron-mediated translation of ferritin mRNA. We conclude that TNF-alpha acts independently of iron in its induction of ferritin H mRNA but requires the presence of iron for this effect to be fully expressed at the protein level.

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.

A mouse liver genomic library screened with a full-length cDNA encoding murine ferritin heavy chain (mFHC) [Torti et al., J. Biol. Chem. 263 (1988) 12638-12644] yielded a functional genomic clone mFHC. The genomic clone isolated included a region of approximately 3 kb containing four exons and three introns. Sequence comparisons of the mouse genomic clone with other genomic clones from rat, human and chicken showed a high degree of similarity among species in the coding regions. Introns and flanking sequences were less conserved. However, comparison of mFHC promoter elements with FHC genes from other species revealed common elements. Analysis of the genomic structure of FHC suggested the presence of pseudogenes. S1 nuclease analysis, however, confirmed that this mouse clone, when transfected into human MRC-5 fibroblasts, was transcribed, indicating that this clone contains an FHC functional gene.

Interleukin 1 alpha (IL-1) and tumor necrosis factor alpha (TNF) are two monokines which play a prominent role in the response to inflammation and injury. We recently observed that TNF leads to an increase in the synthesis of the heavy chain of ferritin, suggesting that TNF may be involved in iron homeostasis (Torti et al. (1988) J. Biol. Chem. 263, 12638-12644). The experiments reported here demonstrate that in cultured human muscle cells, IL-1 induces ferritin H mRNA and protein as effectively as TNF. TNF and IL-1 were additive in their effects on ferritin H expression, and IL-1 induction of ferritin H was not blocked by anti-TNF antibodies. Ferritin H induction was a specific response not observed with beta or gamma interferon, nor with transforming growth factor beta. Both differentiated myotubes as well as myoblasts responded to IL-1 with the induction of ferritin H. These results suggest that monokine-mediated alterations in the subunit composition of the ferritin molecule may be of biological relevance in the response to inflammation and injury.

Ferritin is a ubiquitous and highly conserved protein which plays a major role in iron homeostasis. We have identified and sequenced a full-length cDNA for murine ferritin heavy chain. The isolated cDNA is 819 nucleotides in length. It includes 546 nucleotides which encode a protein of 182 amino acids, a 5' noncoding sequence of 120 nucleotides, and a 3'-noncoding region of 153 nucleotides. The sequence displays a high degree of homology to human ferritin H, and includes a portion of the iron-responsive element conserved in chick, frog, and human ferritin. Tumor necrosis factor (TNF), a cytokine which mediates elements of the stress response, induces expression of ferritin H mRNA. Both mouse TA1 adipocytes and human muscle cells increase expression of ferritin H mRNA 4-6-fold after 48 h exposure to TNF. This increase occurs both prior and subsequent to differentiation of adipocytes and muscle cells, and is accompanied by an increase in the synthesis of the ferritin H subunit. These findings suggest a novel role for TNF in iron metabolism.