Regulation Of Ferritin Research Articles

Serum Ferritin: A Tumor Marker For Renal Cell Carcinoma

Serum ferritin levels in 32 patients with renal cell carcinoma were evaluated preoperatively and postoperatively. Serum ferritin concentration was significantly higher in renal cell carcinoma patients compared to controls (259.10 versus 61.30ng./ml., p <0.001). Furthermore, there was a steady and statistically significant increase in serum ferritin levels with advancing disease stage, as well as a significant decrease in serum ferritin levels after nephrectomy for stages 1 and 2 disease. The intracellular content of ferritin as estimated by polyclonal antibody was dramatically increased in renal cancer tissue compared to normal parenchyma. Although serum ferritin regulation is complex and only partly understood, the present study suggests that serum ferritin may be a useful tumor marker for renal cell carcinoma.

Regulation of ferritin and heme oxygenase synthesis in rat fibroblasts by different forms of iron.

Synthesis of the iron-storage protein ferritin is thought to be regulated at the translational level by the cytosolic content of chelatable iron. This response to iron is regulated by the iron-modulated binding to ferritin mRNAs of a repressor protein, the iron regulatory element-binding protein. From measurements made in a cell-free system, regulation of the iron regulatory element-binding protein has been recently suggested to involve direct interaction with hemin. The following observations on the synthesis of ferritin and of heme oxygenase (HO), the heme-degrading enzyme, in rat fibroblasts or hepatoma cells lead us to conclude that chelatable iron is a direct physiological regulator of ferritin synthesis in intact cells: (i) the inhibitor of heme degradation, tin mesoporphyrin IX, reduces the ability of exogenous hemin to induce ferritin synthesis but enhances HO synthesis; (ii) the iron chelator desferal suppresses the ability of hemin to induce synthesis of ferritin but not of HO; (iii) the heme synthesis inhibitor succinylacetone does not block iron induction of ferritin synthesis; (iv) there is no apparent relationship between the ability of various metalloporphyrins to inactivate the iron regulatory element-binding protein in cell-free extracts and their capacity to induce ferritin synthesis in intact cells; (v) administered inorganic iron significantly induces the synthesis of ferritin but not of HO; (vi) addition of delta-aminolevulinic acid to stimulate heme synthesis represses the ability of inorganic iron to induce ferritin synthesis while activating HO synthesis. Taken together, our results demonstrate that (i) release of iron by HO plays an essential role in the induction of ferritin synthesis by heme and (ii) chelatable iron can regulate ferritin synthesis independently of heme formation.

Transcriptional regulation of ferritin heavy chain messenger RNA expression by thyroid hormone

The effect of 3,5,3′-triiodo-L-thyronine (T3) on the steady state levels of ferritin heavy chain (ferritin H) mRNA in cultured rat glioma C6 cells and various rat tissues was examined. Addition of T3 to cultured C6 cells showed the time and dose-dependent increase in the steady-state level of ferritin H mRNA. Invitro nuclear run-on assay revealed that the stimulatory effect was due to the increase in the transcription rate of ferritin H gene. T3 had no effect on the half live of ferritin H mRNA. In hyperthyroid rats, the level of ferritin H mRNA in the kidney was elevated. On the contrary, that was decreased in hypothyroid rats. The results suggest the involvement of T3 in the regulation of ferritin H gene expression.

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.

Transcriptional regulation of ferritin H messenger RNA levels in FRTL5 rat thyroid cells by thyrotropin.

We examined the effect of thyrotropin (TSH) stimulation of FRTL5 rat thyroid cells on ferritin H mRNA levels. On Northern blot analysis, TSH (in the presence of serum and insulin) increased ferritin H mRNA levels, with an initial response evident after 1 h of stimulation. Ferritin H mRNA levels increased approximately 4-fold over basal levels after 4 h of TSH stimulation and showed little increase thereafter, maintaining a plateau for up to 48 h of TSH stimulation. Inducers of cAMP also increased ferritin H mRNA levels in FRTL5 cells to about the same extent, but the rate of response was not as rapid as with TSH stimulation. In serum-poor medium without insulin, the TSH effect was considerably weaker, increasing only about 2-fold after 24 h of stimulation. Also in serum-poor medium, insulin-like growth factor-I alone had a weak stimulatory effect on ferritin H mRNA levels. TSH and insulin-like growth factor-I had additive effects under these conditions. Nuclear run-on transcription assays were performed using nuclei prepared from FRTL5 cells. In serum-containing medium, TSH increased the transcriptional activity of ferritin H mRNA 3-4-fold without an increase in beta-actin transcriptional activity. The kinetics of TSH stimulation of ferritin H transcriptional activity were similar to the cellular ferritin H mRNA response to TSH stimulation. Dibutyryl-cAMP (dB-cAMP) increased ferritin H transcriptional activity about 4-fold, but not as rapidly as did TSH stimulation. In summary, our data indicate that ferritin H mRNA levels in FRTL5 thyroid cells are transcriptionally regulated by both TSH and dBcAMP stimulation. These data contrast with the predominantly nontranscriptional regulation of ferritin H mRNA levels observed in other tissues. The difference in the kinetics of the response to TSH and dBcAMP is consistent with the concept that not all effects induced by TSH stimulation of thyroid cells are mediated by cAMP as a second messenger.

Translational Regulation of Ferritin Synthesis by Iron

This review starts with a description of certain features of mammalian ferritins and their DNA and RNA structures relevant to translational control of ferritin synthesis. Although the amino acid sequences of the two ferritin subunits (H and L) diverge in about 50% of the coding region, their five alpha-helices and the exon sizes of their genes are compatible with the proposition that they diverged from a single ancestral gene. Of particular note is their long 5'-untranslated regions (5'UTRs) which include a 28-nucleotide sequence almost completely identical in the H- and L-subunits of a range of species. This motif near the cap region of the 5'-UTR, which forms a specific stem-loop structure, provides for regulation of the translation of H- and L-ferritin mRNAs. When intracellular levels of chelatable iron are not in excess, a large reserve of H- and L-mRNAs is present in the cell sap, restrained from translation by a protein with an Mr of about 90-100,000 which binds to the stem-loop structure. When excess iron floods the cytosol, this protein/RNA complex appears to dissociate and the 40S ribosome subunit is now able to initiate ferritin protein synthesis so that the dormant mRNAs become active and are transferred to the polyribosomes. The mechanism whereby the binding protein is regulated in response to iron is currently under investigation. The regulatory protein occurs in the cell sap and is present in several interchangeable forms which appear to differ in the redox state of specific sulphydryls within the protein. Under some circumstances, the abundance of these forms appears to be altered by intracellular iron status. It is unclear how iron influences binding of the regulatory protein to ferritin mRNA. Some investigators consider that iron binds in the form of heme to the regulatory protein, for which they offer in vitro evidence. We have examined the role of heme versus inorganic chelatable iron in the regulation of ferritin and heme oxygenase synthesis in rat fibroblasts and hepatoma cells. By manipulating the flow of iron between the intracellular chelatable iron and heme iron pools we have concluded that chelatable iron can act as a regulator of ferritin synthesis in a manner which is independent of heme formation. This conclusion does not exclude a role for heme in some specialized cell types.

Coordinate Post-Transcriptional Regulation of Ferritin and Transferrin Receptor Expression: The Role of Regulated RNA-Protein Interaction

Excess iron results in an increase in the translation of ferritin mRNA and a decrease in the stability of transferrin receptor (TfR) mRNA. These coordinate regulatory events are mediated by similar sequence/structure motifs that exist within the 5' untranslated region (5'UTR) of the ferritin mRNA and the 3'UTR of the TfR mRNA. We have referred to these motifs as iron-responsive elements (IREs). The IREs from both transcripts interact with a cytoplasmic protein that we have called the IRE-binding protein (IRE-BP). The activity but not the amount of the IRE-BP is dependent on the cellular iron status. The biochemical basis for the altered activity of the IRE-BP appears to be the reversible oxidation-reduction of one or more cysteines in the IRE-BP. The IRE-BP is a 90- to 95-kD cytosolic protein that has been purified to homogeneity by RNA affinity chromatography, and the cDNA corresponding to the IRE-BP has been molecularly cloned. Collectively, our data support a model in which the interaction between the IRE-BP and the ferritin IRE results in attenuation of translation, and similar interaction with TfR mRNA can protect the transcript from rapid degradation mediated by a rapid turnover determinant within the 3'UTR.

Transcriptional regulation of ferritin H and L subunits in adult erythroid and liver cells from the mouse: Unambiguous identification of mouse ferritin subunits and in vitro formation of the ferritin shells

Abstract Ferritin H and L subunits present cell-specific features of structure, function, and transcriptional regulation. Mouse Friend erythroleukemia cells offer an interesting model to analyze the erythroid-specific expression of ferritin genes for comparison with the liver, an iron-storing tissue. cDNA clones for mouse ferritin H and L subunits have been isolated and sequenced. The two subunits have very similar calculated masses, 20.9 and 20.6 kDa for H and L, respectively. Electrophoretic analysis of the subunits encoded by the cDNA 1) allows unambiguous identification of mouse ferritin subunits; 2) clearly shows that mouse H and L chains can make heteropolymers in vitro; and 3) demonstrates that, at least in vitro, free subunits can coexist with subunits polymerized into complete shells. The mouse ferritin gene family displays a variable degree of complexity, ranging from three homologous sequences for the H genes to 10-14 homologous loci for the L genes. Transcription of ferritin genes exhibits tissue-specific difference. Nuclear transcriptional run-off experiments show that the L gene is more actively transcribed in the liver than in Friend erythroleukemia cells at different stages of maturation. The accumulation of the H subunit mRNA which results from dimethyl sulfoxide induction of Friend cells is the consequence of an increase in the transcription rate of the H gene. However, the H gene mRNA is transcribed at a similar rate in the liver and in induced Friend cells although 5-fold more mRNA accumulates in these cells. Therefore, there is a tissue-specific regulation of mouse ferritin expression at both the transcription and mRNA stability levels.

Chromosomal localization of nucleic acid-binding proteins by affinity mapping: assignment of the IRE-binding protein gene to human chromosome 9.

Three human mRNAs are regulated post-transcriptionally by iron via iron-responsive elements (IREs) contained in each mRNA. A cytoplasmic protein (IRE-BP) binds to these cis-acting elements and mediates the translational regulation of ferritin H- and L-chain mRNA and the iron-dependent stability of transferrin receptor (TfR) mRNA. We have taken advantage of the different mobilities of the human and rodent IRE/IRE-BP complexes on non-denaturing polyacrylamide gels to determine the chromosomal localization of the gene encoding the IRE-BP. Utilizing a panel of 34 different human/rodent hybrid cell lines we have assigned the IRE-BP gene to human chromosome 9. This new technique based on nucleic acid/protein interaction may allow determination of the chromosomal localization of other RNA- or DNA-binding proteins.

Induction of ferritin subunit synthesis by iron is regulated at both the transcriptional and translational levels.

Synthesis of the iron storage protein ferritin is induced in rat liver by iron administration, the ferritin L subunit being preferentially stimulated over the H subunit. To examine the basis for this differential regulation of the two subunits, the transcription rates of the L and H genes, the total cellular levels of L and H subunit mRNA, and the distribution of the mRNAs between a stored ribonucleoprotein form and the polysomes were examined in rat liver at several times after iron injection. Iron caused a rapid increase in transcription of the L subunit gene, followed by a rise in L mRNA levels, whereas H subunit gene transcription and H mRNA levels did not increase significantly. Differential transcriptional regulation of ferritin L and H mRNA levels by iron contrasted with the coordinate control of the two subunit mRNAs at the translational level. On giving iron, there was a rapid and synchronous shift of both mRNAs from the ribonucleoprotein fraction onto the polysomes, the same proportion of each mRNA being mobilized. Thus, regulation of ferritin subunit synthesis at these two levels allows both a rapid translational response and a specific transcriptional response to increased intracellular iron levels.

ISH Regulation of ferritin H chain messenger RNA levels in the rat thyroids

Ferritin heavy chain mRNA steady state levels are increased by thyrotropin both in vivo and in two independent thyroid derived permanent cell lines. Maximum induction was achieved 48 hours after thyrotropin addition in the same conditions in which all the thyroid differentiated functions were stimulated. Thyrotropin stimulation of the levels of ferritin heavy chain mRNA seems to be mediated by cyclic AMP since it mimicks the hormone induction.

Transferrin receptor regulation is coupled to intracellular ferritin in proliferating and differentiating HL60 leukemia cells.

Cultured myeloid leukemia cells display transferrin receptors but decrease receptor display after differentiation induction or accumulation of intracellular iron. To determine whether regulation of transferrin receptors and ferritin were linked under these disparate conditions, serum-free and fetal bovine serum (FBS) cultures of HL60 promyelocytic leukemia cells were used to investigate relationships between transferrin receptor display and intracellular ferritin. Using 125I-transferrin binding and immunofluorescence staining for transferrin receptors, HL60 cells cultured in serum-free, transferrin-free medium expressed fewer transferrin receptors and contained increased ferritin when compared to cells cultured with FBS or transferrin supplemented, serum-free medium. When placed in medium containing transferrin, cells previously grown in transferrin-free medium rapidly re-expressed transferrin receptors and decreased their ferritin content. HL60 cells induced to differentiate into granulocytes or macrophages also decreased transferrin receptor display and increased their ferritin content. Transferrin receptor display and ferritin content in both proliferating and differentiating myeloid leukemia cells are inversely related and their regulation is closely linked. Regulation of transferrin receptor display and ferritin synthesis may be important events regulating myeloid cell growth and differentiation.

Regulation of plasma ferritin by the isolated perfused rat liver.

The isolated perfused rat liver has been used to investigate the regulation of plasma ferritin in normal, iron-deficient and iron-overloaded states. Both 125I-labelled and non-labelled rat liver ferritins were rapidly cleared from the perfusion circuit with a half-life of approximately 30 min. Perfusion of livers from normal, iron-loaded or iron-deficient rats with blood obtained from normal, iron-loaded or iron-deficient rats showed that the liver takes up plasma ferritin, and releases ferritin into the perfusate to achieve a perfusate ferritin level appropriate to the iron stores of the animal from which the liver was taken. It is concluded that the liver is capable of both uptake and release of ferritin and that within the liver there resides a mechanism which maintains circulating ferritin concentrations at a level appropriate to body iron stores.

PROCEEDINGS OF THE AUSTRALIAN SOCIETY FOR MEDICAL RESEARCH

PROCEEDINGS OF THE AUSTRALIAN SOCIETY FOR MEDICAL RESEARCH