Increased Transferrin Receptor Research Articles

Increased transferrin receptor expression following 11q23 deletion as a mechanism of malignant progression in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a common adult leukemia characterized by the accumulation of mature neoplastic B-lymphocytes. Typically, CLL follows an indolent course, with most patients surviving for many years. However, 10-20% of CLL patients carry 11q23 chromosomal deletions and often exhibit a more severe disease course, with earlier onset of symptoms, shortened lymphocyte doubling time, poor response to therapy, and shortened survival. The molecular basis for 11q23 deletions resulting in a poor prognosis is currently poorly understood. The tumor suppressor gene, ataxia-telangiectasia mutated (ATM, 11q22.3-23.1), is considered a likely candidate gene whose loss could result in the poor prognosis associated with 11q23 deletion and is mutated in a significant percentage of CLL cases. Recently, recombinant ATM expression in ATM-deficient cells was found to decrease transferrin receptor (TfR) expression, suggesting that deletion of the chromosomal region carrying ATM results in increased TfR expression. TfR imports iron into cells, an event necessary for DNA synthesis and cell growth. Additionally, rapidly growing malignant cells, including lymphomas and CLL, often express high TfR levels. Based on this, we propose that one molecular mechanism by which 11q23 deletions confer a poor prognosis in CLL is via increased TfR expression secondary to ATM loss, resulting in the increased cellular iron import, and hence increased capacity for malignant growth. Our hypothesis may also partially explain why gallium, an atomically iron-like toxic metal that binds to transferrin and the TfR is incorporated into cells and was previously demonstrated to have anti-tumor activity in patients with lymphomas refractory to other chemotherapeutic treatments.

Nickel decreases cellular iron level and converts cytosolic aconitase to iron-regulatory protein 1 in A549 cells

Nickel (Ni) compounds are well-established carcinogens and are known to initiate a hypoxic response in cells via the stabilization and transactivation of hypoxia-inducible factor-1 alpha (HIF-1α). This change may be the consequence of nickel's interference with the function of several Fe(II)-dependent enzymes. In this study, the effects of soluble nickel exposure on cellular iron homeostasis were investigated. Nickel treatment decreased both mitochondrial and cytosolic aconitase (c-aconitase) activity in A549 cells. Cytosolic aconitase was converted to iron-regulatory protein 1, a form critical for the regulation of cellular iron homeostasis. The increased activity of iron-regulatory protein 1 after nickel exposure stabilized and increased transferrin receptor (Tfr) mRNA and antagonized the iron-induced ferritin light chain protein synthesis. The decrease of aconitase activity after nickel treatment reflected neither direct interference with aconitase function nor obstruction of [4Fe–4S] cluster reconstitution by nickel. Exposure of A549 cells to soluble nickel decreased total cellular iron by about 40%, a decrease that likely caused the observed decrease in aconitase activity and the increase of iron-regulatory protein 1 activity. Iron treatment reversed the effect of nickel on cytosolic aconitase and iron-regulatory protein 1. To assess the mechanism for the observed effects, human embryonic kidney (HEK) cells over expressing divalent metal transporter-1 (DMT1) were compared to A549 cells expressing only endogenous transporters for inhibition of iron uptake by nickel. The inhibition data suggest that nickel can enter via DMT1 and compete with iron for entry into the cell. This disturbance of cellular iron homeostasis by nickel may have a great impact on the ability of the cell to regulate a variety of cell functions, as well as create a state of hypoxia in cells under normal oxygen tension. These effects may be very important in how nickel exerts phenotypic selection pressure to convert a normal initiated cell into a cancer cell.

Factors Affecting the Level and Localization of the Transferrin Receptor in Trypanosoma brucei

Transfer of bloodstream-form Trypanosoma brucei variant 221a from calf serum to dog serum-based medium induces acute iron starvation, as the transferrin receptor (Tf-R) of variant 221a binds dog Tf poorly. We show here that transfer to dog serum induces a 3-5-fold increase in Tf-R mRNA and protein within one doubling time (8 h). Because iron stores are still high 8 h after transfer, we infer that the signal for Tf-R overproduction is the decreased availability of cytosolic iron when cellular iron import drops. Up to 30% of the extra Tf-R spills out of the flagellar pocket onto the pellicular surface. Because the 5-fold increase in Tf-R is accompanied by a 5-fold increase in bovine Tf uptake, the up-regulation of Tf-R levels in response to Tf starvation helps the trypanosome to compete for limiting amounts of Tf. We noted that Tf-R levels also vary in calf serum medium. Cells in dense cultures contain up to 5-fold more Tf-R mRNA and protein than in dilute cultures. Only one-tenth of the extra Tf-R reaches the pellicular surface. The increase cannot be explained by a lack of Tf or to cell density sensing but is due to pericellular hypoxia. Our results show that bloodstream-form trypanosomes can regulate the expression of the two Tf-R subunit genes and the localization of their gene products in a flexible manner. This flexibility is made possible by the promoter-proximal position of the two genes in the variant surface glycoprotein expression site.

Increased IRP1 and IRP2 RNA binding activity accompanies a reduction of the labile iron pool in HFE-expressing cells.

Iron regulatory proteins (IRPs), the cytosolic proteins involved in the maintenance of cellular iron homeostasis, bind to stem loop structures found in the mRNA of key proteins involved iron uptake, storage, and metabolism and regulate the expression of these proteins in response to changes in cellular iron needs. We have shown previously that HFE-expressing fWTHFE/tTA HeLa cells have slightly increased transferrin receptor levels and dramatically reduced ferritin levels when compared to the same clonal cell line without HFE (Gross et al., 1998, J Biol Chem 273:22068-22074). While HFE does not alter transferrin receptor trafficking or non-transferrin mediated iron uptake, it does specifically reduce (55)Fe uptake from transferrin (Roy et al., 1999, J Biol Chem 274:9022-9028). In this report, we show that IRP RNA binding activity is increased by up to 5-fold in HFE-expressing cells through the activation of both IRP isoforms. Calcein measurements show a 45% decrease in the intracellular labile iron pool in HFE-expressing cells, which is in keeping with the IRP activation. These results all point to the direct effect of the interaction of HFE with transferrin receptor in lowering the intracellular labile iron pool and establishing a new set point for iron regulation within the cell.

Overexpression of the hereditary hemochromatosis protein, HFE, in HeLa cells induces an iron-deficient phenotype

A transfectant HeLa cell clone expressing HFE under the control of a tetracycline-repressible promoter was generated. HFE expression was fully repressed by the presence of doxycycline, while it was strongly induced by growth in the absence of doxycycline. HFE accumulation was accompanied by a large (∼10-fold) decrease in H- and L-ferritin levels, by a ∼3–4-fold increase in transferrin receptor, and a ∼2-fold increase in iron regulatory protein activity. These indices of cellular iron deficiency were reversed by iron supplementation complexes. The overexpressed HFE immunoprecipitated together with transferrin receptor, indicating a physical association which is the likely cause for the observed ∼30% decrease in 55Fe-transferrin incorporation after 18 h incubation. In the HFE-expressing cells the reduction in transferrin-mediated iron incorporation was partially compensated by a ∼30% increase in non-transferrin iron incorporation from 55Fe-NTA, evident after prolonged, 18 h, incubations. The findings indicate that HFE binding to transferrin receptor reduces cellular iron availability and regulates the balance between transferrin-mediated and non-transferrin-mediated cellular iron incorporation.

Antiproliferative effect of deferiprone on the Hep G2 cell line

Iron is an essential element in cellular metabolism and the growth of all living species, and is involved in DNA replication. The risk of hepatocellular carcinoma development is associated with an increase in iron availability. The aim of the present work was to investigate the effect of an oral iron chelator, deferiprone (CP20), on HepG2 cell-line proliferation in culture. HepG2 cell cultures were maintained in the absence of fetal calf serum (FCS) and in the presence or not (control cultures) of CP20 at the concentrations of 50 or 100 μM; deferoxamine (DFO) was used as an iron chelator reference. Cell proliferation was investigated by the analysis of DNA synthesis using [ 3H] methyl-thymidine incorporation and of the cell cycle by flow cytometry. Iron chelation efficiency in the culture model was studied by analyzing the effect of CP20 on radioactive iron uptake, intracellular ferritin level, and transferrin receptor expression. CP20, at the concentration of 50 or 100 μM, inhibited DNA synthesis after 48 hr of incubation and induced an accumulation of the cells in the S phase of the cell cycle. Iron chelators inhibited cellular iron uptake, decreased intracellular ferritin level, and increased transferrin receptor protein and mRNA levels. Our results show that CP20 as well as deferoxamine inhibit HepG2 cell proliferation and block cell cycle in the S phase.

Effect of Ascorbic Acid Administration on Serum Concentration of Transferrin Receptors

Since Szent-Gyorgyi first purified ascorbic acid (vitamin C) in 1928 (1), a number of associations between ascorbic acid and iron metabolism have been described. At the molecular level, ascorbic acid mobilizes iron from the crystal core of ferritin in vitro by reducing Fe3 to Fe2 (2). Intracellularly, ascorbic acid enhances iron-induced translation of ferritin (3) by favoring the conversion of the iron regulatory protein (IRP) from the RNA binding form to aconitase (3). Ascorbic acid also retards degradation of ferritin by blocking lysosomal autophagy of ferritin and transformation to hemosiderin (4)(5). In humans, the oral administration of ascorbic acid enhances the absorption of non-heme iron from the diet (6) and leads to increases in serum iron in subjects with iron overload and ascorbic acid deficiency (7). Moreover, iron overload states are associated with reduced ascorbic acid concentrations, possibly because of increased catabolism of ascorbic acid (7)(8). The amount of labile iron in the cytosol influences the stability of transferrin receptor mRNA and the translation of ferritin mRNA (9)(10)(11). Posttranscriptional regulation occurs by means of an interaction between IRPs, proteins that sense changes in the chelatable intracellular iron pool, and iron-responsive elements located on untranslated regions of transferrin receptor mRNA and ferritin mRNA (12)(13)(14). In states of iron abundance, an IRP has aconitase activity and does not bind to iron-responsive elements, producing increased transferrin receptor mRNA degradation and increased ferritin mRNA translation. Conversely, when iron is deficient, an IRP loses aconitase activity and binds to iron-responsive elements, causing increased transferrin receptor mRNA stability and a repression of ferritin mRNA translation (15)(16). Thus, intracellularly, the presence of iron leads to inhibition of transferrin receptor expression and promotion of higher ferritin expression, whereas the deprivation of …

Haemoglobin synthesis in erythropoietin-stimulated J2E cells does not require increased numbers of transferrin receptors.

Changes in transferrin-receptor numbers and iron utilisation were monitored during erythropoietin-induced maturation of J2E erythroid cells. Uptake of transferrin and iron doubled 24 h after exposure to erythropoietin, due to a twofold rise in surface transferrin receptors. In addition, a tenfold increase in iron incorporation into haem was observed after erythropoietin stimulation, as iron taken up from transferrin was directed towards haem biosynthesis and away from storage in ferritin. The rise in iron chelation into haem correlated extremely well with haemoglobin synthesis. However, the increase in numbers of transferrin receptors was not essential for haemoglobin synthesis; rather, it was linked with a burst in proliferation stimulated by erythropoietin. We have shown previously that amiloride blocks erythropoietin-enhanced proliferation of J2E cells, but potentiates maturation [Callus, B. A., Tilbrook, P. A., Busfield, S. J. & Klinken, S. P. (1995) Exp. Cell Res. 219, 39-46]. Here we demonstrate that amiloride suppressed the hormone-induced increase in transferrin receptors, whereas the enhanced incorporation of iron into haem was not inhibited. Similarly, when sodium butyrate was used to induce differentiation of J2E cells, proliferation ceased and surface transferrin receptors remained unaltered, while haemoglobin production was accelerated. It was concluded from these experiments that the erythropoietin-stimulated rise in transferrin receptors during the final stages of J2E cell maturation is linked to cell division, and is not essential for haemoglobin synthesis.

Regulation of Cellular Iron Metabolism by Erythropoietin: Activation of Iron-Regulatory Protein and Upregulation of Transferrin Receptor Expression in Erythroid Cells

AbstractErythropoietin (Epo) is the central regulator of red blood cell production and acts primarily by inducing proliferation and differentiation of erythroid progenitor cells. Because a sufficient supply of iron is a prerequisite for erythroid proliferation and hemoglobin synthesis, we have investigated whether Epo can regulate cellular iron metabolism. We present here a novel biologic function of Epo, namely as a potential modulator of cellular iron homeostasis. We show that, in human (K562) and murine erythroleukemic cells (MEL), Epo enhances the binding affinity of iron-regulatory protein (IRP)-1, the central regulator of cellular iron metabolism, to specific RNA stem-loop structures, known as iron-responsive elements (IREs). Activation of IRP-1 by Epo is associated with a marked increase in transferrin receptor (trf-rec) mRNA levels in K562 and MEL, enhanced cell surface expression of trf-recs, and increased uptake of iron into cells. These findings are in agreement with the well-established mechanism whereby high-affinity binding of IRPs to IREs stabilizes trf-rec mRNA by protecting it from degradation by a specific RNase. The effects of Epo on IRE-binding of IRPs were not observed in human myelomonocytic cells (THP-1), which indicates that this response to Epo is not a general mechanism observed in all cells but is likely to be erythroid-specific. Our results provide evidence for a direct functional connection between Epo biology and iron metabolism by which Epo increases iron uptake into erythroid progenitor cells via posttranscriptional induction of trf-rec expression. Our data suggest that sequential administration of Epo and iron might improve the response to Epo therapy in some anemias.

Divergent effects of alpha 1-antitrypsin on the regulation of iron metabolism in human erythroleukaemic (K562) and myelomonocytic (THP-1) cells.

The acute-phase protein alpha 1-antitrypsin (alpha 1-AT) has been shown to inhibit the binding of transferrin to its cell-surface receptor. Here we demonstrate that in human erythroleukaemic cells (K562) alpha 1-AT enhances the binding affinity of iron-regulatory protein (IRP), the central regulator of cellular iron metabolism, to iron-responsive elements. Activation of IRP by alpha 1-AT is associated with a marked increase in transferrin receptor (trf-rec) mRNA levels in K562 and enhanced cell-surface expression of transferrin-binding sites, whereas ferritin production is decreased, although ferritin mRNA levels remain unchanged. In agreement with the well-established mechanism of cellular iron regulation, alpha 1-AT seems to modulate trf-rec and ferritin expression primarily post-transcriptionally/translationally by influencing IRP activity. In contrast, alpha 1-AT produces only minor changes in IRP activity, and subsequently in trf-rec expression and ferritin synthesis in THP-1 cells. Moreover the effects of alpha 1-AT on iron homeostasis in K562 cannot be overcome by the addition of iron salts, whereas concomitant treatment of THP-1 with iron and alpha 1-AT results in the same metabolic changes as the addition of iron alone. Because alpha 1-AT blocks transferrin binding on K562 as well as on THP-1 cells, it is suggested, on the basis of the results presented here, (1) that erythroid and monocytic cells might differ in their dependence on transferrin-mediated iron supply and (2) that THP-1 might be able to acquire iron by a transferrin-independent iron uptake system. alpha 1-AT might therefore be involved in the diversion of iron traffic between various cellular compartments under inflammatory conditions.

Effect of modulation of the transferrin receptor on gallium-67 uptake and cytotoxicity in lymphoma cell lines

Gallium-67 is a radionuclide that accumulates in haematological malignancies and is used for diagnostic purposes. Uptake of 67Ga into the cell occurs via the transferrin receptor, which is differentially expressed during the various cell cycle phases. With the aim of selectively increasing 67Ga uptake, we studied whether the transferrin receptor (TfR) expression could be modulated in the U937 and U715 lymphoma cell lines by cytostatic drugs inducing cell cycle phase accumulation. We tested clinically relevant drugs such as 1-beta-D-arabinofuranosylcytosine (Ara-C), hydroxyurea and methotrexate. Cytotoxicity was determined by testing the clonogenic capacity of the lymphoma cell lines. All three drugs induced an increase in S-phase content, TfR expression and 67Ga uptake in U937 and U715 single cells. The combinations of drugs and 67Ga resulted in an additive effect on the clonogenic capacity. In U937 spheroids, cultured by the fibrin clot technique, we found an accumulation in the S-phase too as well as an increase of the transferrin receptor expression after Ara-C preincubation. As in single cells 67Ga uptake was increased without synergistic effects on the clonogenic capacity. In conclusion, priming with drugs induces increased transferrin receptor expression and 67Ga uptake. Inhibition of clonogenic capacity was additive rather than synergistic.

Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro.

Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin receptors. This effect is rapid and dose dependent, with maximal down-regulation elicited with 30 min of treatment and with an IC50 approximately 25 nM wortmannin. Wortmannin-treated cells display an increased endocytic rate constant for transferrin internalization and decreased exocytic rate constants for transferrin recycling. In addition to these effects in vivo, wortmannin is a potent inhibitor (IC50 approximately 15 nM) of a cell-free assay that detects the delivery of endocytosed probes into a common compartment. Inhibition of the in vitro assay involves the inactivation of a membrane-associated factor that can be recruited onto the surface of vesicles from the cytosol. Its effects on the cell-free assay suggest that wortmannin inhibits receptor sorting and/or vesicle budding required for delivery of endocytosed material to "mixing" endosomes. This idea is consistent with morphological changes induced by wortmannin, which include the formation of enlarged transferrin-containing structures and the disruption of the perinuclear endosomal compartment. However, the differential effects of wortmannin, specifically increased transferrin receptor internalization and inhibition of receptor recycling, implicate a role for phosphatidylinositol 3-kinase activity in multiple sorting events in the transferrin receptor's membrane traffic pathway.

T-cell activation in human intestinal mucosa: The role of superantigens

Background : Superantigens are a class of potent T-cell mitogens that activate T cells using specific antigen receptor V regions. Superantigens have been implicated in the pathogenesis of several autoimmune diseases, including inflammatory bowel disease. The present study examines the role of Superantigens in the human gastrointestinal immune response. Methods : Human intestinal epithelial cells and T lymphocytes were obtained from surgical specimens and cultured in the presence or absence of exogenous superantigens. Activation of T cells and V region usage were measured by thymidine incorporation and by cell staining using a panel of monoclonal antibodies. Results : Neither epithelial cells from normal nor diseased intestinal mucosa expressed virally encoded, endogenous superantigens. However, 50% of epithelial cell preparations could present exogenous bacterial superantigens to T cells. In the other 50%, a defect in this function was observed, which did not represent production of suppressive factors or absence of accessory cytokines. Mucosal T lymphocytes proliferated in response to superantigens in vitro, expressing increased transferrin receptor, interleukin-2 receptor, and HLA-DR. Conclusions : A superantigen-driven mucosal immune response may occur in health and in chronic inflammatory states. The intestinal epithelial cell may mediate this response, through presentation of superantigens to mucosal T lymphocytes.

Regulation of heme biosynthesis: distinct regulatory features in erythroid cells.

Our previous research has demonstrated that in hemoglobin-synthesizing cells, as compared with nonerythroid cells, a step in iron transport from transferrin localized between the transferrin receptor and ferrochelatase is rate-limiting for the synthesis of heme. In this communication we report our more recent studies on the mechanisms involved in the regulation of the transferrin receptors and ferrochelatase in differentiating erythroid cells. Our studies indicate that transferrin receptor gene expression is regulated differently in hemoglobin synthesizing as compared with uninduced murine erythroleukemia (MEL) cells: 1) With nuclear run-on assays our experiments showed increased transferrin receptor mRNA transcription cells of MEL following induction of erythroid differentiation with dimethylsulfoxide (DMSO). 2) DMSO treatment of MEL cells does not increase iron-responsive element binding protein (IRE-BP) activity which is, however, increased in uninduced MEL cells by Fe chelators. 3) Following induction of MEL cells there is an increase in the stability of transferrin receptor mRNA whose level is only slightly affected by iron excess. Using murine ferrochelatase cDNA as a probe, two ferrochelatase transcripts having lengths of 2.9 kb and 2.2 kb were found in extracts of mouse liver, kidney, brain, muscle and spleen, the 2.9 kb transcript being more abundant in nonerythroid tissues and the 2.2 more predominant in spleen. In MEL cells, the 2.9 ferrochelatase transcript is also more abundant; however, following induction of erythroid differentiation by DMSO there is a preferential increase in the 2.2 kb transcript which eventually predominates. With mouse reticulocytes, the purest immature erythroid cell population available, over 90% of the total ferrochelatase mRNA is present as the 2.2 kb transcript. Our further experiments indicate that the 2.2 kb transcript results from the utilization of the upstream polyadenylation signal and suggest that the preferential utilization of the upstream polyadenylation signal may be an erythroid-specific characteristic of ferrochelatase gene expression. These results provide further evidence for the idea that iron metabolism and heme synthesis are controlled by distinct mechanisms in erythroid versus nonerythroid cells.

The role of transferrin in heme transport

Porphyrin accumulation by proliferating cells, e.g., those associated with cancers or wounds, tends to correlate with increased transferrin receptor density. To determine whether transferrin might be implicated in porphyrin transport, fluorescence and absorption spectroscopy were used to study the interaction of porphyrins with transferrin. A single high-affinity binding site for heme and other porphyrins (Kd ∼ 20–25 nM) was detected by fluorescence spectroscopy. Difference spectroscopy revealed three additional heme-binding sites. These sites were distinct from the iron-binding sites: 1) Apotransferrin and diferric transferrin bound porphyrins with equal affinity; 2) 59Fe was not displaced from transferrin by porphyrins. Murine erythroleukemia cells incubated with [ 59Fe]hemin-[ 125I]transferrin internalized both labels concomitantly. Accumulation of [ 59Fe]hemin could be blocked by a 100-fold excess of diferric transferrin but not by apotransferrin. These results indicate that cells can internalize exogenous heme, and possibly porphyrins, bound to transferrin via its receptor.

Gene transfer to respiratory epithelial cells via the receptor-mediated endocytosis pathway.

Gene-based therapies for a variety of inherited and acquired pulmonary diseases will require the development of vectors capable of safe and efficient transfer of DNA to the respiratory epithelium. The present study examined the feasibility of delivering DNA to respiratory epithelial cells by the receptor-mediated endocytosis pathway. This strategy employs molecular conjugates consisting of a cognate moiety, in this case human transferrin, covalently linked to a DNA-binding moiety, such as a cationic polyamine. Complexes were formed between transferrin-polylysine conjugates (hTfpL) and plasmid DNA carrying the firefly luciferase reporter gene (pRSVL). The conjugate-DNA complexes were added directly to cells in tissue culture and incubated for 24 h, after which cell lysates were analyzed for luciferase enzyme activity by luminometry. An immortalized human respiratory epithelial cell line (HBE1) treated with the transferrin-polylysine-DNA complexes exhibited luciferase enzyme activity significantly augmented over background levels. This respiratory epithelial cell line exhibited greater susceptibility to gene transfer by the transferrin-polylysine conjugates than did non-respiratory epithelial cell lines known to possess high levels of transferrin receptors. Effective gene transfer was shown to require both the DNA-binding moiety and cognate moiety for the cell surface receptor. Specific internalization of the conjugates by the transferrin pathway was verified by competition for the transferrin receptor. In addition, treatment with agents that either increased transferrin receptor number or decreased lysosomal degradation markedly augmented gene expression mediated by the conjugates. Thus, respiratory epithelial cells possess receptors for transferrin that can be exploited to accomplish gene transfer by the receptor-mediated endocytosis pathway.

Response of rat mammary gland transferrin receptors to maternal dietary iron during pregnancy and lactation

Mammalian milk iron (MFe) concentration decreases during lactation. In human attempts to increase MFe through supplementing the mother with iron during lactation have failed and no correlation between maternal iron status or intake and MFe has been determined, suggesting a regulatory mechanism. In contrast MFe in lactating rats can be affected by very high or low amounts of ingested iron. We previously described a transferrin receptor (TfR) mechanism that controls iron entry into rat mammary tissue. In this study lactating rats were used to determine effects of varying dietary iron during gestation and lactation on mammary TfR and MFe. Dams fed low-iron diets had a small increase in TfR, lower hematological indices (p less than 0.005), and lower MFe (p less than 0.005) than did controls or dams fed high iron. Dams fed the high-iron diet had a significant increase in TfR without a concomitant increase in MFe. There was no correlation between MFe and TfR. These findings suggest that control of MFe lies after iron entry into mammary tissue.

Characterization of Rat Transferrin Receptor cDNA: The Regulation of Transferrin Receptor mRNA in Testes and in Sertoli Cells in Culture

A 3.4 kilobase cDNA complementary to rat transferrin receptor mRNA has been isolated from an adult rat testis cDNA library. The rat transferrin receptor nucleotide sequence was shown to be 82% similar to the human transferrin receptor sequence over the amino acid coding region and over 90% similar in the sequences known to be responsible for iron regulation in the human mRNA. The mRNA was shown by Northern blot analysis to be regulated by iron levels in Sertoli cells in culture. Iron depletion resulted in at least a 5-fold increase in receptor message in Sertoli cells, as well as in an actively growing testicular cell line (S10-7). The level of transferrin receptor mRNA in cultured Sertoli cells was not influenced by hormones; however, chronic administration of testosterone or FSH to hypophysectomized rats resulted in increased transferrin receptor mRNA levels in the testis. Northern blot analysis of mRNAs from testes of rats synchronized at various stages of the cycle of the seminiferous epithelium showed that transferrin receptor mRNA was differentially regulated throughout the cycle. Northern blots of mRNA from germinal cell populations derived from synchronized tests showed that the message was regulated in the nongerminal cell components of the tubule, most likely the Sertoli cell. The comparison of transferrin receptor mRNA levels in normal testes and testes from hypophysectomized rats, as well as in isolated germinal cells and cultured Sertoli cells, suggested that transferrin receptor mRNA levels were considerably higher in Sertoli cells than in other cell types of the seminiferous tubules.

A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor from human placenta

We describe a simple method for the affinity purification of specific RNA-binding proteins. DNA sequences corresponding to the protein-binding site of the RNA are subcloned into an in vitro transcription vector between the T7 viral promoter and a poly(A) track. A polyadenylated RNA transcript is bound to poly(U)-Sepharose and subsequently incubated with a cellular extract prepurified on heparin-agarose. Specifically adsorbed proteins are recovered in high yield and purity from the affinity matrix by high salt elution. Using this method we isolated the iron regulatory factor (IRF), a cytoplasmic protein which binds to specific palindromic elements in the 5' and 3' untranslated sequences of ferritin and transferrin receptor mRNA, respectively. Activation and binding of this regulatory factor correlates with increased transferrin receptor mRNA stability and inhibition of ferritin translation. The purified factor from human placenta migrates as a monomer in gel chromatography, but is present in equimolar amounts of two proteins with molecular weights of 95 and 100 kDa when analysed by SDS/PAGE. The two proteins are highly related as judged by the identity of their isoelectric points and their specificity to form RNA-protein complexes.

Increase of transferrin receptors and iron uptake in regenerating motor neurons.

After injury, motor neurons exhibit a number of metabolic and protein changes that are assumed to be part of an inherent neuronal regeneration program, which, when activated, eventually leads to functional restitution. The mechanisms underlying this regeneration are unclear, but it may be expected that factors supporting neuronal growth or survival play an important role in the restoration of neuronal integrity. A number of neuronal growth-associated proteins have been identified, but their functional roles remain unclear. This paper shows that axotomy results in a strong increase in transferrin receptors (TfRs) in regenerating motor neurons and that this phenomenon is functionally associated with an elevated uptake of exogenous iron. The association of TfR expression in regenerating motor neurons with direct uptake of iron into the brain provides evidence that iron uptake into neural tissue may be related to neuronal metabolic activation. We suggest that the enhanced capacity of regenerating motor neurons to bind transferrin and to take up iron plays an important role in neuronal repair.