Number Of Transferrin Receptors Research Articles

Intracellular heme coordinately modulates globin chain synthesis, transferrin receptor number, and ferritin content in differentiating Friend erythroleukemia cells

The effect of succinylacetone (SA), a highly specific inhibitor of ALA- dehydratase and heme synthesis, on hemoglobin (Hb) production, transferrin receptor (TfR), and ferritin expression was analyzed in differentiating Friend leukemia cells (FLC). This compound exerted a pronounced inhibitory effect not only on heme and Hb synthesis, but also on all the remaining above-mentioned parameters. In particular, SA induced: (1) a reduction of the level of alpha-globin mRNA; (2) a decreased number of exposed TfR molecules, without modification of their affinity for the ligand; (3) a reduced level of TfR RNA, without significant change of TfR gene transcription rate; and (4) a lower ferritin content. The addition of exogenous hemin to differentiating FLC exerted opposite effects, and particularly induced an increase of both the number of TfRs and ferritin content. These findings suggest that in erythroid cells optimal heme synthesis is required to coordinately sustain globin chains synthesis and TfR/ferritin production; thus, the intracellular heme level may represent a key regulatory factor in the Hb synthesis pathway.

Intracellular Heme Coordinately Modulates Globin Chain Synthesis, Transferrin Receptor Number, and Ferritin Content in Differentiating Friend Erythroleukemia Cells

The effect of succinylacetone (SA), a highly specific inhibitor of ALA-dehydratase and heme synthesis, on hemoglobin (Hb) production, transferrin receptor (TfR), and ferritin expression was analyzed in differentiating Friend leukemia cells (FLC). This compound exerted a pronounced inhibitory effect not only on heme and Hb synthesis, but also on all the remaining above-mentioned parameters. In particular, SA induced: (1) a reduction of the level of alpha-globin mRNA; (2) a decreased number of exposed TfR molecules, without modification of their affinity for the ligand; (3) a reduced level of TfR RNA, without significant change of TfR gene transcription rate; and (4) a lower ferritin content. The addition of exogenous hemin to differentiating FLC exerted opposite effects, and particularly induced an increase of both the number of TfRs and ferritin content. These findings suggest that in erythroid cells optimal heme synthesis is required to coordinately sustain globin chains synthesis and TfR/ferritin production; thus, the intracellular heme level may represent a key regulatory factor in the Hb synthesis pathway.

Distribution and number of transferrin receptors in Parkinson's disease and in MPTP-treated mice

Transferrin is a glycoprotein that functions primarily to deliver iron to the cell. Recent studies suggest that the transferrin receptor mediates the intracellular delivery and transport of iron bound to transferrin in the CNS. Iron-catalyzed free radical generation has been proposed as a possible cause of nigral cell death in Parkinson's disease. Our hypothesis is that abnormal iron handling by the transferrin receptor may contribute to the formation of free radical species which catalyze the lipid peroxidation of nigral cell membranes. We have assessed the number of transferrin receptors on membrane fractions prepared from the human striatum from control subjects and patients with Parkinson's disease. Equilibrium-binding studies demonstrated a reversible, saturable, and high-affinity transferrin binding site ( K D = 3 nM) in human brain membranes. Regional binding assays indicate that the number of transferrin receptors in the putamen was reduced significantly in Parkinson's disease. The density of transferrin receptors was unaltered in membranes prepared from the caudate nuclei and the globus pallidus. To address the possibility that transferrin receptors are located on dopaminergic terminals, we have examined the distribution and number of transferrin receptors in the striatum of MPTP-treated mice using in vitro autoradiographic methods. In these experiments, the loss of dopaminergic terminals in the striatum was visualized by differential [ 3H]mazindol uptake site autoradiography. A marked reduction in the density of both transferrin receptors and [ 3H]mazindol binding sites was observed in the mouse striatum 7 days post-MPTP treatment. Quantitative densitometric analysis demonstrated a recovery in [ 125I]ferrotransferrin binding in the striata of mice sacrificed at later survival times. The time-course for the recovery of striatal transferrin receptors preceded the rise in [ 3H]mazindol binding sites. Taken together, these observations suggest that transferrin receptors may be located, in part, on dopaminergic terminals. The recovery of [ 125I]ferrotransferrin binding in the striatum may reflect early sprouting of dopaminergic fibers. Alternatively, transferrin receptors may be up-regulated in surviving dopaminergic terminals as a cellular adaptation to increased metabolic activity.

Recycling kinetics and transcytosis of transferrin in primary cultures of bovine brain microvessel endothelial cells

Primary cultures of bovine brain microvessel endothelial cells (BMECs) were used to examine the cycling kinetics of ferrotransferrin (Tf) and to provide evidence for a transcytotic pathway in vitro. Binding of 125I-Tf to BMECs grown on matrix-coated plastic was measured in the presence of saponin to calculate the total number of transferrin receptors (TfRs). Nonlinear regression analysis of the binding isotherm showed that there were 100,000 high-affinity receptors per cell and that expression was maximum at cell confluence. Binding of Tf at 4 degrees C indicated that there was a large intracellular receptor pool comprising 85-90% of the total cellular receptors. Accumulation of Tf at 37 degrees C, inhibited at low temperature and in the presence of metabolic poisons, occurred with an initial rate coefficient of 0.030 min-1 and this decreased by 83% after 60 min. Concomitant accumulation of 59Fe from Tf-59Fe was linear. In the absence of externally added ligand, 80% of the accumulated 125I-Tf was released into the medium with a rate coefficient of 0.017 min-1 and this was inhibited at low temperature. In the presence of the weak base primaquine, the accumulation of Tf and 59Fe and the efflux of Tf were decreased. Moreover, phorbol myristate acetate (PMA) caused a 30% increase in surface TfRs and an 82% increase in Tf accumulation, although the size of the recycling pool remained unchanged. Despite the low numbers of TfR expressed by post-confluent cells, filter-grown BMEC monolayers were used to measure transcytosis of Tf. A small portion of the Tf that was accumulated from the apical side entered a transcytotic pathway. Most of the Tf and all of an accumulated fluid-phase tracer were recycled towards the apical side. These results showed that cultured BMECs cycle Tf-TfR complexes slowly and vectorially and suggested that the large intracellular receptor pool may facilitate steady state accumulation and regulate transcellular transport of iron.

Apical and basolateral transferrin receptors in polarized BeWo cells recycle through separate endosomes.

Contrary to most other epithelia, trophoblasts in the human placenta, which form the physical barrier between the fetal and the maternal blood circulation, express high numbers of transferrin receptors on their apical cell surface. This study describes the establishment of a polarized trophoblast-like cell line BeWo, which exhibit a high expression of transferrin receptors on the apex of the cells. Cultured on permeable filter supports, BeWo cells formed a polarized monolayer with microvilli on their apical cell surface. Across the monolayer a transepithelial resistance developed of approximately 600 omega.cm2 within 4 d. Depletion of Ca2+ from the medium decreased the resistance to background levels, showing its dependence on the integrity of tight junctions. Within the same period of time the secretion of proteins became polarized. In addition, the compositions of integral membrane proteins at the apical and basolateral plasma membrane domains were distinct as determined by domain-selective iodination. Similar to placental trophoblasts, binding of 125I-labeled transferrin to BeWo monolayers revealed that the transferrin receptor was expressed at both plasma membrane domains. Apical and basolateral transferrin receptors were found in a 1:2 surface ratio and exhibited identical dissociation constants and molecular weights. After uptake, transferrin recycled predominantly to the domain of administration, indicating separate recycling pathways from the apical and basolateral domain. This was confirmed by using diaminobenzidine cytochemistry, a technique by which colocalization of endocytosed 125I-labeled and HRP-conjugated transferrin can be monitored. No mixing of the two types of ligands was observed, when both ligands were simultaneously internalized for 10 or 60 min from opposite domains, demonstrating that BeWo cells possess separate populations of apical and basolateral early endosomes. In conclusion, the trophoblast-like BeWo cell line can serve as a unique model to compare the apical and basolateral endocytic pathways of a single ligand, transferrin, in polarized epithelial cells.

Effect of diferric transferrin and culture time on transferrin-receptor number and distribution in syncytiotrophoblast in vitro

Effect of diferric transferrin and culture time on transferrin-receptor number and distribution in syncytiotrophoblast in vitro

Insulin and transferrin restore important cellular functions of human fetal kidney in serum-free organ culture

A model previously developed in our laboratory to culture human fetal kidneys in serum-free chemically defined medium was used to evaluate the direct influence of potential regulators on nephrogenesis. The aim of the present work was to verify the effects of insulin and transferrin, two hormones considered as essential in other serum-free culture systems. Explants of renal cortex from human fetuses (15-21 weeks) were cultured for 2 and 5 days in serum-free Leibovitz's L-15 medium (37 degrees C, 95% air - 5% CO2). The addition of transferrin (5 micrograms/mL) had no effect, but insulin (30, 60, and 125 mU/mL) increased DNA and protein syntheses in a dose-dependent manner. The influence of insulin (125 mU/mL) was potentiated by the addition of transferrin and the combination of the two stimulated DNA synthesis by threefold on day 2 when compared with controls and by sixfold on day 5 of culture. After 5 days, synthesis was restored to values observed at day 0. Transferrin did not modify the insulin effect on protein synthesis, since the latter was already maximally stimulated as early as day 2 of culture and at levels well above that of uncultured explants (day 0). The activities of four hydrolases considered as markers of brush border differentiation were not importantly changed by any of the hormones, supplemented alone or in combination. The results indicate that proliferation rather than differentiation is the parameter mostly influenced by these two hormones. The combination of insulin plus transferrin restores cellular functions of human fetal kidney explants cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Positive modulation of hemoglobin, heme, and transferrin receptor synthesis by murine interferon-alpha and -beta in differentiating Friend cells. Pivotal role of heme synthesis.

Administration of highly purified preparations of murine interferon (IFN)-alpha 1, -alpha 4, -alpha 6, or -beta to Friend leukemia cells induced to differentiate by dimethyl sulfoxide leads to a 100% increase of benzidine-positive (B+) cells. Different efficiencies for the two IFN species have been observed; a 10-fold higher dose of IFN-alpha is needed for stimulation of hemoglobin production and inhibition of cell growth as compared with IFN-beta. Both species of IFN induce a substantial increase in heme, hemoglobin, and transferrin receptor levels. In vitro run-on transcription assays indicate that IFN-beta moderately stimulates transcription of the alpha-globin gene but not the transferrin receptor gene. It is postulated that IFN induces the enhancing effect on differentiation via a marked increase of heme synthesis and number of transferrin receptors, which in turn leads to an enhancement of globin chain synthesis. In this regard, the negative feedback reported in a variety of other cell types for the regulation of transferrin receptor expression by heme does not seem to be operative in maturing Friend erythroleukemia cells, which present evidence for a positive mechanism.

The Relationship between 67Ga Uptake and the Number of Transferrin Receptors in Cultured Cells

The Relationship between <SUP>67</SUP>Ga Uptake and the Number of Transferrin Receptors in Cultured Cells

Effect of aluminium on iron uptake and transferrin-receptor expression by human erythroleukaemia K562 cells

Incubation of human erythroleukaemia K562 cells with Al-transferrin inhibited iron uptake from 59Fe-transferrin by about 80%. The inhibition was greater than that produced by a similar quantity of Fe-transferrin. Preincubation of cells for 6 h with either Al-transferrin or Fe-transferrin diminished the number of surface transferrin receptors by about 40% compared with cells preincubated with apo-transferrin. Al-transferrin did not compete significantly with Fe-transferrin for transferrin receptors and, when cells were preincubated for 15 min instead of 6 h, the inhibitory effect of Al-transferrin on receptor expression was lost. Both forms of transferrin also decreased the level of transferrin receptor mRNA by about 50%, suggesting a common regulatory mechanism. Aluminium citrate had no effect on iron uptake or transferrin-receptor expression. AlCl3 also had no effect on transferrin-receptor expression, but at high concentration it caused an increase in iron uptake by an unknown, possibly non-specific, mechanism. Neither Al-transferrin nor AlCl3 caused a significant change in cell proliferation. It is proposed that aluminium, when bound to transferrin, inhibits iron uptake partly by down-regulating transferrin-receptor expression and partly by interfering with intracellular release of iron from transferrin.

Modulation of transferrin-receptor activity and recycling after induced differentiation of BeWo choriocarcinoma cells

BeWo human choriocarcinoma cells normally grow as cytotrophoblast cells. However, in the presence of 100 microM-forskolin or 5 mM-theophylline, these cells form syncytia similar to morphologically well differentiated syncytiotrophoblasts. We have examined the effect of syncytia formation on transferrin-receptor activity and recycling. Although cellular proliferation stops upon growth in the presence of forskolin or theophylline, the number of cell-surface transferrin-receptors unexpectedly increased 2-fold, whereas the total cellular number increased at most 15%. The rate of biosynthesis of the transferrin receptor as well as class I MHC glycoprotein did not change measurably during syncytium formation. The biosynthesis of human chorionic gonadotropin increased 35-fold after 30 h of growth in the presence of theophylline. The redistribution of the transferrin receptor in syncytia is maintained by a decreased rate constant of endocytosis (0.141 min-1 compared with 0.231 min-1 for control cells) and an increased rate constant of externalization (0.122 min-1 compared with 0.060 min-1 for control cells). These altered rates of endocytosis and externalization resulted in an increased rate of iron accumulation in the syncytia. Furthermore, the recycling time of the transferrin receptor decreased in cells grown in the presence of theophylline (14.6 min compared with 21.2 min in control cells).

Iron absorption in the iron-deficient rat

Iron absorption in the iron-deficient rat was compared with that in the normal rat to better understand the regulation of this dynamic process. It was found that: Iron uptake by the iron-deficient intestinal mucosa was prolonged as a result of slower gastric release, particularly when larger doses of iron were employed. The increased mucosal uptake of ionized iron was not the result of increased adsorption, but instead appeared related to a metabolically active uptake process, whereas the increased mucosal uptake of transferrin iron was associated with increased numbers of mucosal cell membrane transferrin receptors. Mucosal ferritin acted as an iron storage protein, but its iron uptake did not explain the lower iron absorption in the normal rat. Iron loading the mucosal cell (by presenting a large iron dose to the intestinal lumen) decreased absorption for 3 to 4 days. Iron loading of the mucosal cell from circulating plasma transferrin was proportionate to the plasma iron concentration. Mucosal iron content was the composite of iron loading from the lumen and loading from plasma transferrin versus release of iron into the body. These studies imply that an enhanced uptake-throughout mechanism causes the increased iron absorption in the iron-deficient rat. Results were consistent with the existence of a regulating mechanism for iron absorption that responds to change in mucosal cell iron, which is best reflected by mucosal ferritin.

Transferrin receptor distribution and regulation in the rat small intestine

Abstract A combination of biochemical quantitation and immunohistochemistry has been used to examine in detail transferrin receptor distribution and expression in the rat small intestine and its relationship to iron absorption. Receptor numbers were quantitated by transferrin binding to preparations of basolateral or brush-border membranes. Receptors were demonstrated on the basolateral membranes of the gut cells, but not on the brush-border fraction. Apotransferrin demonstrated little binding to basolateral membranes at physiological pH. Dietary or parenteral iron loading of animals produced a significant decline in transferrin binding, whereas binding was increased in iron deficiency. These data were confirmed by immunohistochemical studies using a monoclonal antibody to the transferrin receptor. When iron absorption was increased threefold following acute hemolysis and without a decrease in body iron stores, there was no change in transferrin receptor number. These data indicate that intestinal transferrin receptors may be regulated by body iron stores but suggest that they are not directly involved in iron absorption.

Transferrin receptor distribution and regulation in the rat small intestine: Effect of iron stores and erythropoiesis

A combination of biochemical quantitation and immunohistochemistry has been used to examine in detail transferrin receptor distribution and expression in the rat small intestine and its relationship to iron absorption. Receptor numbers were quantitated by transferrin binding to preparations of basolateral or brush-border membranes. Receptors were demonstrated on the basolateral membranes of the gut cells, but not on the brush-border fraction. Apotransferrin demonstrated little binding to basolateral membranes at physiological pH. Dietary or parenteral iron loading of animals produced a significant decline in transferrin binding, whereas binding was increased in iron deficiency. These data were confirmed by immunohistochemical studies using a monoclonal antibody to the transferrin receptor. When iron absorption was increased threefold following acute hemolysis and without a decrease in body iron stores, there was no change in transferrin receptor number. These data indicate that intestinal transferrin receptors may be regulated by body iron stores but suggest that they are not directly involved in iron absorption.

Transferrin Receptor Expression in Rat Liver: Immunohistochemical and Biochemical Analysis of the Effect of Age and Iron Storage

Hepatic transferrin receptors were studied in normal male rats at 1 to 59 wk after weaning, using immunohistochemical and biochemical techniques. The number of transferrin receptors measured and the intensity of the staining in situ decreased rapidly during the first 10 wk of life and more slowly thereafter. Immunohistochemistry further demonstrated changes in the topographical and (sub)cellular localization of the transferrin receptor. In the young rat livers, staining was almost exclusively present on hepatocytes in acinar zone 2 + 3 in a honeycomb to sinusoidal pattern. With aging, a panacinar heterogeneous and mainly sinusoidal staining of hepatocytes was more frequent. Kupffer cell positivity was more obvious as compared with the young rat livers. The observed changes in transferrin receptor expression may partly be explained by age-dependent alterations in DNA synthesis and proliferative potential of the liver cells. A series of rats were iron loaded with carbonyl iron up to 39 wk and "unloaded" by administration of a normal diet during 20 wk. In these animals, serial histochemical studies showed predominantly parenchymal (7 to 14 wk), mixed parenchymal and reticuloendothelial (39 wk) and almost exclusive reticuloendothelial siderosis (59 wk). In the siderotic livers transferrin receptor numbers tended to be lower than in the controls with significant differences after 14 and 39 wk. Immunohistochemistry showed decreased parenchymal but increased reticuloendothelial transferrin receptor expression with iron load. After the period of unloading, parenchymal transferrin receptors were virtually absent despite the negligible siderosis of these cells. In contrast, siderotic reticuloendothelial cells were intensely positive. These findings support down-regulation of parenchymal transferrin receptor resulting from iron storage. However, the positivity of siderotic reticuloendothelial cells and the absence of re-emergence of parenchymal receptors in conditions of minimal parenchymal and prominent reticuloendothelial siderosis need further elucidation.

A rapid redistribution of the transferrin receptor to the cell surface of HL-60 cells and K562 cells upon treatment with dimethyl sulfoxide due to slowing of endocytosis

Treatment of two human leukemia cell lines with 1.25% dimethyl sulfoxide at 37 °C results in a rapid increase in the number of transferrin receptors on the cell surface detected by fluoroscein-labeled anti-transferrin receptor antibodies. Both HL-60 cells, a human myeloid cell line, and K562 cells, a human erythroid—myeloid cell line, showed a 25–65% increase in cell surface transferrin binding in parallel experiments. Scatchard plot analysis of the data indicates that the number of receptors increases while the affinity of transferrin for the receptor remains the same. This rapid increase in the number of receptors at the cell surface appears to be due to a slowing of endocytosis rather than an increase in externalization of the receptor.

Mechanism of Decrease in Transferrin Receptor Synthesis by Interferon-α Treated Human Lymphoblastoid Cells

To clarify the mechanism of antiproliferative action of interferon-α (IFN-α) in hematological malignancy, we examined the transferrin receptor system in the lymphoblastoid cell line, Daudi cells treated with IFN-α. When cells were cultured with 10(4)U/ml of IFN-α, the number of surface transferrin receptors was decreased to 60% of that seen in the control culture. This decrease was not neutralized by co-incubation with the iron chelator, desferrioxamine (10-200 μM), suggesting that the change in the level of chelatable iron did not account for the decrease in transferrin receptor numbers. When determined by metabolic labeling using (35)S-methionine, IFN-α markedly decreased the rate of transferrin receptor biosynthesis. Uptake of iron and the cellular ferritin content also decreased by 50% when incubated with 10(4)U/ml of IFN-α. These data indicate that IFN-α inhibits transferrin receptor biosynthesis in an iron-independent fashion and the subsequent cellular iron-deficiency state may play a role in the antiproliferative action of IFN-α.

Change in transferrin receptor distribution in regenerating rat liver

Partial hepatectomy results in an increase in the ability of the liver cells to bind 125I-labeled transferrin to surface receptors. Scatchard analysis of the binding of transferrin in regenerating rat liver indicates that this increase was due to an increase in the number of transferrin receptors on the cell surface. When we measured total cellular transferrin receptor number, we found that control and regenerating livers had identical amounts of receptor number. The increase in the surface receptor number is apparently due to the translocation of intracellular transferrin receptor to the cell surface.

Analysis by partitioning in aqueous two-phase systems of the loss of transferrin-binding capacity during maturation of rat reticulocytes.

A decrease in the number of binding sites for 125I-transferrin, without an apparent modification of the association constant, has been observed during the maturation of reticulocytes into erythrocytes. As an experimental model, different red cell populations from phenylhydrazinic anaemic rates (95% to 12% reticulocyte-rich) have been used. The fractionation by multiple partition in two-phase systems of these red cell populations has been applied here to show the relationship between number of transferrin receptors and rate of reticulocyte maturation.

The effects of inhibition of heme synthesis on the intracellular localization of iron in rat reticulocytes

These studies assessed the fate and localization of incoming iron in 6-8-day rat reticulocytes during inhibition of heme synthesis by succinylacetone. Succinylacetone inhibition of heme synthesis increased iron uptake by increasing the rate of receptor recycling without affecting receptor KD for transferrin, transferrin uptake, or total receptor number. Its net effect was to amplify the number of surface transferrin receptors by recruitment of receptors from an intracellular pool. Despite increased iron influx in inhibited cells, only 2-4% of total incoming iron was diverted into ferritin. The majority of incoming iron (65-80%) in succinylacetone-inhibited cells was recovered in the stroma, where ultrastructural and enzymic analyses revealed it to be accumulated mainly in mitochondria. Intramitochondrial iron (70-75%) was localized mainly in the inner membrane fraction. Removal of succinylacetone restored heme synthesis, utilizing iron accumulated within mitochondria for its support. Thus, inhibition of heme synthesis in rat reticulocytes results in accumulation of incoming iron in a functional mobile intramitochondrial precursor iron pool used directly for heme synthesis. Under normal conditions, there is no significant intracellular or intramitochondrial iron pool in reticulocytes, which are therefore dependent upon continuous delivery of transferrin-bound iron to maintain heme synthesis. Ferritin plays an insignificant role in iron metabolism of reticulocytes.