Binding and receptor-mediated endocytosis of erythropoietin in Friend virus-infected erythroid cells

The binding of labeled erythropoietin (EP) to cell surface receptors and subsequent processing of the hormone within the cell was studied in erythroid cells procured from the spleens of mice infected with the anemia strain of Friend virus. These immature erythroid cells respond to EP in culture to differentiate into reticulocytes and erythrocytes. Radiolabeled EP (both iodinated and tritiated) binds to 800-1000 cell surface receptors on these cells at 4 degrees C. Using 125I-EP, we found that 300 of these cell surface receptors have a higher affinity for EP (Kd = 0.09 nM) than the remaining receptors (Kd = 0.57 nM). The number of molecules of EP bound per cell increased about 2-fold when binding was carried out at 37 degrees C. Treatment of the cell surface with pronase or removal of surface-bound EP with a low pH wash revealed that radiolabeled EP is internalized by the cells at 37 degrees C. Pulse chase experiments showed that degradation products of radiolabeled EP are released into the medium with a corresponding loss of label from the interior of the cell. Inhibitors of lysosomal function greatly reduced this degradation of 125I-EP. Since 180 of the 300 high affinity receptors and very few of the low affinity receptors are occupied at the concentration of EP which elicits the maximum biological response in these cells, we suggest that interaction of EP with the high affinity receptors are necessary for the full biological effect of the hormone. A different murine erythroleukemia cell line which does not differentiate in response to EP was found to have only the lower affinity binding sites for the hormone.     

Erythropoietin stimulates 45Ca2+ uptake in Friend virus-infected erythroid cells

It has been shown previously in this laboratory that in vitro infection of mouse bone marrow cells with the anemia strain of Friend leukemia virus leads to growth of large bursts of erythroid cells which are arrested in development prior to hemoglobin synthesis but can respond to erythropoietin (EP) to complete the late stage of erythroblast differentiation. In this study, the effect of EP on the metabolism of 45Ca2+ in these cells was examined. At 4 degrees C, an increased rate of 45Ca2+ uptake and efflux as well as an increase in the steady state level of 45Ca2+ in treated cells was observed. Exchange of 45Ca2+ from preloaded cells at 4 degrees C indicated that treatment with EP increased the size of a rapidly exchanging pool of 45Ca2+ from 5 to 12% of total 45Ca2+ in the cell. The effect of treatment with EP can be seen as increased exchange of extracellular 45Ca2+ with cellular Ca2+; however, an effect of EP on the net level of Ca2+ in these cells cannot be excluded. This investigation demonstrates one of the earliest effects of EP on erythroid cells and suggests that alterations in Ca2+ metabolism may contribute to the progression of erythroid cells to their final development.     

Changes in number, mobility, and topographical distribution of lectin receptors during maturation of chick erythroid cells

Plant lectins have been used to probe changes in cell surface characteristics that accompny differentiation in a complete series of chick erythroid cells. Dramatic differences in lectin receptor mobility were observed between the most immature cells of the series, the proerythroblasts, and cells at the next stage of maturation, the erythroblasts. Both concanavalin A and Ricinus communis agglutinin form caps on proerythroblasts, whereas they develop a patchy distribution on erythroblasts. Erythroid cells at later developmental stages show a homogeneous distribution of surface-bound R. communis agglutinin. Concanavalin A also shows a uniform distribution on the cell periphery, but appears to be concentrated in a ring above the perinuclear region of the cell. In addition to changes in mobility of lectin receptors, a large reduction (50-70%) in the number of lectin receptors per cell accompanies maturation of proerythroblasts to erythroblasts. Pretreatment of the cells with neuraminidase results in enhanced binding of R. communis agglutinin to proerythroblasts. The number of additional R. communis agglutinin receptors exposed by enzyme treatment remains relatively constant during subsequent cell maturation.     

Selective synthesis and modification of nuclear proteins during maturation of avian erythroid cells.

The synthesis of the nuclear proteins of duck erythroid cells at different stages of maturation has been investigated. Synthesis of histone fractions H1, H2a, H2b, H3, and H4 is restricted to the erythroblasts, while synthesis of H5 can be detected even at later stages of maturation after DNA synthesis has ceased. The synthesis of nonhistone nuclear proteins (NHNP), on the other hand, occurs in cells at all stages of maturation although their rates of synthesis decline as the cells mature. The same size classes of NHNP appear to be synthesized in erythroblasts and in early- and midpolychromatic erythrocytes. In late polychromatic erythrocytes the synthesis of a new group of NHNP of molecular weights ranging from 54,000 to 130,000 was observed. This group of proteins does not accumulate in the mature erythrocyte, indicating that their relative proportions are very small.Turnover of histone-bound phosphate was found to occur mainly at the erythroblast stage, except for histone H2a which was actively phosphorylated even at more advanced stages of maturation. Phosphorylation of most of the histones appears to be coupled to histone (and coordinate DNA) synthesis.Incorporation of radioactive acetate into histones occurs at all stages, but the rate of acetylation decreases four- to fivefold with maturation. Although the RNA synthetic activity of erythroid cells also decreases with age, experiments involving the use of RNA polymerase inhibitors suggest that the mechanisms that control RNA synthesis and histone acetylation are not tightly coupled.     

Transferrin endocytosis and iron uptake by developing erythroid cells in the chicken (Gallus domesticus)

Summary The mechanism of iron uptake by avian erythroid cells was investigated using cells from 7 and 15-day chicken embryos, and chicken serum transferrin and conalbumin (ovotransferrin) labelled with¹²⁵I and⁵⁹Fe. Endocytosis of the protein was determined by incubation of the cells with Pronase at 4°C to distinguish internalized from surface-bound protein.Iron was taken up by the cells by receptor-mediated endocytosis of transferrin or conalbumin. The receptors had the same affinity for serum transferrin and conalbumin. Endocytosis of diferric transferrin and conalbumin and exocytosis of apo-protein occurred at the same rates, indicating that iron donation to the cells occurred during the process of intracellular cycling of the protein. The recycling time was approximately 4 min. The rate of endocytosis of diferric protein varied with incubation temperature and at each temperature the rate of endocytosis was sufficient to account for the iron accumulated by the cells. These results and experiments with a variety of inhibitors confirmed the role of endocytosis in iron uptake.The mean cell volumes, receptor numbers and iron uptake rates of 7-day embryo cells were approximately twice those of 15-day embryo cells but the protein recycling times were approximately the same. Hence, the level of transferrin receptors is probably the main determinant of the rate of iron uptake during development of chicken erythroid cells.Transferrins from a variety of mammalian species were unable to donate iron to the chicken cells, but toad (Bufo marinus) transferrin could do so at a slow rate. The mechanism of iron uptake by developing chicken erythroid cells appears to be similar to that described for mammalian cells, although receptor numbers and iron uptake rates are lower than those reported for mammalian cells at a similar stage of development.Abbreviations BSS Hanks balanced salt solution - PBS phosphate buffered saline - MCV mean corpuscular volume - CCCP carbonyl cyanide-M-chlorophenyl hydrazone     

Early transport changes during erythroid differentiation of Friend leukemic cells

Early transport changes occurring during Friend erythroleukemic cell differentiation are reported. A decrease in the rate of 86Rb transport was observed beginning approximately five hours after stimulation with 1.5% dimethylsulfoxide (DMSO), a potent inducer of Friend cell differentiation. By 12 to 14 hours after DMSO addition, the transport rate had stabilized at close to 60% of control level. This decrease in the rate of 86Rb transport preceded a previously reported decrease in cell volume. Other chemical inducers of Friend cells, such as hypoxanthine and ouabain, also caused early decreases in 86Rb influx. In contrast, xanthine, which does not induce Friend cell differentiation, also did not affect 86Rb influx. The transport of two amino acid analogues, alpha-aminoisobutyric acid and 2-aminobicyclo [2,2,1]-heptane-2-carboxylic acid, which differ in their mode of uptake, was also measured following induction by DMSO. The transport rates of both compounds decreased after a 12-hour exposure to DMSO. In contrast, the uptake of 3H-colchicine, a drug which diffuses passively across the cell membrane, was not significantly affected. Studies with several variant cell lines which do not synthesize hemoglobin in response to DMSO indicate that these non-inducible cells can be divided into two classes--those that demonstrate early changes in transport very similar to the changes observed in inducible cell lines and those which exhibit only small changes in transport. Results obtained using a revertant clone have helped to distinguish between those transport changes which are associated with the induction of hemoglobin synthesis and those which are not. In addition, these early transport changes may be useful in defining the stage in the differentiation process at which a particular variant line is blocked.     

Transferrin receptors and iron uptake during erythroid cell development

Experiments were performed to determine the level of transferrin receptors and rate of transferrin-bound iron uptake by various immature erythroid cell populations. Developing erythroid cells from the rat and mouse foetal liver at various stages of gestation were studied. In addition Friend leukaemic cells grown in culture were examined. The transferrin receptor level of Friend cells was similar to that of erythroid cells from the mouse foetal liver. During erythroid cell development the transferrin receptor level increased from about 300,000 per cell at the early normoblast stage to reach a maximum of about 8000,000 per cell on intermediate normoblasts. Further maturation of intermediate normoblasts was accompanied by a decline in the number of transferrin receptors, reaching a level of 105,000 in the circulating reticulocyte. The rate of iron uptake from transferrin during erythroid cell development was found to correlate closely with the number of transferrin receptors. In each of the immature erythroid cell populations studied the rate of iron uptake was about 36 iron atoms per receptor per hour. These results indicate that the level of transferrin receptors may be the major factor which determines the rate of iron uptake during erythroid cell development.     

Changes in cyclic nucleotide levels during erythroid differentiation in Friend leukemia cells

Changes in cyclic nucleotide levels were observed in Friend leukemia cells exposed to the inducers of erythroid differentiation dimethylsulfoxide (DMSO), sodium butyrate and hypoxanthine. The most extensive changes in cAMP levels occurred during the initial 24 h growth period. In uninduced cultures, cAMP concentration increased 35-fold compared with a 7-fold increase in the presence of inducer. During the same period. cGMP levels rose about 3-fold in uninduced cells compared with a 20-fold increase in induced cells.     

Early and late volume changes during erythroid differentiation of cultured Friend leukemic cells.

Friend erythroleukemic cells (FLC) can be induced to differentiate in vitro by addition of dimethylsulfoxide (DMSO). We have studied the kinetics of induction by measuring cell volume, volume coefficient of variation and cell doubling time. Two distinct volume changes (early and late) are observed after the addition of the inducing agent. The early change occurs after ten hours and consist of a 10-20% decrease in volume compared to an untreated control population. This shift persists for two days and its magnitude is proportional to both the concentration of DMSO and the number of differentiated cells seen on day 5. FLC lines which induce weakly or not all with DMSO exhibit a reduced or absent early volume shift. Inclusion of a local anaesthetic in the culture prevents the appearance of differentiated cells and also counteracts the early volume shift. The exact time of the early volume change is a function of cell growth rate and appears to be cell cycle related. Synchronized cell populations exposed to DMSO during G2 and S phase undergo one round of mitosis before expression of the volume change whereas cells in G2-M express the change only after a second mitosis. A later, more gradual decrease in volume is observed in those cultures which begin to produce hemoglobin. It occurs after approximately five doubling times and coincides with the first appearance of hemoglobin-containing cells. Volume distribution parameters indicate that only a proportion of the population becomes smaller in size.     

HS1 interacts with Lyn and is critical for erythropoietin-induced differentiation of erythroid cells

Erythroid cells terminally differentiate in response to erythropoietin binding its cognate receptor. Previously we have shown that the tyrosine kinase Lyn associates with the erythropoietin receptor and is essential for hemoglobin synthesis in three erythroleukemic cell lines. To understand Lyn signaling events in erythroid cells, the yeast two-hybrid system was used to analyze interactions with other proteins. Here we show that the hemopoietic-specific protein HS1 interacted directly with the SH3 domain of Lyn, via its proline-rich region. A truncated HS1, bearing the Lyn-binding domain, was introduced into J2E erythroleukemic cells to determine the impact upon responsiveness to erythropoietin. Truncated HS1 had a striking effect on the phenotype of the J2E line-the cells were smaller, more basophilic than the parental proerythoblastoid cells and had fewer surface erythropoietin receptors. Moreover, basal and erythropoietin-induced proliferation and differentiation were markedly suppressed. The inability of cells containing the truncated HS1 to differentiate may be a consequence of markedly reduced levels of Lyn and GATA-1. In addition, erythropoietin stimulation of these cells resulted in rapid, endosome-mediated degradation of endogenous HS1. The truncated HS1 also suppressed the development of erythroid colonies from fetal liver cells. These data show that disrupting HS1 has profoundly influenced the ability of erythroid cells to terminally differentiate.     

Alterations in the protein synthetic apparatus of Friend erythroleukemia cells during their erythroid differentiation

The changes in rate of protein synthesis and cell division and the distribution of polyribosomes and globin mRNA on the polyribosomes of Friend erythroleukemia (FL) cells exposed to 2% DMSO and maintained at low cell density, were examined at different times after exposure to DMSO. The rate of protein synthesis and the capacity of cells to divide declined in concert to 50% of the level found in untreated cell cultures at 24 hours after exposure. Thereafter these rates recovered to 70% of the rate found in untreated control cultures until 96 hours post-exposure and then irreversibly declined as the cells lost the capacity to divide. The proportion of ribosomes present as polyribosomes in cells exposed to DMSO paralleled the capacity of these cells to synthesize protein. The distribution of polyribosomes analyzed by sedimentation in sucrose gradients demonstrated that a discrete, abundant class of polyribosomes composed of pentamers to heptamers appeared as early as 48 hours after exposure to DMSO. The appearance of an abundant class of polyribosomes was correlated with globin synthesis by demonstrating that a discrete class of polyribosomes arises in cells treated with the inducers hexamethylene bisacetamide and hemin.     

Decreased deoxy- -glucose transport in Friend cells during exposure to inducers of erythroid differentiation

Various chemicals will induce Friend cells to undergo erythroid differentiation. Concurrent with this differentiation is a large increase in the synthesis of globin messenger RNA (mRNA) and hemoglobin, occurring several days after the initial exposure of the cells to inducer. An earlier change associated with the Friend cell erythroid differentiation is a decrease in the transport of the glucose analogue, 2-deoxy- -glucose. A substantial transport drop is seen within the first 12 h of exposure to inducer, and this lowered transport rate remains as a characteristic of the induced cell. This change was seen when three different inducers were tested (dimethylsulfoxide, tetramethyl urea and hexamethylene bisacetamide). However, when non-inducible cells (chick embryo and human fibroblasts, and mouse L cells) were exposed to these inducers, no similar decrease in 2-deoxy- -glucose transport was observed. During induction, this decrease in 2-deoxy- -glucose transport was associated with a 3-fold drop in the transport V_max (4.6-1.54 nmoles 2-deoxy- -glucose/10⁶ cells/min) while no change in the transport K_m was observed. -Glucose uptake was less than 3% of the 2-deoxy- -glucose uptake at similar hexose concentrations in both induced and uninduced cells. Further, cytochalasin B (CB) (20 μM) inhibited greater than 90% of the deoxy- -glucose uptake in both cell types. This indicated, most if not all of the total sugar transported was by a carrier-mediated process.     

Expression of transferrin receptors during erythroid maturation

A monoclonal antibody, F111/2Dl, produced after immunisation of C3H/He mice with the human erythroleukemia cell line, K562, is described. It detects cell surface determinants of similar distribution to those characterised by the OKT-9 monoclonal antibody, which has been shown to identify the transferrin receptor. The F111/2Dl antibody, as well as OKT-9, has been used to investigate the distribution of transferrin receptors during erythroid maturation in normal bone marrow and peripheral blood, and on the K562 cell line during erythroid differentiation, induced in vitro.