Restricted expression of an MHC alloantigen in cells of the erythroid series: A specific marker for erythroid differentiation

The spectrum of reactivity with various types of cells of a monoclonal antibody (CH-4) which detects a private MHC antigen of chickens was analysed. CH-4 agglutinates only RBCs that possess the B² (MHC) haplotype. A new rosetteforming cell (RFC) assay was devised to detect individual cells (excluding RBCs) that possess the CH-4 specificity on their cell surfaces. RBCs that have CH-4 chemically coupled to their surfaces attach to, and form rosettes with, B² antigen-bearing cells. Most non-RBC RFC were detected in active erythropoietic organs (adult bone marrow and embryonic spleen), and none were found in organs where erythropoiesis does not occur: adult thymus and bursa. Preincubation of bone marrow cells with CH-4 plus complement almost completely inhibits their capacity to form CFU-E without affecting their ability to form GM-CFU. In addition, CH-4 plus complement does not inhibit the capacity of B²/B² lymphocytes to induce a graft-versus-host reaction under conditions where anti-B² lymphocyte alloantisera are completely inhibitory. Our results strongly suggest that CH-4 monoclonal antibodies detect a private specificity on a gene product of the B-G locus whose expression is restricted to erythroid stem cells and erythrocytes.     

The expression and synthesis of the band 3 protein initiates the formation of a stable membrane skeleton in murine Rauscher-transformed erythroid cells.

While the temporal sequences of the synthesis and assembly of membrane skeletal proteins has been studied during erythroid maturation, relatively little is known about the events which initiate the assembly of membrane skeleton at the early stages of mammalian erythroid commitment. To investigate the early events that initiate the assembly of the membrane skeleton in mammalian erythroid cells, we have studied the synthesis and assembly of membrane skeletal proteins in murine Rauscher erythroleukemia virus-transformed cells. These cells are blocked in differentiation at around the early progenitor (burst forming unit-erythroid, BFUe) cell stage but can be induced to differentiate in vitro. Pulse-labeling studies reveal that Rauscher cells actively synthesize alpha spectrin, beta spectrin, ankyrin and band 4.1 proteins. However, the synthesis of the band 3 protein and its mRNA are barely detectable in these cells. The peripheral membrane skeletal components assemble only transiently in the membrane skeleton and turn over rapidly, resulting in about 20-fold lower steady state levels than are found in mature erythrocytes. Upon induction with erythropoietin and dimethyl sulfoxide, the mRNA level and synthesis of band 3 are increased about 50-fold. In contrast, the synthesis of spectrin, ankyrin and band 4.1 is increased only about 1.5 to 2.0-fold. However, after induction, the fraction of these proteins assembled on the membrane is increased, their half-lives on the membrane are nearly doubled with a concomitant 4 to 5-fold increase in their steady-state levels. These results suggest that the synthesis of peripheral membrane proteins is detected at the earliest stages of erythroid commitment and increases only slightly during further differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anion transporter: highly cell-type-specific expression of distinct polypeptides and transcripts in erythroid and nonerythroid cells

Affinity-purified antibodies and cDNA probes specific for the chicken erythrocyte anion transporter (also referred to as band 3) have been used to demonstrate that this protein is expressed in a highly cell-type-specific manner in the avian kidney. Indirect immunofluorescence analysis indicates that this polypeptide is present in only a small subset of total kidney cells and is predominantly localized to the proximal convoluted tubule of this organ. Chicken erythrocytes synthesize and accumulate two structurally and serologically related band 3 polypeptides. The polypeptide that accumulates in kidney membranes has an apparent molecular weight greater than either of its erythroid counterparts. This diversity is also reflected at the RNA level, as the single band 3 mRNA species detected during various stages of erythroid development is distinct in size from that found in kidney cells. Genomic DNA blot analysis suggests that both the erythroid and kidney band 3 RNAs arise from a single gene. Furthermore, of the adult tissues we have examined that are known to express ankyrin and spectrin polypeptides, only kidney accumulates detectable levels of the band 3 mRNA and polypeptide. These observations suggest that a subset of kidney cells use an anion transport mechanism analogous to that of erythrocytes and that band 3 is expressed in a noncoordinate manner with other components of the erythroid membrane skeleton in nonerythroid cells.     

Regulation of differentiation in normal and transformed erythroid cells

Summary Studies are described employing two erythropoietic systems to elucidate regulatory mechanisms that control both normal erythropoiesis and erythroid differentiation of transformed hemopoietic precursors. Evidence is provided suggesting that normal erythroid cell precursors require erythropoietin as a growth factor that regulates the number of precursors capable of differentiating. Murine erythroleukemia cells proliferate without need of erythropoietin; they show a variable, generally low, rate of spontaneous differentiation and a brisk rate of erythropoiesis in response to a variety of chemical agents. Present studies suggest that these chemical inducers initiate a series of events including cell surface related changes, alterations in cell cycle kinetics, and modifications of chromatin and DNA structure which result in the irreversible commitment of these leukemia cells to erythroid differentiation and the synthesis of red-cell-specific products. Presented in the formal symposium on Mechanisms of Cellular Control at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. These studies were supported in part by grants and contracts from the National Institutes of Health (GM-14552, CA-13696, CA-18314, NO1-CB-4008 and NO1-CP-1008) and the National Science Foundation (NSF-PCM-75-08696). E.F. and R.C.R. are fellows of the Schultz Foundation; A.B. was supported in part as an American Cancer Society Scholar; J.E.S. was supported by a USPHS Medical Scientist Training Grant; and M.T. and G.M.M. are Hirschl Trust Scholars.     

Disialoganglioside GD3 synthase expression recruits membrane transglutaminase 2 during erythroid differentiation of the human chronic myelogenous leukemia K562 cells

By employing proteomics analysis tool, we examined the effects of GD3 synthase expression on the differentiation properties of chronic myelogenous leukemia (CML)-derived leukemia cells K562. Forced expression of GD3 synthase induced erythroid differentiation as determined by an increase in glycophorin A expression and synthesis of hemoglobins. The proteomic analysis revealed that 15 proteins were increased by GD3 synthase. In contrast, we observed three protein gel spots decreased in contents in the cell membranes of GD3 synthase-transfected K562 cells. Among the increased proteins, membrane transglutaminase 2 (TG2) was specifically increased in the cell membrane of GD3 synthase-transfected K562 cells. Then, we generated the GD3 synthase-transfected cells in the K562 cells. Interestingly, the TG2 level was increased in GD3 synthase-transfected cells compared with vector- and plasma membrane-associated ganglioside sialidase (Neu3)-transfected cells. In addition, its ability to be photoaffinity-labeled with [alpha-(32)P]GTP was also increased in the GD3 synthase- and TG2-transfected cells. Moreover, small interfering RNA (siRNA) analysis for the GD3 synthase showed the decrease or abolishment of the membrane TG2. Finally, GD3 synthase-transfected cells accelerated the erythroid differentiation. Therefore, we propose that the recruitment of TG2 into membranes by GD3 might play an important role in the erythroid differentiation in K562 cells.     

Emodin can induce K562 cells to erythroid differentiation and improve the expression of globin genes

In China, the traditional Chinese medicine “YiSui ShenXu Granule” has been used for treating β-thalassemia over 20 years and known to be effective in clinic. Several purified components from “YiSui ShenXu Granule” are tested in K562 cells to reveal its effect on globin expression and erythroid differentiation, and one of the purified components, emodin, was demonstrated to increase the expression of α-, ε-, γ-globin, CD235a, and CD71 in K562 cells. Moreover, the increase of their expression is emodin concentration-dependent. The mRNA and microRNA (miRNA) expression profiles are further analyzed and 417 mRNAs and 35 miRNAs with differential expression between untreated and emodin-treated K562 cells were identified. Among them, two mRNAs that encode known positive regulators of erythropoiesis, ALAS2, and c-KIT respectively, increased during emodin-induced K562 erythroid differentiation, meanwhile, two negative regulators, miR-221 and miR-222, decreased during this process. These results indicate that emodin can improve the expression of globin genes in K562 cells and also induce K562 cells to erythroid differentiation possibly through up-regulating ALAS2 and c-KIT and down-regulating miR-221 and miR-222.     

Biogenesis of the avian erythroid membrane skeleton: receptor-mediated assembly and stabilization of ankyrin (goblin) and spectrin

Ankyrin is an extrinsic membrane protein in human erythrocytes that links the alpha beta-spectrin-based extrinsic membrane skeleton to the membrane by binding simultaneously to the beta-spectrin subunit and to the transmembrane anion transporter. To analyse the temporal and spatial regulation of assembly of this membrane skeleton, we investigated the kinetics of synthesis and assembly of ankyrin ( goblin ) with respect to those of spectrin in chicken embryo erythroid cells. Electrophoretic analysis of Triton X-100 soluble and cytoskeletal fractions show that at steady state both ankyrin and spectrin are detected exclusively in the cytoskeleton. In contrast, continuous labeling of erythroid cells with [35S]methionine, and immunoprecipitation of ankyrin and alpha- and beta-spectrin, reveals that newly synthesized ankyrin and spectrin are partitioned into both the cytoskeletal and Triton X-100 soluble fractions. The soluble pools of ankyrin and beta-spectrin reach a plateau of labeling within 1 h, whereas the soluble pool of alpha-spectrin is substantially larger and reaches a plateau more slowly, reflecting an approximately 3:1 ratio of synthesis of alpha- to beta-spectrin. Ankyrin and beta-spectrin enter the cytoskeletal fraction within 10 min of labeling, and the amount assembled into the cytoskeletal fraction exceeds the amount present in their respective soluble pools within 1 h of labeling. Although alpha-spectrin enters the cytoskeletal fraction with similar kinetics to beta-spectrin and ankyrin, and in amounts equimolar to beta-spectrin, the amount of cytoskeletal alpha-spectrin does not exceed the amount of soluble alpha-spectrin even after 3 h of labeling. Pulse-chase labeling experiments reveal that ankyrin and alpha- and beta-spectrin assembled into the cytoskeleton exhibit no detectable turnover, whereas the Triton X-100 soluble polypeptides are rapidly catabolized, suggesting that stable assembly of the three polypeptides is dependent upon their association with their respective membrane receptor(s). The existence in the detergent-soluble compartment of newly synthesized ankyrin and alpha- and beta-spectrin that are catabolized, rather than assembled, suggests that ankyrin and spectrin are synthesized in excess of available respective membrane binding sites, and that the assembly of these polypeptides, while rapid, is not tightly coupled to their synthesis. We hypothesize that the availability of the high affinity receptor(s) localized on the membrane mediates posttranslationally the extent of assembly of the three cytoskeletal proteins in the correct stoichiometry, their stability, and their spatial localization.     

Regulated expression of the c-myb and c-myc oncogenes during erythroid differentiation

We have investigated the expression of the genes c-myb, c-myc, and alpha globin in murine erythroid cells at different stages of development, in viral-induced erythroleukemias, as well as in two mouse erythroleukemia cell lines that can be induced to terminally differentiate when exposed to dimethylsulfoxide. We find that there is a reciprocal correlation between the cell's production of messenger RNA for c-myb and globin. c-myc message shows a similar but less dramatic decrease coincident with globin RNA production. Initially with the administration of an inducing agent, dimethylsulfoxide, there is a rapid decrease of myc and myb mRNA, which is followed by signs of differentiation in the induced culture. We conclude that these oncogenes function in early maturational stages of development of these cells. In the erythroleukemic state these genes are down-regulated by forced differentiation and may play a direct role in influencing the state of differentiation of these cells.     

Regulation of differentiation in normal and transformed erythroid cells.

Studies are described employing two erythropoietic systems to elucidate regulatory mechanisms that control both normal erythropoiesis and erythroid differentiation of transformed hemopoietic precursors. Evidence is provided suggesting that normal erythroid cell precursors require erythropoietin as a growth factor that regulates the number of precursors capable of differentiating. Murine erythroleukemia cells proliferate without need of erythropoietin; they show a variable, generally low, rate of spontaneous differentiation and a brisk rate of erythropoiesis in response to a variety of chemical agents. Present studies suggest that these chemical inducers initiate a series of events including cell surface related changes, alterations in cell cycle kinetics, and modifications of chromatin and DNA structure which result in the irreversible commitment of these leukemia cells to erythroid differentiation and the synthesis of red-cell-specific products.     

Inducible gene expression of activin A/erythroid differentiation factor in HL-60 cells.

Treatment of HL-60 cells with 12-O-tetradecanoyl-phorbol 13-acetate (TPA) for 48 h induced expression of mRNA of beta A chain of activin A/erythroid differentiation factor. Under the same condition, interferon-gamma caused a slight increase in beta A chain mRNA, whereas 1 alpha, 25-dihydroxyvitamin D3, dimethylsulfoxide and all-trans-retinoic acid failed to induce this mRNA in HL-60 cells. Furthermore, 4 h-treatment with TPA or lipopolysaccharide (LPS) induced a marked increase in beta A chain mRNA levels in interferon-gamma-pretreated HL-60 cells. In the cells pretreated with 1 alpha, 25-dihydroxyvitamin D3, TPA and LPS induced as little increase in beta A chain mRNA as in the control cells. Neither alpha nor beta B chain mRNA was detected in any sample. These results indicate that interferon-gamma has a priming effect on the activation of activin A/erythroid differentiation factor gene by TPA or LPS in HL-60 cells.     

Taurine transporter gene expression in peripheral mononuclear blood cells of type 2 diabetic patients

Taurine acts as antioxidant, cell osmolyte, modulator of glucose metabolism, and plays a role in the retinal function. It is 10³-fold more concentrated in the intracellular than in the extracellular milieu due to a specific taurine-Na-dependent transporter (TauT), which is upregulated by hypertonicity, low extracellular taurine, or oxidative stress and acutely downregulated ‘in vitro’ by high glucose concentrations. Aim of this study was to investigate whether TauT expression was modified in mononuclear peripheral blood cells (MPC) of type 2 diabetic patients with or without micro/macrovascular complications. Plasma taurine, as well as other sulphur-containing aminoacids (assayed by HPLC) and TauT gene expression (assayed by real-time PCR analysis) were measured in MPC of 45 controls and of 81 age-and-sex matched type 2 diabetic patients with or without micro/macrovascular complications. Median value (interquartile range) of plasma taurine was significantly lower in diabetic patients than in controls [28.7 (13.7) μmol/l vs. 46.5 (20.3) μmol/l; P?<?0.05], while median TauT expression, in arbitrary units, was significantly higher in diabetics than in controls [3.8 (3.9) vs. 1 (1.3); P?<?0.05) and was related to HbA1c only in controls (r?=?0.34; P?<?0.05). Patients with retinopathy (n?=?25) had lower TauT expression than those who were unaffected [3.1 (2.8) vs. 4.1 (3.4); P?<?0.05], while persistent micro/macroalbuminuria was associated with unchanged TauT expression. A trend toward reduction in TauT expression was observed in patients with macroangiopathy [n?=?27; 3.3 (2.5) vs. 4 [3.7]; P?=?NS]. In conclusion, TauT gene is overexpressed in MPC of type 2 diabetic patients, while presence of retinopathy is specifically associated with a drop in TauT overexpression, suggesting its possible involvement in this microangiopathic lesion.     

Terminal differentiation of yolk-sac erythroid cells of the Syrian hamster in vitro

Summary The developmental fate of Syrian hamster yolk-sac (primitive) erythroid cells was examined in vitro. Highly purified yolk-sac erythroid cells at the polychromatophilic stage, obtained from the peripheral blood of embryos at day 10 of gestation, showed morphological and biochemical changes in our modified semi-solid culture system. Several morphological changes observed in the primitive erythroid cell cultures, such as nuclear condensation, approach of nuclei to the periphery of cells, development by cells of an extended pear-like shape, enucleation, and an increase in haemoglobin content, were quite similar to those of the terminal differentiation of fetal liver or adult bone marrow (definitive) erythroid cells. In addition, the transition of molecular species of haemoglobin from the embryonic to the fetal/adult pattern was also observed in our culture system. Thus we provide evidence, by the in vitro culture of yolk-sac erythroid cells, that primitive erythroid cells undergo terminal differentiation in a manner similar to that of definitive erythroid cells.     

Changes in surface-membrane components during the differentation of rabbit erythroid cells.

The membrane components of rabbit bone-marrow-bound erythroid cells were characterized and compared with those of circulating rabbit erythroid cells. By the criteria of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, radioiodination with lactoperoxidase and binding of radioiodinated lectins, the two circulating forms of erythroid cells (the reticulocyte and erythrocyte) have the same surface components. In contrast, bone-marrow-bound nucleated erythroid cells have a unique set of membrane surface components which are completely different from those found on circulating cells. Of the ten Coomassie-Blue-staining proteins present in nucleated erythroid-cell plasma-membrane preparations, eight are accessible at the extracellular surface, and all of these are lectin-binding glycoproteins. Bone-marrow erythroid cells separated according to age by velocity sedimentation were also studied. The changeover in surface components occurs after the last nucleated stage of the erythroid cells (the orthochromatic normoblast). We discuss the alterations in membrane surface components observed during the differentiation of the erythroid-cell series in relation to the transition from bone-marrow-bound to circulating forms of these cells. We suggest that the change in membrane surface components may be linked to the loss of the nucleus from the normoblast and the entry of the erythroid cell into the circulation.