BiP binding keeps ATF6 at bay

The erythropoietin receptor transduces signals leading to the growth, differentiation, and survival of red blood cell precursors via interaction with Janus kinase 2 (JAK2). This interaction was thought to occur only at the plasma membrane. Recent evidence, however, shows that JAK2 assembles with newly synthesized erythropoietin receptors in the endoplasmic reticulum, and that this assembly is essential for efficient expression of the receptors at the cell surface.     

Primitive erythropoiesis of mouse teratocarcinoma stem cells PCC3/A/1 in serum-free medium

Mouse teratocarcinoma stem cells PCC3/A/1 differentiated into various types of cells, such as red cells, when they were grown in serum-free medium containing transferrin and bovine serum albumin on a KCF cell feeder layer. These red cells were stained well with 2,7-diaminofluorene (DAF), and therefore were erythroid cells. They were nucleated and contained embryonic globin chains, immunologically identified with antiembryonic hemoglobin antisera after acid urea Triton X-100 polyacrylamide gel electrophoresis (UT-PAGE). The addition of erythropoietin to the culture medium enhanced the production of both embryonic and adult globin chains. The addition of interleukin-3 also enhanced the production of embryonic globin chains, but not the production of adult globin chains. These results indicated that primitive erythropoiesis of PCC3/A/1 teratocarcinoma cells did not require exogenous addition of any hematopoietic factor such as erythropoietin or interleukin-3. This culture system will be a new model system for investigating the factors regulating the primitive erythropoiesis in yolk sac blood islands.     

Chronic psychological stress activates BMP4‐dependent extramedullary erythropoiesis

Psychological stress affects different physiological processes including haematopoiesis. However, erythropoietic effects of chronic psychological stress remain largely unknown. The adult spleen contains a distinct microenvironment favourable for rapid expansion of erythroid progenitors in response to stressful stimuli, and emerging evidence suggests that inappropriate activation of stress erythropoiesis may predispose to leukaemic transformation. We used a mouse model to study the influence of chronic psychological stress on erythropoiesis in the spleen and to investigate potential mediators of observed effects. Adult mice were subjected to 2 hrs daily restraint stress for 7 or 14 consecutive days. Our results showed that chronic exposure to restraint stress decreased the concentration of haemoglobin in the blood, elevated circulating levels of erythropoietin and corticosterone, and resulted in markedly increased number of erythroid progenitors and precursors in the spleen. Western blot analysis revealed significantly decreased expression of both erythropoietin receptor and glucocorticoid receptor in the spleen of restrained mice. Furthermore, chronic stress enhanced the expression of stem cell factor receptor in the red pulp. Moreover, chronically stressed animals exhibited significantly increased expression of bone morphogenetic protein 4 (BMP4) in the red pulp as well as substantially enhanced mRNA expression levels of its receptors in the spleen. These findings demonstrate for the first time that chronic psychological stress activates BMP4‐dependent extramedullary erythropoiesis and leads to the prolonged activation of stress erythropoiesis pathways. Prolonged activation of these pathways along with an excessive production of immature erythroid cells may predispose chronically stressed subjects to a higher risk of leukaemic transformation.     

Activation of erythropoietin signaling by receptor dimerization

The hormone erythropoietin (Epo) is essential for red blood cell development. Epo binds a high affinity receptor on the surface of erythroid progenitor cells, stimulating receptor dimerization and activation of the intracellular signal transduction pathways that support erythroid cell survival, proliferation and differentiation. Biochemical and structural analysis of the erythropoietin receptor (EpoR) is revealing the molecular mechanisms of EpoR function, leading the way to the development of small molecule Epo mimetics. This review focuses on the role EpoR dimerization plays in receptor function.     

Stimulation of erythropoiesis in teratocarcinoma cells by erythropoietin and “Friend inducers”

Murine teratocarcinoma cells (PCC3/A1) formed erythroid cells in the form of blood islands when they were grown in organ culture. Addition of dimethyl sulfoxide (DMSO), N′N-dimethylacetamide and erythropoietin enhanced the formation of blood islands. An additive stimulatory effect was observed when expiants were incubated with DMSO and erythropoietin. In all of these cultures, the formed erythroblasts showed the characteristics of primitive erythroid cells, regardless of the nature of treatment. Small, enucleated red cells were occasionally observed. These results are compared with the characteristics of erythropoiesis in normal adults, embryos and in murine erythroleukemia.     

Inactivation of erythropoietin leads to defects in cardiac morphogenesis.

Erythropoietin is an essential growth factor that promotes survival, proliferation, and differentiation of mammalian erythroid progenitor cells. Erythropoietin(-/-) and erythropoietin receptor(-/-) mouse embryos die around embryonic day 13.5 due, in part, to failure of erythropoiesis in the fetal liver. In this study, we demonstrated a novel role of erythropoietin and erythropoietin receptor in cardiac development in vivo. We found that erythropoietin receptor is expressed in the developing murine heart in a temporal and cell type-specific manner: it is initially detected by embryonic day 10.5 and persists until day 14.5. Both erythropoietin(-/-) and erythropoietin receptor(-/-) embryos suffered from ventricular hypoplasia at day 12-13 of gestation. This defect appears to be independent from the general state of hypoxia and is likely due to a reduction in the number of proliferating cardiac myocytes in the ventricular myocardium. Cell proliferation assays revealed that erythropoietin acts as a mitogen in cells isolated from erythropoietin(-/-) mice, while it has no effect in hearts from erythropoietin receptor(-/-) animals. Erythropoietin(-/-) and erythropoietin receptor(-/-) embryos also suffered from epicardium detachment and abnormalities in the vascular network. Finally, through a series of chimeric analysis, we provided evidence that erythropoietin acts in a manner which is non-cell-autonomous. Our results elucidate a novel role of erythropoietin in cardiac morphogenesis and suggest a combination of anemia and cardiac failure as the cause of embryonic lethality in the erythropoietin(-/-) and erythropoietin receptor(-/-) animals.     

The Notch2–Jagged1 interaction mediates stem cell factor signaling in erythropoiesis

Stem cell factor (SCF), the ligand for the c-kit receptor, is essential for the production of red blood cells during development and stress erythropoiesis. SCF promotes erythroblast proliferation and survival, while delaying erythroid differentiation through mechanisms that are largely unknown. In cultures of primary human differentiating erythroblasts, we found that SCF induces an increase in the expression of Notch2, a member of the Notch family implicated in the control of cell growth and differentiation. Functional inhibition of either Notch or its ligand Jagged1 inhibited the effects of SCF on erythroid cell expansion. SCF also induced the expression of Hes-1 and GATA-2, which may contribute to transduce Notch2 signals in response to SCF. Transduction of primary erythroid precursors with a dominant-negative Notch2 mutant inhibited both basal and SCF-mediated erythroblast expansion, and counteracted the effects of SCF on erythroblast differentiation. These findings provide a clue to understand the effects of increased proliferation and delayed differentiation elicited by SCF on the erythroid compartment and indicate Notch2 as a new player in the regulation of red cell differentiation.     

CNTO 530: molecular pharmacology in human UT-7EPO cells and pharmacokinetics and pharmacodynamics in mice

CNTO 530 is a 58 kD antibody Fc domain fusion protein, created using Centocor's MIMETIBODY platform, that contains two EMP1 sequences as a pharmacophore. CNTO 530 has no sequence homology with EPO but acts as a novel erythropoietin receptor agonist. In UT-7(EPO) cells, CNTO 530 caused protein phosporylation of the erythropoietin receptor associated signaling pathway (Jak2, STAT5, AKT and ERK1/2). CNTO 530 also rescued these cells from apoptosis and mediated proliferation. In mice, pharmacokinetic analysis showed that CNTO 530 was slowly cleared from circulation with a t(1/2) approximately 40 h. Pharmacodynamic analysis in mice showed that a single sc dose of CNTO 530 caused a long-lived stimulation of erythropoiesis that translated into increases in red blood cell counts and hemoglobin values that were maintained for at least 28 d. In conclusion, CNTO 530 is a long-lived EPO-R agonist that stimulates erythropoiesis in a manner similar to epoetin-alpha. These data suggest that CNTO 530 may be an effective treatment of anemia in humans.     

Development of erythropoiesis in the mouse

Hematopoiesis consists of a small number of stem cells which rise to a progeny of several distinct lineages. Among these, cells of the erythroid lineage emerge early in the mammalian embryo and provide indispensable functions throughout gestation and postnatal life. Knowledge of the existence of two distinct erythroid lineages, traditionally known as primitive and definitive, has more than a century. In the last decade, it has become evident that the ontogeny of erythropoiesis is more complex than a simple two-steps model. This review summarizes the development and differentiation of the erythroid lineage cells during gestation, and also the characteristics of erythropoiesis during the infant stage, discussing which issues remain open and which need further research.     

A novel way to induce erythroid progenitor self renewal: cooperation of c-Kit with the erythropoietin receptor

Red blood cells are of vital importance for oxygen transport in vertebrates. Thus, their formation during development and homeostasis requires tight control of both progenitor proliferation and terminal red cell differentiation. Self renewal (i.e. long-term proliferation without differentiation) of committed erythroid progenitors has recently been shown to contribute to this regulation. Avian erythroid progenitors expressing the EGF receptor/c-ErbB (SCF/TGFalpha progenitors) can be induced to long-term proliferation by the c-ErbB ligand transforming growth factor alpha and the steroids estradiol and dexamethasone. These progenitors have not yet been described in mammals and their factor requirements are untypical for adult erythroid progenitors. Here we describe a second, distinct type of erythroid progenitor (EpoR progenitors) which can be established from freshly isolated bone marrow and is induced to self renew by ligands relevant for erythropoiesis, i.e. erythropoietin, stem cell factor, the ligand for c-Kit and the glucocorticoid receptor ligand dexamethasone. Limiting dilution cloning indicates that these EpoR progenitors are derived from normal BFU-E/CFU-E. For a detailed study, mEpoR progenitors were generated by retroviral expression of the murine Epo receptor in bone marrow erythroblasts. These progenitors carry out the normal erythroid differentiation program in recombinant differentiation factors only. We show that mEpoR progenitors are more mature than SCF/TGFalpha progenitors and also do no longer respond to transforming growth factor alpha and estradiol. In contrast they are now highly sensitive to low levels of thyroid hormone, facilitating their terminal maturation into erythrocytes.     

The accumulation of basic nonhemoglobin proteins during the differentiation of primitive (embryonic) red cells of amphibia

The preferential accumulation of hemoglobin is a characteristic of the differentiation of definitive (adult) red blood cells. Since primitive (embryonic or larval) red blood cells have many properties which contrast with definitive red cells, the accumulation of red cell proteins was analyzed during the differentiation of primtive red cells to determine whether or not hemoglobin was the only protein which showed a substantial increase in amount. Primitive red cells of amphibia were used because the mature circulating cell retains large amounts (13%) of specific, well characterized, nonhemoglobin proteins (CP). Preparations of primitive red cells enriched in immature cells were obtained from the circulation of bullfrog tadpoles recovering from phenylhydrazine-induced anemia. The amount of CP, determined by electrophoresis, imunodiffusion, and ion-exchange chromatography, was compared for red cells from normal animals and anemic animals. Mature cells contained three to six times the amount of CP and three times the amount of hemoglobin found in the population of red cells enriched in immature cells. The accumulation of CP during maturation of primitive red cells indicates that differentiation of primitive red cells is less restrictive than differentiation of definitive red cells. Since the primitive red cell is less specialized than the definitive red cell, it is possible that primitive red cells have several roles in the developing animal, in contrast to the single role of synthesizing and maintaining hemoglobin in the adult animal.     

Fetal anemia and apoptosis of red cell progenitors in Stat5a-/-5b-/- mice: a direct role for Stat5 in Bcl-X(L) induction.

The erythropoietin receptor (EpoR) is essential for production of red blood cells; a principal function of EpoR is to rescue committed erythroid progenitors from apoptosis. Stat5 is rapidly activated following EpoR stimulation, but its function in erythropoiesis has been unclear since adult Stat5a-/-5b-/- mice have normal steady-state hematocrit. Here we show that Stat5 is essential for the high erythropoietic rate during fetal development. Stat5a-/-5b-/- embryos are severely anemic; erythroid progenitors are present in low numbers, show higher levels of apoptosis, and are less responsive to Epo. These findings are explained by a crucial role for Stat5 in EpoR's antiapoptotic signaling: it mediates the immediate-early induction of Bcl-X(L) in erythroid cells through direct binding to the Bcl-X promoter.     

Insulin-like growth factor I and erythropoiesis.

The bone marrow, the primary site of hematopoiesis, is a self-renewing system consisting of a unique micro-environment that promotes the differentiation and proliferation of the various hematopoietic cell lines. While many critical factors necessary for red cell production have been identified, the regulation of erythropoiesis has not been completely elucidated. In addition to multi-lineage growth factors (e.g. interleukin 3 or 4) and lineage-specific hematopoietic growth factors (e.g. erythropoietin), several lines of evidence suggest a key role for insulin-like growth factor I (IGF-I). First, growth hormone stimulates erythropoiesis and IGF-I is known to mediate many of growth hormone's actions (somatomedin hypothesis). Second, factors in bovine serum and in serum from an anephric human with erythropoietic activity distinct from erythropoietin have been identified as IGFs. Third, IGF receptors are found on both erythrocyte precursors as well as mature erythrocytes. Fourth, in vitro IGF-I stimulates erythropoiesis in bone marrow cultures. Fifth, IGF-I administration to neonatal or hypophysectomized animals results in increased erythropoiesis in vivo. Recent studies indicate that IGF-I at physiologic concentrations stimulates erythropoiesis and that growth hormone's action is indirect, occurring via IGF-I. The physiologic source of IGF-I for the bone marrow may be delivery from the serum (an endocrine mechanism) or synthesis within the bone marrow by stromal or other cells (a paracrine mechanism). Our recent studies have shown that mouse bone marrow stromal cells secrete both IGF-I and IGF binding proteins (IGFBPs). The role of IGFBPs in erythropoiesis is not known, but they might modulate the local concentration of IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)