Ras signaling plays an important role in erythropoiesis. Its function has been extensively studied in erythroid and nonerythroid cell lines as well as in primary erythroblasts, but inconclusive results using conventional erythroid colony-forming unit (CFU-E) assays have been obtained concerning the role of Ras signaling in erythroid differentiation. Here we describe a novel culture system that supports terminal fetal liver erythroblast proliferation and differentiation and that closely recapitulates erythroid development in vivo. Erythroid differentiation is monitored step by step and quantitatively by a flow cytometry analysis; this analysis distinguishes CD71 and TER119 doublestained erythroblasts into different stages of differentiation. To study the role of Ras signaling in erythroid differentiation, different H-ras proteins were expressed in CFU-E progenitors and early erythroblasts with the use of a bicistronic retroviral system, and their effects on CFU-E colony formation and erythroid differentiation were analyzed. Only oncogenic H-ras, not dominant-negative Hras, reduced CFU-E colony formation. Analysis of infected erythroblasts in our newly developed system showed that oncogenic H-ras blocks terminal erythroid differentiation, but not through promoting apoptosis of terminally differentiated erythroid cells. Rather, oncogenic H-ras promotes abnormal proliferation of CFU-E progenitors and early erythroblasts and supports their erythropoietin ( IntroductionWithin the fetal liver and the adult bone marrow, hematopoietic cells are formed continuously from a small population of pluripotent stem cells that generate progenitors committed to one or a few hematopoietic lineages. In the erythroid lineage, the earliest committed progenitors identified ex vivo are the slowly proliferating erythroid burst-forming units (BFU-Es). 1,2 These early BFU-E cells divide and further differentiate through the "mature" BFU-E stage into rapidly dividing erythroid colony-forming units (CFUEs). Neither of these 2 types of progenitors is identified by morphology but instead by the colonies they produce in colonyformation assays. BFU-E colonies take 15 days (human) or 7 to 10 days (mouse) to form in culture, whereas CFU-E colonies take 7 days (human) or 2 days (mouse). 1,2 CFU-E progenitors undergo 3 to 5 divisions as they differentiate through several morphologically defined stages: proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts, and orthochromatophilic erythroblasts (OEs). Finally, the OEs extrude their nuclei (enucleation) and become reticulocytes, which further expel all organelles and detach from their microenvironment to form mature circulating erythrocytes. As erythroid differentiation proceeds, erythroblasts display a gradual decrease in cell size, increase in chromatin condensation, and increase in hemoglobin concentration. 3 Several cytokines and their receptors are important for erythroid differentiation. Among them, erythropoietin (Epo) and its specific receptor (EpoR) are crucial for pro...
We have constructed a genetic map of the mouse genome containing 4,006 simple sequence length polymorphisms (SSLPs). The map provides an average spacing of 0.35 centiMorgans (cM) between markers, corresponding to about 750 kb. Approximately 90% of the genome lies within 1.1 cM of a marker and 99% lies within 2.2 cM. The markers have an average polymorphism rate of 50% in crosses between laboratory strains. The markers are distributed in a relatively uniform fashion across the genome, although some deviations from randomness can be detected. In particular, there is a significant underrepresentation of markers on the X chromosome. This map represents the two-thirds point toward our goal of developing a mouse genetic map containing 6,000 SSLPs.
Erythropoietin (Epo) is essential for the production of mature red blood cells, and recombinant Epo is commonly used to treat anemia, but how Epo is degraded and cleared from the body is not understood. Glycosylation of Epo is required for its in vivo bioactivity, although not for in vitro receptor binding or stimulation of Epo-dependent cell lines; Epo glycosylation actually reduces the affinity of Epo for the Epo receptor (EpoR). Interestingly, a hyperglycosylated analog of Epo, called novel erythropoiesis-stimulating protein (NESP), has a lower affinity than Epo for the EpoR but has greater in vivo activity and a longer serum half-life than Epo. We hypothesize that a major mechanism for degradation of Epo in the body occurs in cells expressing the Epo receptor, through receptor-mediated endocytosis of Epo followed by degradation in lysosomes, and therefore investigated the trafficking and degradation Erythropoietin (Epo)2 and the erythropoietin receptor (EpoR) are required for the production of mature red blood cells (1); recombinant Epo is commonly used to treat anemia in cases of chronic renal failure, cancer, and AIDS. Epo contains one O-linked and three N-linked carbohydrate chains, each having 2-4 branches that often end in a negatively charged sialic acid. These carbohydrate chains are not required for receptor binding in vitro or stimulation of growth of EpoR-expressing cultured cells but are required for the in vivo bioactivity of Epo to increase red blood cell mass (2). Heterogeneous branching of Epo N-linked carbohydrates results in Epo isoforms with different sialic acid contents up to a maximum of 14. Epo isoforms with higher sialic acid content have a lower affinity for EpoR but a longer serum half-life and are more effective for stimulating the production of red blood cells in vivo (3).How Epo is cleared from the circulation and degraded in the body is not understood (4). In addition, we do not know how the sialic acid content of Epo has such a large effect on its serum half-life and in vivo bioactivity. Epo, both produced endogenously or injected, can be cleared from the circulation by filtration into urine. However, only a small fraction of an injected dose of Epo is excreted intact in urine (5); most of the Epo is degraded in the body, and the degradation products of Epo are excreted in urine (6). Where and how this degradation of Epo occurs is not known, and the clearance of Epo from the circulation and degradation in vivo may occur through more than one mechanism. It is possible that extracellular proteases and enzymes could degrade or modify Epo or that Epo could be taken up through unknown mechanisms and degraded in cells that do not express EpoR. However, there is a strong correlation between the affinity of Epo or Epo analogs for the EpoR and the in vivo lifetime of the ligand. In particular, a hyperglycosylated analog of Epo, called novel erythropoiesis-stimulating protein (NESP), was engineered to contain two additional N-linked carbohydrate chains and a maximum of 22 sialic acids (7...
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