The mechanism whereby the hormone erythropoietin (EP) directs terminal differentiation of erythroblasts into erythrocytes will be studied. A system of in vitro erythropoiesis which is uniquely suited to the study of the effects of EP has been developed in this laboratory. The erythroblasts used in this system are developmentally synchronized in that they have not begun hemoglobin synthesis and yet they do undergo complete erythroid differentiation including hemoglobin synthesis and enucleation in vitro when exposed to EP. These erythroblasts are obtained in sufficient numbers and purity to allow serial biochemical analyses during EP-mediated terminal erythroid differentiation. How EP initiates globin transcription in erythroblasts will be investigated by determining whether an activator protein binds to the promoter region of globin genes and how the release, activation or synthesis of such an activator occurs in the cell which is exposed to EP. Also, whether the product of the adenovirus early gene, E1A, can initiate globin transcription will be determined. EP-induced changes in synthesis and accumulation of non-globin proteins will be determined in several subcellular fractions of the erythroblasts. How the EP--induced post-translational modifications of phosphorylation and glycosylation affect the association of the cytoskeleton and plasma membrane will be determined in the developing erythroblasts. Monoclonal antibodies will be raised against proteins of the erythroblasts both before and after EP treatment. These antibodies will be used as reagents in the study of those proteins which characterize erythroid cells before the later, terminal stages of differentiation as well as those proteins whose synthesis and accumulation are specifically induced by EP. The dependence of erythroblasts upon EP for survival in vitro will be explored in terms of EP's effects on glucose transport, energy flux,and Ca++ ion flux. The research described here will help in understanding how EP controls the later stages of normal erythroid cell development and should shed light on diseases such as polycythemia vera and erythroleukemia as well as anemia due to chronic disease or aplasia. With the recent molecular cloning of EP and the availability of recombinant hormone for possible clinical applications, the knowledge of how the hormone causes its effects on erythroid cells may become important clinically as well as in the laboratory.
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