An understanding of the mechanisms controlling erythropoiesis is of great importance to efforts directed toward the regulation of red cell production in both inherited and acquired anemias. In this regard the hormone erythropoietin stands out as a prime regulator of this differentiation process. The erythropoietin-responsive Rauscher murine erythroleukemia cell line provides an excellent model system with which to examine the cellular physiology and biochemistry of the erythropoietin response. These cells can be grown continuously in vitro and undergo terminal erythroid differentiation, including characteristic morphological changes and the synthesis of erythroid specific markers such as hemoglobin and spectrin, in response to erythropoietin as well as synthetic inducers. Rauscher cells will be used to answer several fundamental questions about the mechanism of action of erythropoietin, specifically regarding the transducer mechanism(s) operative in the erythropoietin response. A kinetic and clonal analysis of induction will demonstrate whether the hormone activates a """"""""master switch"""""""" of commitment or whether its presence is important throughout erythroid differentiation. Using immobillized hormone it will be determined if erythropoietin must enter the cell and perhaps function by a """"""""first messenger"""""""" mechanism or if it is fully active when interacting with only the cell surface. Several subcellular pathways will be examined by a series of experiments using specific pharmacological inhibitors of each, thus providng unambiguous evidence for or against that system's involvement in the hormone's action. They include ion channel activation, changes in membrane electrochemical gradients, enzyme activation and the role of receptor clustering and cytoskeletal systems. In addition the modulation of the erythropoietin response by Beta-adrenergic agents will be explored. Finally, a library fo monoclonal antibodies which recognize progenitor and lineage-specific antigens on normal hematopoietic cells will be expanded, fully characterized, and used to prepare homogenous CFU-E and other progenitor populations, thus permitting an examination of erythropoietin's action on nontransformed erythroid cells. Answers to these fundamental questions will provide new directions for the investigation of erythropoietic regulation.
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