ERYTHROPOIETIN & REGULATION OF ERYTHROPOIESIS

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The aim of this research is to enhance understanding of the regulation of erythropoiesis and the application of this knowledge to clinical anemias and polycythemias. We have purified human colony forming units-erythroid (CFU-E) and have identified erythropoietin (EPO) receptors on these cells. We now propose to purify blood burst-forming units-erythroid (BFU-E) and determine the time course of the evolution of EPO receptors during BFU-E development and differentiation. We will also determine the effect of interleukin-3 (IL-3) granulocyte colony-stimulating factor, granulocytemacorphage colony-stimulating factor, and insulin-like growth factor-I (IGF-I) on down- or up-modulation of EPO receptors. We have also found that while tumor necrosis factor (TNF) and IL-1 depress normal human bone marrow CFU-E development, purified CFU-E are not inhibited by these factors. Thus, TNF and IL-1 are apparently acting on accessory cells to produce a factor that is released and inhibits CFU-E. We will determine the identity of these intermediate accessory cells, purify the factor that is being released, and measure the effect of this factor on human CFU-E EPO receptors to determine if TNF and/or IL-1 down- module EPO receptors as a mechanism for producing the anemia. We will also further characterize the EPO receptor during erythroid differentiation. A heterobifunctional, photoreactive, cleavable, iodinated cross-linker will provide radioactive EPO receptors which will be characterized for molecular size, isoelectric point, glycosylation and phosphorylation capacity. The EPO receptor gene will be obtained and will be used to measure the effect of EPO and IGF-I on EPO receptor mRNA in highly purified murine CFU-E as they differentiate. These studies will enhance our knowledge of the events that are controlled by EPO and they will enhance our under-standing of the anemia of chronic disorders. They should also provide a firm base of information for further understanding a wide variety of additional clinical anemia and polycythemias that may occur through the malfunction of normal EPO control mechanisms.