The aim of this research is to enhance understanding of the regulation of erythropoiesis and apply this knowledge to clinical anemias. Human burst-forming units-erythroid (BFU-E) and colony-forming units- erythroid (CFU) have been highly purified and the evolution of BFU- erythropoietin (EPO) receptors as well as response to EPO, stem cell factor (SCF), interleukin-3 (IL-3) and insulin-like growth factor I (IGF-I) have been well characterized in a serum-free medium. It has been shown that Fas (CD95) ligand (FasL) is constitutively present in human erythroid progenitors and when interferongamma (IFNgamma) induces the presence of Fas this produces an autocrine and/or paracrine apoptosis which can be overcome with high concentrations of SCF and EPO. In addition, withdrawal of EPO activates the Jun N-terminal kinases (JNKs) and p38 MAP kinase which also produce apoptosis. These cells now will be used to study further the mechanism by which IFNgamma produces apoptosis, by delineating its effect on JNKs/p38 MAP kinase activation, and the mechanism by which EPO inhibits IFNgamma-induced apoptosis. This will determine how IFNgamma- induced apoptosis is regulated in primary human erythroid progenitors and how EPO modulates this process and prevents apoptosis. Preliminary experiments demonstrate that IFNgamma activates JNKs and p38 MAP kinase and that specific antisense oligonucleotides block this and block IFNgamma-induced apoptosis. The role of one death resistance pathway Usurpin (which is the same as FLIP, CASH, CASPER) also will be studied. We have found that Usurpin is present in the normal human CFU-E, but is greatly decreased in myelodysplasia (MDS) marrow cells, which have enhanced apoptosis. We will determine if hyperexpression of Usurpin reduces apoptosis in MDS marrow cells. We also have demonstrated a major synergistic activation of MAP kinase (ERK1/2) by EPO and SCF, which is essential for expanded erythropoiesis, and that SCF and EPO prevent apoptosis through different pathways. We will further investigate these transduction systems by determining the role of specific signaling molecules in EPO- and SCF- stimulated erythropoiesis. These experiments have direct application to understanding the anemia of chronic disease and the increased apoptosis in MDS, which produces a marked decrease in human blood cells.
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