ERYTHROPOIETIN AND THE REGULATION OF ERYTHROPOIESIS

这项研究的目的是加强对红细胞生成调节的理解，并将这一知识应用于临床贫血。人红系(BFU-E)和红系集落形成单位(CFU)已经得到了高度的纯化，在无血清培养条件下，BFU-EPO受体的进化以及对EPO、干细胞因子(SCF)、白介素3(IL-3)和胰岛素样生长因子I(IGF-I)的反应已经被很好地表征。已有研究表明，Fas(CD95)配体(FasL)存在于人红系祖细胞中，当干扰素(IFN-γ)诱导Fas的存在时，会产生自分泌和/或旁分泌的细胞凋亡，而高浓度的SCF和EPO可以克服这一现象。此外，停用EPO还会激活Jun氨基末端激酶(JNKs)和p38MAPK，从而导致细胞凋亡。这些细胞现在将被用来通过描绘其对JNKs/p38 MAP激酶激活的影响以及EPO抑制IFNGamma诱导的细胞凋亡的机制来进一步研究IFNGamma诱导细胞凋亡的机制。这将决定在原代人类红系祖细胞中如何调控IFNGamma诱导的细胞凋亡，以及EPO如何调控这一过程并防止细胞凋亡。初步实验表明，IFNGamma可激活JNKs和p38MAP，特定的反义寡核苷酸可阻断JNKs和p38MAPK，从而阻断IFNGamma诱导的细胞凋亡。一个死亡抗性途径Usurpin(与Flip、Cash、Casper相同)的作用也将被研究。我们发现Usurpin在正常人CFU-E中存在，但在骨髓发育不良(MDS)骨髓细胞中显著减少，从而促进了细胞凋亡。我们将确定Usurpin的高表达是否减少了MDS骨髓细胞的凋亡。我们还证明了EPO和SCF对MAP激酶(ERK1/2)的主要协同激活，这是扩大红细胞生成所必需的，并且SCF和EPO通过不同的途径阻止细胞凋亡。我们将通过确定特定的信号分子在EPO和SCF刺激的红细胞生成中的作用来进一步研究这些转导系统。这些实验直接应用于了解慢性病的贫血和MDS中细胞凋亡的增加，这会导致人类血细胞的显著减少。

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.