ERYTHROPOIETIN AND THE REGULATION OF ERYTHROPOIESIS

本研究的目的是提高对红细胞生成调控的认识，并将这些知识应用于临床贫血。人红细胞爆发形成单位（BFU-E）和红细胞集落形成单位（CFU）已被高度纯化，并且BFU-红细胞生成素（EPO）受体的进化以及对EPO、干细胞因子（SCF）、白细胞介素-3（IL-3）和胰岛素样生长因子I（IGF-I）的应答已在无血清培养基中被充分表征。已经表明Fas（CD 95）配体（FasL）组成性地存在于人红系祖细胞中，并且当干扰素γ（IFN γ）诱导Fas的存在时，这产生自分泌和/或旁分泌细胞凋亡，其可以用高浓度的SCF和EPO克服。此外，EPO的撤销激活Jun N-末端激酶（JNK）和p38 MAP激酶，其也产生细胞凋亡。这些细胞现在将用于进一步研究IFN γ产生细胞凋亡的机制，通过描述其对JNKs/p38 MAP激酶激活的作用，以及EPO抑制IFN γ诱导的细胞凋亡的机制。这将确定IFN γ诱导的细胞凋亡在原代人红系祖细胞中是如何调节的，以及EPO如何调节该过程并防止细胞凋亡。初步实验表明，IFN γ激活JNKs和p38 MAP激酶，特异性反义寡核苷酸阻断了这一点，并阻断了IFN γ诱导的细胞凋亡。还将研究一种死亡抵抗途径Usurpin（与FLIP、CASH、CASPER相同）的作用。我们已经发现Usurpin存在于正常人CFU-E中，但在骨髓增生异常（MDS）骨髓细胞中大大减少，这增强了细胞凋亡。我们将确定Usurpin的过表达是否减少MDS骨髓细胞的凋亡。我们还证明了EPO和SCF对MAP激酶（ERK 1/2）的主要协同激活作用，这对扩大的红细胞生成至关重要，SCF和EPO通过不同的途径防止细胞凋亡。我们将通过确定EPO和SCF刺激的红细胞生成中特定信号分子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疾病的贫血和MDS中细胞凋亡的增加，这导致人类血细胞的显着减少。

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.