Mechanisms of erythroid differentiation

描述(申请人提供)：影响成熟红细胞产生的疾病对公众健康有重大影响。我们的长期目标之一是了解调节红系终末分化的信号转导途径。为此，我们采用了一种称为Friend病的红系分化的病毒/小鼠模型。在之前的资助期间，我们定位克隆了一种宿主因子FV2，它是感染Friend病的易感性所必需的。我们确定FV2编码受体酪氨酸激酶StK(SF-StK)的截短版本。除了对Friend病的机制有明确的影响外，这一结果还开始对红系分化的调节产生洞察力。感染了Friend病毒贫血诱导株(FVA细胞)的红细胞经历了依赖促红细胞生成素(EPO)的终末分化。没有促红细胞生成素的FVA细胞会发生凋亡。在这里，我们提出的证据表明，促红细胞生成素受体(EPOR)信号不仅在FVA细胞存活中发挥作用，而且也是诱导终末分化所必需的。此外，转录因子信号转导和转录激活因子-5a和-5b(Stat5ab)也是必需的。我们的目标是阐明STAT5下游诱导分化的途径。在FVA细胞中，我们假设Stat5激活了靶基因的表达，这些基因反馈SF-STK和/或EPOR信号来诱导末端分化。为了验证这一假设，我们提出了一系列实验。我们的第一个目标是定义Stat5依赖的途径。我们建议用微阵列识别直接的Stat5靶标；识别SF-StK和EPOR下游信号通路中Stat5依赖的变化；识别细胞蛋白质和翻译后修饰中Stat5依赖的变化；识别蛋白质相互作用中Stat5依赖的变化；以及定位翻译后修饰的位置。我们的第二个目标是研究特定的Stat5靶点在FVA细胞分化中的作用。我们建议研究已知的Stat5靶点，顺式和SOCS3，在来自顺式/-小鼠的FVA细胞和来自SOCS3/-胎肝细胞移植接受者的FVA细胞中的作用。我们还建议研究一个潜在的Stat5靶点，胰岛素受体底物-2(Irs2)，在来自Irs2-/-小鼠的FVA细胞中的作用。IRS2激活一些与远端EPOR相同的信号通路。因此，我们将确定在正常的红系细胞中，Irs2与远端EPOR是否存在功能冗余。

DESCRIPTION (provided by applicant): Diseases that affect the production of mature erythrocytes have a major impact on public health. One of our long term goals is to understand the signal transduction pathways that regulate terminal erythroid differentiation. Towards that end, we have adopted a viral/murine model of erythroid differentiation known as Friend disease. In the previous funding period, we positionally cloned a host factor Fv2 that is required for susceptibility to Friend disease. We determined that Fv2 encodes a truncated version of the receptor tyrosine kinase Stk (Sf-Stk). Besides clear implications for the mechanism of Friend disease, this result is beginning to yield insights into the regulation of erythroid differentiation. Erythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells) undergo Erythropoietin (Epo)-dependent terminal differentiation. FVA cells deprived of Epo undergo apoptosis. Here, we present evidence that Erythropoietin receptor (Epor) signaling not only has a role in FVA cell survival, but is also required for the induction of terminal differentiation. Further, the transcription factors Signal Transducer and Activator of Transcription-5a and -5b (Stat5ab) are required. Our objective is to elucidate the pathway, downstream of Stat5, which induces differentiation. In FVA cells, we hypothesize that Stat5 activates the expression of target genes, which feedback on Sf-Stk and/or Epor signaling to induce terminal differentiation. To test this hypothesis, we propose a series of experiments. Our first aim is to define the Stat5-dependent pathway. We propose to identify direct Stat5 targets with microarrays; to identify Stat5-dependent changes in the signaling pathways downstream of Sf-Stk and Epor; to identify Stat5-dependent changes in cellular proteins and posttranslational modifications; to identify Stat5-dependent changes in protein interactions; and to map sites of post-translational modifications. Our second aim is to examine the role of specific Stat5 targets in FVA cell differentiation. We propose to study the role of known Stat5 targets, Cis and Socs3, in FVA cells derived from Cis-/- mice, and FVA cells derived from Socs3-/- fetal liver cell transplant recipients. We also propose to study the role of a potential Stat5 target, Insulin receptor substrate-2 (Irs2), in FVA cells derived from Irs2-/- mice. Irs2 activates some of the same signaling pathways as the distal Epor. So, we will determine whether Irs2 is functionally redundant with the distal Epor in normal erythroid cells.