Mechanisms of erythroid differentiation

红系分化机制

• 批准号: 6989400
• 负责人: PAUL A NEY
• 金额: $ 25.5万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-15 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6989400
• 关键词: Friend leukemia; Friend virus; binding proteins; biological signal transduction; cell differentiation; cell proliferation; cell transformation; disease /disorder model; erythropoiesis; erythropoietin; gene expression; genetically modified animals; growth factor receptors; laboratory mouse; mass spectrometry; microarray technology; phenotype; posttranslational modifications; protein protein interaction; protein structure; proteomics; receptor expression; site directed mutagenesis; transcription factor; viral leukemogenesis; virus genetics; virus protein

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.

描述（由申请人提供）：影响成熟红细胞生成的疾病对公共卫生有重大影响。我们的长期目标之一是了解调控终末红细胞分化的信号转导途径。为此，我们采用了一种称为Friend病的病毒/鼠红细胞分化模型。在上一个资助期，我们定位克隆了一个宿主因子Fv 2，它是对Friend病易感性所必需的。我们确定，Fv 2编码受体酪氨酸激酶Stk（Sf-Stk）的截短版本。除了对Friend病的机制有明确的意义外，这一结果开始对红细胞分化的调节产生深刻的见解。用Friend病毒的贫血诱导株（FVA细胞）感染的成红细胞经历促红细胞生成素（Epo）依赖的终末分化。缺乏Epo的FVA细胞发生凋亡。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细胞存活的作用，但也需要诱导终末分化。此外，还需要转录因子信号转导和转录激活因子-5a和-5b（Stat 5ab）。我们的目标是阐明Stat 5下游诱导分化的途径。在FVA细胞中，我们假设Stat 5激活靶基因的表达，所述靶基因反馈Sf-Stk和/或Epor信号传导以诱导终末分化。为了验证这一假设，我们提出了一系列的实验。我们的第一个目标是确定Stat 5依赖性途径。我们建议用微阵列识别直接Stat 5靶点;识别Sf-Stk和Epor下游信号通路中Stat 5依赖的变化;识别细胞蛋白和翻译后修饰中Stat 5依赖的变化;识别蛋白质相互作用中Stat 5依赖的变化;以及映射翻译后修饰的位点。我们的第二个目的是研究特定Stat 5靶点在FVA细胞分化中的作用。我们建议研究已知的Stat 5靶标Cis和Socs 3在来自Cis-/-小鼠的FVA细胞和来自Socs 3-/-胎肝细胞移植受体的FVA细胞中的作用。我们还建议研究一个潜在的Stat 5目标，胰岛素受体底物-2（Irs 2），在FVA细胞来自Irs 2-/-小鼠的作用。Irs 2激活一些与远端Epor相同的信号通路。因此，我们将确定Irs 2在正常红系细胞中是否与远端Epor功能冗余。