Molecular Analysis of Erythropoiesis In Vivo

促红细胞生成素受体，EPOR，是一种同源二聚体细胞因子受体，对红细胞的形成至关重要。此外，它还是红细胞产生率的主要动态平衡调节剂，动态范围宽。编码EPOR信号定量特征的分子机制在很大程度上尚不清楚，但可能涉及早期红系祖细胞的凋亡控制。这些机制的失调可能导致致癌转化。这一建议的目的是确定编码对EPOR信号的定量反应的分子机制，以及其失调易患白血病。将采取以下措施：1.催乳素受体PrlR是一种非造血细胞因子受体，可以在体外拯救EPOR-/-祖细胞并支持其向红细胞完全分化。这一发现和类似的发现表明，同源二聚体细胞因子受体通过一个类似的“通用”程序发出信号。这一计划的组成部分将使用体外红细胞分化试验进行鉴定。这些可能与其他细胞因子-受体调节系统有关，如乳腺上皮2。尽管PrlR和EPOR在功能上有明显的相似性，但它们在细胞质信号域上几乎没有同源性。小鼠的基因打靶将被用来产生“敲入”模型，该模型允许在体内对尚未怀疑的适应进行严格的测试。这两种受体在组织动态平衡的数量调控上不同的假设将得到检验。3.申请人目前正在开发一种新的流式细胞术分析方法，该方法可以从新鲜移植的造血组织中分析和分离特定成熟阶段的红系前体细胞。与表达微阵列和其他方法一起，这将被用来识别EPOR基因靶点和调节红细胞生成率的细胞功能。这一方法最近通过观察EPOR激活的转录因子Stat 5的突变小鼠而得到验证。这表明早期红细胞中抗凋亡的Stat5-bclxl通路是红系造血率的决定因素。4.利用逆转录病毒表达克隆技术筛选新的红系抗细胞凋亡基因。凋亡机制的组成部分将被评估为红细胞生成率和/或白血病转化的候选调节剂。

The receptor for erythropoietin, EpoR, is a homodimeric cytokine receptor essential for the formation of red cells. In addition, it is the principal homeostatic regulator of red cell production rate, through a wide dynamic range. The molecular mechanisms that encode quantitative features of EpoR signaling are largely unknown, but may involve the control of apoptosis in early erythroid progenitors. Dysregulation of these mechanisms may lead to oncogenic transformation. The purpose of this proposal is to identify molecular mechanisms that encode the quantitative response to EpoR signaling, and whose dysregulation predisposes to leukemia. The following approaches will be taken: 1. The receptor for prolactin, PrlR, a non-hematopoietic cytokine receptor, can rescue EpoR-/- progenitors and support their full differentiation into red cells in vitro. This and similar findings suggest that homodimeric cytokine receptors signal through a similar, 'generic' program. Components of this program will be identified using in-vitro red-cell differentiation assays. These may be relevant to other cytokine- receptor regulated systems such as mammary epithelium 2. In spite of apparent functional similarity, PrlR and EpoR share little homology in their cytoplasmic signaling domains. Gene targeting in mice will be used to generate 'knock-in' models that allow stringent testing in vivo of as yet unsuspected adaptations by these. The hypothesis that these two receptors differ in their quantitative regulation of tissue homeostasis will be tested. 3. A novel flow-cytometry assay that allows analysis and separation of erythroid precursor populations at specific maturation stages from freshly explanted hematopoietic tissue is currently being developed by the applicant. Together with expression microarrays and other approaches, this will be used to identify EpoR gene targets and cellular functions that modulate erythropoietic rate. This approach was recently validated by looking at mice mutant for the EpoR-activated transcription factor Stat 5. This identify the anti-apoptotic Stat5-bcl-xL pathway in early erythroblasts as a determinant of erythropoietic rate. 4. Retroviral expression cloning will be used to identify new erythroid anti-apoptotic genes. Components of the apoptotic machinery will be assessed as candidate modulators of erythropoietic rate and/or leukemogenic transformation.