Functions of pRb in Stress Erythropoiesis

描述（由申请人提供）：Rb肿瘤抑制因子（pRb）在应激红细胞生成中起关键作用。我们已经表明，在应激条件下，如溶血性贫血，骨髓移植或肿瘤发生，需要pRb调节成红细胞扩增和协调细胞周期退出与去核。pRb的缺失导致再生障碍性贫血和骨髓和脾脏干细胞和祖细胞的耗竭。然而，解释pRb在应激红细胞生成中的关键作用的潜在机制尚不清楚。我们假设Rb肿瘤抑制因子调节成红细胞中对氧化应激和DNA损伤水平敏感的分化检查点。我们将确定氧化应激和/或DNA损伤是否影响成红细胞退出细胞周期、通过去核分化和成熟的能力，以及这样做的能力是否依赖于功能性pRb（目的1）。此外，我们将表征Rb缺失对DNA修复和氧化应激的关键调节剂（包括红细胞抗氧化剂）表达的影响。我们还提出，E2 f-2是关键的E2 f目标的pRb在有丝分裂后的成红细胞，并通过了解E2 f-2是如何调节和确定生理相关的靶基因，我们将了解为什么pRb是应激红细胞生成的关键。我们将确定上游信号通路，促进E2 f-2的表达，并需要诱导生长停滞的成红细胞（目标2）。我们将通过识别结合并激活E2 f-2启动子的转录因子来表征这些信号通路如何影响E2 f-2的转录调控。最后，我们将鉴定和验证在分化成红细胞中受E2 f-2和/或pRb调控的基因，这些基因解释了pRb/E2 f-2在调节氧化应激、DNA损伤和红细胞成熟中所起的作用（目的3）。因此，通过研究成红细胞管理氧化应激、修复DNA损伤和经历检查点阻滞的能力如何影响其分化潜能，以及这反过来如何受pRb和E2 f-2的调节，我们将阐明贫血应激后对贫血的增殖反应如何减弱，以及人类再生障碍性贫血、骨髓纤维化和其他血液疾病如何发展。

DESCRIPTION (provided by applicant): The Rb tumor suppressor (pRb) plays a critical role in stress erythropoiesis. We have shown that under stress conditions, such as hemolytic anemia, bone marrow transplant or tumorigenesis, pRb is required to regulate erythroblast expansion and to coordinate cell cycle exit with enucleation. Loss of pRb resulted in aplastic anemia and depletion of stem cells and progenitors from bone marrow and spleen. However, the underlying mechanisms that explain the critical role of pRb in stress erythropoiesis are not known. We hypothesize that the Rb tumor suppressor regulates a differentiation checkpoint in erythroblasts that is sensitive to oxidative stress and levels of DNA damage. We shall determine whether oxidative stress and/or DNA damage affects the ability of erythroblasts to exit cell cycle, differentiate and mature by enucleating and whether the ability to do so is dependent on functional pRb (Aim 1). Furthermore, we shall characterize the effects of Rb loss on expression of key modulators of DNA repair and oxidative stress, including red cell antioxidants. We also propose that E2f-2 is the key E2f target of pRb in post-mitotic erythroblasts and that by understanding how E2f-2 is regulated and by identifying physiologically relevant target genes, we shall understand why pRb is critical for stress erythropoiesis. We shall identify the upstream signaling pathways that promote expression of E2f-2 and are required to induce growth arrest of erythroblasts (Aim 2). We shall characterize how these signaling pathways impinge upon transcriptional regulation of E2f-2 by identifying the transcription factors that bind to and activate the E2f-2 promoter. Finally, we shall identify and validate genes that are regulated by E2f-2 and/or pRb in differentiating erythroblasts that explain aspects of the role played by pRb/E2f-2 in modulating oxidative stress, DNA damage and maturation of red cells (Aim 3). Thus by examining how the ability of erythroblasts to manage oxidative stress, repair DNA damage and undergo checkpoint arrest affects their differentiation potential, and how this in turn is regulated by pRb and E2f-2, we shall shed light on how the proliferative response to anemia is attenuated following anemic stress and how aplastic anemia, myelofibrosis and other blood disorders develop in humans.