Functions of pRb in Stress Erythropoiesis

描述(申请人提供)：RB肿瘤抑制因子(PRB)在应激性红细胞生成中起关键作用。我们已经证明，在应激条件下，如溶血性贫血、骨髓移植或肿瘤发生，pRb需要调节红细胞的扩张，并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的干细胞和祖细胞的枯竭。然而，解释pRB在应激性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肿瘤抑制因子调节红细胞的分化检查点，该检查点对氧化应激和DNA损伤水平敏感。我们将确定氧化应激和/或DNA损伤是否影响红细胞通过去核退出细胞周期、分化和成熟的能力，以及这种能力是否依赖于功能性的pRb(目标1)。此外，我们将表征RB缺失对DNA修复和氧化应激关键调节因子表达的影响，包括红细胞抗氧化剂。我们还提出，E2F-2是pRB在有丝分裂后红细胞中的关键E2F靶点，通过了解E2F-2是如何调控的，并通过识别生理相关的靶基因，我们将理解为什么pRB在应激性红细胞生成中起关键作用。我们将确定促进E2F-2表达并诱导红细胞生长停滞所需的上游信号通路(目标2)。我们将通过鉴定与E2F-2启动子结合并激活E2F-2启动子的转录因子来表征这些信号通路如何影响E2F-2的转录调控。最后，我们将确定和验证在红细胞分化中受E2F-2和/或pRb调控的基因，这些基因解释了pRb/E2F-2在调节氧化应激、DNA损伤和红细胞成熟方面所起的作用(目标3)。因此，通过研究红细胞管理氧化应激、修复DNA损伤和经历检查点停止的能力如何影响其分化潜力，以及这反过来如何受到pRB和E2F-2的调节，我们将阐明贫血应激后对贫血的增殖反应是如何减弱的，以及再生障碍性贫血、骨髓纤维化和其他血液疾病在人类中是如何发展的。

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.