Regulation of erythroid progenitors in steady-state and stress erythropoiesis by the VDR transcription factor

项目摘要 红细胞生成是由红系祖细胞产生红细胞的过程。在……下面 正常情况下，每天产生的红细胞超过1000亿。然而，这个数字必须在 对缺氧、出血、溶血引起的贫血或其他减少的情况的反应 血液中的红细胞数量或血红蛋白水平。世界卫生组织估计超过16亿 世界各地的人们都患有贫血症，导致严重的发病率和死亡率。调控机制 对贫血或应激性红细胞生成的反应尚不完全清楚。类固醇信号转导 激素受体，特别是糖皮质激素受体，对于红系反应是必需的。 压力。Baron实验室之前的工作已经确定了维生素D3受体(VDR)转录因子 作为红系祖细胞体外增殖的调节剂。VDR是类固醇激素家族中的一员 转录因子。维生素D3配体的结合激活VDR并诱导其移位到 核，在那里它招募转录共调节复合体。VDR信号通路一直是 主要研究骨骼和癌症。通过这一途径对红细胞生成的调节基本上是 未被开发的。这项建议的目的是阐明VDR信号调节的机制 红血球生成。我的初步数据表明，VDR信号调节细胞细胞控制基因。我会先来 测试VDR信号是否通过调节细胞周期维持祖细胞并延缓其分化。我 将决定胎肝红系祖细胞中VDR的激活是否会影响细胞周期长度。vbl.使用 针对CDK4/6的小分子抑制剂，我会问是否在培养的胎肝中阻断它们的功能 红系祖细胞模仿VDR激活的效果。我将确定VDR信号是否会改变 细胞周期蛋白D-CDK4/6复合靶蛋白Rb的表达和/或磷酸化状态。接下来我要测试一下 VDR信号在体内应激性红细胞生成中起作用的假说。我将使用 VDR基因敲除小鼠，测试VDR在应激反应中的作用。VDR缺失突变体、杂合型和野生型 将检查小鼠对静脉切开术或苯肼诱导的应激性红细胞生成的反应。 将分析应激和非应激小鼠外周血中红系参数的变化 计数、红细胞压积和血红蛋白)、脾大小(小鼠应激性红细胞生成的部位)和红系 成人骨髓和脾分离细胞的祖细胞潜能。此外，细胞分裂的速度 以及关键细胞周期、红系调节因子和应激性红细胞生成相关基因的表达 分析过了。这些研究的主要发现将使用原代人类CD34+红系祖细胞进行验证 在常氧和低氧条件下培养，并加入VDR shRNA慢病毒或对照 下调VDR的表达。成功完成本提案中概述的目标可以确定 调节红细胞生成的新机制和途径，并导致治疗贫血的新方法。

Project Summary Erythropoiesis is the process by which red blood cells (RBC) are produced from erythroid progenitors. Under normal conditions more than 100 billion RBC are produced each day. However, this number must increase in response to anemic conditions caused by hypoxia, hemorrhage, hemolysis, or other conditions that reduce RBC number or hemoglobin levels in the blood. The World Health Organization estimates more than 1.6 billion people suffer from anemia worldwide leading to significant morbidity and mortality. Mechanisms regulating erythropoiesis in response to anemia, or stress erythropoiesis, are not fully understood. Signaling by steroid hormone receptors, specifically the glucocorticoid receptor, are necessary for an erythropoietic response to stress. Previous work by the Baron laboratory has identified the Vitamin D3 receptor (VDR) transcription factor as a regulator of erythroid progenitor proliferation in vitro. VDR is a member of the steroid hormone family of transcription factors. Binding of the Vitamin D3 ligand activates VDR and induces its translocation into the nucleus, where it recruits transcriptional coregulatory complexes. The VDR signaling pathway has been studied mostly in bone and in cancers. The regulation of erythropoiesis by this pathway has been essentially unexplored. The aim of this proposal is to elucidate the mechanism by which VDR signaling regulates erythropoiesis. My preliminary data suggest that VDR signaling regulates cell cylce control genes. I will first test whether VDR signaling maintains progenitors and delays their differentiation by regulating the cell cycle. I will determine whether activation of VDR in fetal liver erythroid progenitors influences cell cycle length. Using small molecule inhibitors specific for Cdk4/6, I will ask whether blocking their function in cultured fetal liver erythroid progenitors mimics the effects of VDR activation. I will determine if VDR signaling alters level of expression and/or the phosphorylation status of the cyclin D-Cdk4/6 complex target protein Rb. Next I will test the hypothesis that VDR signaling plays a role in stress erythropoiesis in vivo using mouse models. I will use a Vdr knockout mouse to test the role of VDR in response to stress. Vdr null mutant, heterozygous, and wild type mice will be examined for their response to phlebotomy or phenylhydrazine-induced stress erythropoiesis. Stressed and unstressed mice will be analyzed for changes in erythroid parameters in peripheral blood (RBC counts, hematocrit, and hemoglobin), size of the spleen (the site of stress erythropoiesis in mice), and erythroid progenitor potential of cells isolated from adult bone marrow and spleen. In addition, the rate of cell division and expression of key cell cycle, erythroid regulators, and genes associated with stress erythropoiesis will be analyzed. Key findings from these studies will be validated using primary human CD34+ erythroid progenitors cultured under normoxic and hypoxic conditions in the presence of VDR shRNA lentiviruses or controls to knock down expression of VDR. Successful completion of the objectives outlined in this proposal could identify novel mechanisms and pathways that regulate erythropoiesis and lead to new approaches to treat anemia.