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

VDR 转录因子对稳态和应激红细胞生成中红细胞祖细胞的调节

• 批准号: 9395696
• 负责人: Brad Reinholt
• 金额: $ 3.57万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2018-01-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9395696
• 关键词: Adult; Adverse effects; Affect; Anemia; Binding; Biological; Blood; Bone Marrow; CCND2 gene; CD34 gene; Calcitriol; Cell Count; Cell Cycle; Cell Cycle Regulation; Cell Nucleus; Cell division; Cells; Cholecalciferol; Complex; Cyclin D1; Cyclin-Dependent Kinases; Data; Dexamethasone; Differentiation and Growth; Erythrocyte Transfusion; Erythrocytes; Erythroid; Erythropoiesis; Erythropoietin; Family; Fetal Liver; Generations; Genes; Genetic Transcription; Globin; Glucocorticoid Receptor; Glucocorticoids; Glycoproteins; Health system; Hematocrit procedure; Hemoglobin; Hemoglobin concentration result; Hemolysis; Hemorrhage; Hormones; Human; Hypoxia; In Vitro; Knockout Mice; Laboratories; Lead; Length; Ligands; Malignant Neoplasms; Modeling; Morbidity - disease rate; Mus; Nuclear; Output; Pathway interactions; Patients; Phase; Phosphorylation; Play; Population; Process; Production; Proliferating; Receptor Activation; Receptor Signaling; Recruitment Activity; Red Blood Cell Count; Regulation; Regulator Genes; Regulatory Pathway; Retinoblastoma Protein; Role; Signal Pathway; Signal Transduction; Site; Spleen; Stem cells; Stress; Subfamily lentivirinae; Surface; System; Target Populations; Testing; Tissues; Transfusion; Up-Regulation; Venous blood sampling; Vitamin D3 Receptor; Wild Type Mouse; Work; World Health Organization; bone; global health; improved; in vivo; knock-down; member; mortality; mouse model; mutant; new therapeutic target; novel; novel strategies; peripheral blood; phenylhydrazine; progenitor; response; self-renewal; small hairpin RNA; small molecule inhibitor; steroid hormone; steroid hormone receptor; transcription factor

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.

项目摘要 红细胞生成是由红系祖细胞产生红细胞（RBC）的过程。下 正常情况下，每天产生超过1000亿个RBC。然而，这一数字必须增加， 对由缺氧、出血、溶血或其他降低 血液中的红细胞数量或血红蛋白水平。世界卫生组织估计， 全世界的人患有贫血，导致显著的发病率和死亡率。的调节机制 对贫血反应的红细胞生成或应激性红细胞生成尚未完全了解。类固醇信号传导 激素受体，特别是糖皮质激素受体，是红细胞生成反应所必需的， 应力Baron实验室先前的工作已经确定了维生素D3受体（VDR）转录因子 作为体外红系祖细胞增殖的调节剂。VDR是类固醇激素家族的一员， 转录因子维生素D3配体的结合激活VDR并诱导其易位到 细胞核，在那里它招募转录共调节复合物。VDR信号通路已经被 主要研究骨骼和癌症。通过该途径调节红细胞生成基本上是 未开发的该提案的目的是阐明VDR信号调节的机制， 红细胞生成我的初步数据表明，VDR信号调节细胞周期控制基因。我会先 测试VDR信号是否通过调节细胞周期来维持祖细胞并延迟其分化。我 将确定胎肝红系祖细胞中VDR的激活是否影响细胞周期长度。使用 针对Cdk 4/6的小分子抑制剂，我将询问是否在培养的胎肝中阻断其功能 红系祖细胞模拟VDR活化的作用。我将确定VDR信号是否会改变 细胞周期蛋白D-Cdk 4/6复合物靶蛋白Rb的表达和/或磷酸化状态。接下来我将测试 VDR信号传导在使用小鼠模型的体内应激红细胞生成中起作用的假设。我会用 VDR基因敲除小鼠来测试VDR在应激反应中的作用。VDR无效突变体、杂合子和野生型 检查小鼠对放血术或苯肼诱导的应激性红细胞生成的反应。 分析应激和非应激小鼠外周血（RBC）中红细胞参数的变化 计数、血细胞比容和血红蛋白）、脾的大小（小鼠中应激性红细胞生成的部位）和红细胞 从成人骨髓和脾分离的细胞的祖细胞潜能。此外，细胞分裂的速度 关键细胞周期、红细胞调节因子和与应激红细胞生成相关的基因的表达， 分析了这些研究的关键发现将使用原代人CD 34+红系祖细胞进行验证 在常氧和低氧条件下在VDR shRNA慢病毒或对照存在下培养， 敲低VDR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。