NOVEL REGULATORS OF LATE STAGE BONE MARROW ERYTHROBLAST DEVELOPMENT

晚期骨髓成红细胞发育的新型调节因子

• 批准号: 8292179
• 负责人: DON Michael WOJCHOWSKI
• 金额: $ 21.49万
• 依托单位: MAINEHEALTH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8292179
• 关键词: Acquired Immunodeficiency Syndrome; Adhesions; Adult; Affect; Aging; Anemia; Attention; Binding; Bone Marrow; CBL gene; CD34 gene; Cell Communication; Cell Polarity; Cell surface; Cells; Cellular Structures; Cellular biology; Complex; Coupled; Development; Disease; Employee Strikes; Erythro; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Event; GATA1 gene; Gene Expression Profile; Genetic; Growth and Development function; Health; Hour; Human; Inherited; Investigation; Island; Kidney Diseases; Knock-out; Knockout Mice; Maps; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Molecular; Molecular Mechanisms of Action; Mus; Myeloproliferation; Nature; Pathway interactions; Physiological; Population; Process; Production; Proteomics; Regulation; Reticulocytes; Role; Serum; Sickle Cell Anemia; Staging; Stromal Cells; System; T-Cell Development; TRAF6 gene; Testing; Transcript; Transgenic Mice; Work; base; beta-1 Globin; cancer therapy; cell growth; chemotherapy; clinically relevant; cohort; in vivo; insight; mouse model; novel; progenitor; public health relevance; selective expression; tissue oxygenation; tumorigenesis; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Late stage erythroblast development is emerging as a sharply regulated process. New insight into key regulators has been won via aggressive genetic and cell biology approaches as recently applied to primary erythroid cell systems (with attention paid to the step-wise nature of erythroblast formation). Examples include Bcl11a and TR2/TR4 repressors of beta-globins (1, 2); miR-150, miR-144/451 as erythro-megakaryocytic transcript decay factors (3-5); and EMP plus ICAM4 as erythroblastic island bridging factors (6, 7). In ongoing studies of murine bone marrow (BM) erythropoiesis, the PI has observed a striking in vivo expansion capacity for a selective cohort of late-stage BM erythroblasts, specifically a KitnegCD71highTer119neg "stage E2" population. Towards characterizing this dynamic new cohort, we've optimized serum-free systems for murine BM erythroblast development, and have purified "stage E2" erythroblasts, their "stage-E1" KitposCD71highTer119neg progenitors, and "stage-E3" KitnegCD71highTer119pos progeny. Upon profiling each, we discovered two E3 ubiquitin ligase adaptors to be selectively expressed at high, sustained levels in late-stage murine and human BM erythroblasts (and subject to GATA1 regulation), TRIB3 pseudokinase and SPRY1. We've further constructed conditional Trib3-/- and Spry1-/- mouse models - and in each have observed selectively skewed erythropoiesis at steady-state; and markedly compromised erythropoiesis during anemia. In Aim #1, we now propose to define specific stages, and physiological conditions, during which TRIB3 and SPRY1 regulate erythroblast development. Effects on cellular pathways also will be investigated; and co-IP plus mass spectrometry approaches will be used to identify TRIB3 and SPRY1 complexed E3 ubiquitin ligases plus coupled erythroid targets. In Aim #2, we will apply transcriptome and phosphoproteome approaches to test the hypothesis that the above "stage E2" erythroblasts are dynamically advantaged in their expansion capacities via stage-specific expression of cell surface adhesion factors that mediate their selective interactions with a supporting BM stromal cell component. Studies promise to provide new insight into factors that (dys)regulate late-stage erythroblast and red cell production; and may uncover rational targets for new anti-anemia agents (as urgently needed in chemotherapy contexts)(8-10). PUBLIC HEALTH RELEVANCE: Hour-to-hour bone marrow production of red blood cells (RBC) is essential for tissue oxygenation. Faltered RBC formation results in health-compromising anemia as associated with cancer therapies, renal disease, aging, AIDS, and inherited RBC disorders (eg, sickle cell anemia). Proposed work promises to discover and characterize novel regulators of erythroblast and RBC production as potentially drugable E3 ligase complexes; and as a new RBC progenitor niche in bone marrow with unique expansion potential.

描述(由申请人提供)：晚期红细胞发育是一个严格调控的过程。通过最近应用于初级红系细胞系统的积极的遗传学和细胞生物学方法，已经获得了对关键调控因素的新见解(注意到红细胞形成的阶梯性)。例子包括β-珠蛋白的BCL11A和TR2/TR4抑制物(1，2)；作为红细胞巨核细胞转录衰退因子的miR-150，miR-144/451(3-5)；以及作为红系岛桥因子的EMP和ICAM 4(6，7)。在正在进行的小鼠骨髓(BM)红细胞生成的研究中，PI观察到了一组选择性晚期BM红细胞的显著体内扩增能力，特别是KitenerCD71HighTer119neg“Stage E2”群体。为了描述这一动态的新队列，我们优化了小鼠骨髓红细胞发育的无血清系统，并纯化了“E2阶段”红细胞、其“阶段-E1”KitposCD71HighTer119-neg祖细胞和“阶段-E3”KitenerCD71HighTer119pos后代。在对每个E3泛素连接酶适配器进行分析时，我们发现两个E3泛素连接酶适配器在晚期小鼠和人类BM红细胞(受GATA1调控)、TRIB3假激酶和SPRY1中有选择性地高水平表达。我们进一步构建了条件Trib3-/-和Spry1-/-小鼠模型，并在每个模型中观察到稳定状态下选择性地倾斜的红细胞生成；以及在贫血期间显著降低的红细胞生成。在目标1中，我们现在建议定义特定的阶段和生理条件，在此期间TRIB3和SPRY1调节红细胞的发育。还将研究对细胞通路的影响；共IP+质谱学方法将用于鉴定TRIB3和SPRY1复合的E3泛素连接酶和偶联的红系靶标。在目标#2中，我们将应用转录组和磷蛋白组学方法来检验这一假设，即上述“E2期”红细胞通过调节其与支持的骨髓基质细胞成分的选择性相互作用的细胞表面黏附因子的阶段特异性表达，在其扩增能力方面具有动态优势。研究有望为调节晚期红细胞和红细胞生成的因素提供新的见解；并可能发现新的抗贫血药物的合理靶点(如化疗环境中迫切需要的)(8-10)。 与公共健康相关：每小时骨髓产生的红细胞(RBC)对于组织氧合是必不可少的。红细胞形成受阻会导致与癌症治疗、肾脏疾病、衰老、艾滋病和遗传性红细胞疾病(如镰状细胞性贫血)相关的危害健康的贫血。拟议的工作有望发现并表征红细胞和红细胞产生的新调节因子，作为潜在的可药物E3连接酶复合体；以及作为具有独特扩展潜力的骨髓中新的红细胞祖细胞利基。