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）：晚期成红细胞发育正在成为一个严格调控的过程。通过最近应用于原代红细胞系统的积极的遗传和细胞生物学方法（关注有红细胞形成的逐步性质），人们对关键调节因子有了新的认识。示例包括 β-珠蛋白的 Bcl11a 和 TR2/TR4 阻遏蛋白 (1, 2)； miR-150、miR-144/451 作为红巨核细胞转录衰减因子 (3-5)；和 EMP 加 ICAM4 作为成红细胞岛桥接因子 (6, 7)。在正在进行的小鼠骨髓 (BM) 红细胞生成研究中，PI 观察到一组选择性的晚期 BM 红细胞，特别是 KitnegCD71highTer119neg“E2 期”群体具有惊人的体内扩增能力。为了表征这个动态的新群体，我们优化了用于小鼠 BM 成红细胞发育的无血清系统，并纯化了“E2 期”成红细胞、其“E1 期”KitposCD71highTer119neg 祖细胞和“E3 期”KitnegCD71highTer119pos 后代。在对每个E3泛素连接酶进行分析后，我们发现了两个E3泛素连接酶接头TRIB3假激酶和SPRY1在晚期小鼠和人类骨髓成红细胞（并受GATA1调节）中选择性地以高、持续的水平表达。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倾斜的红细胞生成；贫血期间红细胞生成明显受损。在目标 1 中，我们现在建议定义 TRIB3 和 SPRY1 调节红细胞发育的特定阶段和生理条件。还将研究对细胞途径的影响； co-IP 加质谱方法将用于鉴定 TRIB3 和 SPRY1 复合 E3 泛素连接酶以及偶联的红细胞靶标。在目标 #2 中，我们将应用转录组和磷酸化蛋白质组方法来检验以下假设：上述“E2 阶段”成红细胞通过细胞表面粘附因子的阶段特异性表达，介导其与支持性 BM 基质细胞成分的选择性相互作用，从而在其扩增能力方面具有动态优势。研究有望为调节晚期成红细胞和红细胞生成（失调）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标（化疗中迫切需要的）(8-10)。 公共卫生相关性：骨髓每小时产生红细胞 (RBC) 对于组织氧合至关重要。红细胞形成障碍会导致危害健康的贫血，这与癌症治疗、肾病、衰老、艾滋病和遗传性红细胞疾病（例如镰状细胞贫血）有关。拟议的工作有望发现并表征成红细胞和红细胞生成的新型调节剂，作为潜在可药物化的 E3 连接酶复合物；作为骨髓中新的红细胞祖细胞利基，具有独特的扩张潜力。