NOVEL REGULATORS OF LATE STAGE BONE MARROW ERYTHROBLAST DEVELOPMENT

描述（由申请人提供）：晚期成红细胞发育是一个受到严格调控的过程。通过最近应用于原代红系细胞系统的积极的遗传和细胞生物学方法（注意成红细胞形成的逐步性质），已经赢得了对关键调控因子的新见解。实例包括β-球蛋白的Bcl 11 a和TR 2/TR 4阻遏物（1，2）;作为红-巨核细胞转录物衰减因子的miR-150、miR-144/451（3-5）;以及作为成红细胞岛桥接因子的EMP加ICAM 4（6，7）。在正在进行的小鼠骨髓（BM）红细胞生成研究中，PI观察到晚期BM成红细胞的选择性队列（特别是KitchenCD 71 highTer 119阴性“E2期”群体）具有显著的体内扩增能力。为了表征这种动态的新队列，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”KitposCD 71 highTer 119 neg祖细胞和“E3期”KitposCD 71 highTer 119 pos子代。在分析每一个后，我们发现两个E3泛素连接酶衔接子在晚期鼠和人BM成红细胞中以高的、持续的水平选择性表达（并且受到GATA 1调节），TRIB 3假激酶和SPRY 1。我们进一步构建了条件性Trib 3-/-和Spry 1-/-小鼠模型，并在每种模型中观察到稳定状态下选择性偏斜的红细胞生成;以及贫血期间红细胞生成明显受损。在目标#1中，我们现在提出定义TRIB 3和SPRY 1调节成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;将使用co-IP加质谱法来鉴定TRIB 3和SPRY 1复合的E3泛素连接酶加偶联的红细胞靶点。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期”成红细胞通过介导其与支持BM基质细胞组分的选择性相互作用的细胞表面粘附因子的阶段特异性表达而动态地抑制其扩增能力。研究承诺提供新的洞察因素（dys）调节晚期成红细胞和红细胞的生产;并可能发现新的抗贫血药物的合理目标（在化疗环境中迫切需要）（8-10）。 公共卫生相关性：每小时骨髓产生的红细胞（RBC）对组织氧合至关重要。红细胞形成异常会导致与癌症治疗、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）相关的危及健康的贫血。拟议的工作有望发现和表征成红细胞和红细胞生产的新调节剂作为潜在的药物E3连接酶复合物;并作为骨髓中具有独特扩增潜力的新的红细胞祖细胞生态位。

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.