NOVEL REGULATORS OF LATE STAGE BONE MARROW ERYTHROBLAST DEVELOPMENT

描述（由申请人提供）：后期红母细胞发育是一个受到严格调控的过程。通过积极的遗传和细胞生物学方法获得了对关键调节因子的新见解，这些方法最近应用于原代红细胞系统（关注红母细胞形成的阶梯式性质）。例子包括β -珠蛋白的Bcl11a和TR2/TR4抑制因子（1,2）；miR-150、miR-144/451作为红巨核细胞转录物衰减因子（3-5）；EMP + ICAM4作为红母细胞岛桥因子（6,7）。在正在进行的小鼠骨髓（BM）红细胞生成的研究中，PI已经观察到选择性的晚期BM红母细胞群体，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队列，我们优化了小鼠BM红母细胞发育的无血清系统，并纯化了“E2期”红母细胞，它们的“e1期”kitposcd71highter119pos祖细胞和“e3期”KitnegCD71highTer119pos后代。通过对它们的分析，我们发现了两个E3泛素连接酶接头在晚期小鼠和人BM红母细胞（并受GATA1调节），TRIB3假激酶和SPRY1中选择性地以高水平持续表达。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扭曲的红细胞生成；在贫血期间红细胞功能明显受损。在目标1中，我们现在建议定义TRIB3和SPRY1调节红母细胞发育的特定阶段和生理条件。对细胞通路的影响也将被研究；和co-IP +质谱方法将用于鉴定TRIB3和SPRY1络合E3泛素连接酶加上偶联的红系靶标。在Aim #2中，我们将应用转录组学和磷酸化蛋白质组学方法来验证上述“E2期”红母细胞通过细胞表面粘附因子的阶段特异性表达在其扩张能力上具有动态优势，该细胞表面粘附因子介导其与支持的BM基质细胞组分的选择性相互作用。研究有望提供对调节晚期红母细胞和红细胞生成的因素的新见解；并可能发现新的抗贫血药物的合理靶点（在化疗环境中迫切需要）（8-10）。

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.