Integrated signaling of eicosanoids in HSC formation and regeneration

描述（由申请人提供）：造血系统疾病是美国发病率和死亡率的常见原因。在脊椎动物中，确定性造血干细胞（hsc）在其一生中产生个体的每种成熟血细胞谱系。造血干细胞形成或分化的缺陷可导致儿童毁灭性疾病或白血病。胚胎中第一批造血干细胞起源于环绕背主动脉的区域，称为主动脉-性腺中肾（AGM）区。大多数参与HSC形成的基因在脊椎动物中是高度保守的，并且在成人中继续调节HSC的自我更新和分化。转录因子Runx1是人类白血病染色体突变的常见靶标，在哺乳动物的HSC鉴定中是绝对必需的；runx1的表达在斑马鱼中是保守的，在受精后36小时（hpf）在AGM中出现。通过对斑马鱼runx1表达修饰剂的化学筛选，我们发现与类二十烷酸产生和信号传导相关的化合物可调节HSC数量。二十烷类前列腺素E2 （PGE2）可以调节脊椎动物胚胎HSC的形成和成体稳态。相比之下，相关的二十烷类物质大麻素（CB）在HSC发育和pge2介导的修复中的功能尚未得到解决。我们的长期目标是了解各种因素相互作用影响造血的分子和细胞机制。在斑马鱼和小鼠中，我们的目的是表征类二十烷酸交叉调节对HSC形成、增殖和迁移在发育和损伤后恢复过程中的功能影响。我们的中心假设是两个相关的类二十烷酸亚类，PGE2和CBs，在胚胎发生和再生过程中单独或协同作用于hsc。这一假设来源于我们自己的筛选结果、随后的初步数据以及之前发表的研究。我们工作的基本原理是，详细了解CB配体对pge2介导的HSC生长和增殖的影响，将使治疗骨髓衰竭状态、化疗后恢复或HSC移植后的靶向治疗方法成为可能。在specific Aim 1中，我们计划研究CB激动剂/拮抗剂对斑马鱼造血发育过程中HSC规格和增殖的影响。我们将研究这种活性在骨髓损伤期间和之后的保存情况。在Specific Aim 2中，我们计划分析CB激动剂和PGE2对hsc的联合作用。我们假设PGE2和cb激动剂联合使用可以使AGM中的HSC数量增加到比单独使用这些化合物更高的水平。然后，我们将在斑马鱼和小鼠移植模型以及人类脐带血试验中检查类二十烷酸之间的相互作用是否与指导损伤后骨髓恢复有关。这些结果有望更深入地揭示炎症介质在骨髓再生中的相互作用，并与输血生物学中直接临床结果的组合靶向相关。

DESCRIPTION (provided by applicant): Disorders of the hematopoietic system are a common cause of morbidity and mortality in the United States. In vertebrates, definitive hematopoietic stem cells (HSCs) produce each of the mature blood cell lineages of an individual throughout its lifetime. Defects in HSC formation or differentiation can lead to devastating diseases in childhood or to leukemia. The first HSCs arise in the embryo from an area encompassing the dorsal aorta, termed the aorta-gonad mesonephros (AGM) region. Most genes involved in HSC formation are highly conserved across vertebrates, and continue to regulate HSC self-renewal and differentiation in the adult. The transcription factor Runx1, a frequent target of chromosomal mutation in human leukemia, is absolutely required for HSC specification in mammals; runx1 expression is conserved in zebrafish, appearing robustly by 36 hours post fertilization (hpf) in the AGM. Through a zebrafish chemical screen for modifiers of runx1 expression, we have found that compounds related to eicosanoid production and signaling modulate HSC number. The eicosanoid prostaglandin E2 (PGE2) can regulate embryonic HSC formation and adult homeostasis across vertebrate species. In contrast, the function of related eicosanoids, cannabinoids (CB), in HSC development and PGE2-mediated repair has not been addressed. Our long-term goal is to understand the molecular and cellular mechanisms by which various factors interact to affect hematopoiesis. Our objective here is to characterize the functional implications of eicosanoid cross-regulation on HSC formation, proliferation and migration during development and recovery after injury, in both zebrafish and mice. Our central hypothesis is that two related eicosanoid subclasses, PGE2 and CBs, act individually and in concert to exert specific effects on HSCs during embryogenesis and regeneration. This hypothesis has been derived from our own screening results and subsequent preliminary data as well as previously published studies. The rationale for our work is that a detailed understanding of the impact of CB ligands on PGE2-mediated HSC growth and proliferation will enable targeted therapeutic approaches for the treatment of bone marrow failure states, recovery from chemotherapy, or after HSC transplant. In Specfic Aim 1, we plan to investigate the effects of CB agonists/antagonists on HSC specification and proliferation during hematopoietic development in zebrafish. We will examine the conservation of this activity during and after marrow injury. In Specific Aim 2, we plan to analyze the combined effects of CB agonists and PGE2 on HSCs. We postulate that PGE2 and CB-agonists together can increase HSC number in the AGM to a higher level than each of these compounds alone. We will then examine if the interaction between eicosanoids is relevant in directing enhanced marrow recovery after injury in zebrafish and murine transplantation models and in assays with human cord blood. These results are expected to reveal deeper insight into interactive roles of inflammatory mediators in marrow regeneration and have relevance for combinatorial targeting to direct clinical outcome in transfusion biology. PUBLIC HEALTH RELEVANCE: Hematopoietic stem cells form the foundation of our blood and immune system; the formation and function of these cells are carefully controlled in the body. The proposed research will help to identify mechanisms that regulate the birth and propagation of these stem cells. This work has great relevance for the development of novel agents to regulate leukemia, and for recovery from chemotherapy and bone marrow transplant.