Novel regulators of stress erythropoiesis

说明(申请人提供)：贫血与终末期肾脏疾病、化疗和放射治疗、炎症、骨髓发育不良、铁调节失调和血红蛋白疾病有关，会导致慢性加速的、但有压力的、通常是受损的红细胞生成。对这种应激性红细胞生成的关键调节因素的具体了解对于深入了解新的贫血治疗方法具有基本的重要性。重组人促红细胞生成素本身可以治疗，但有很大的高血压副作用，会引发溶栓事件，是一种高成本的重组糖蛋白，可能会使某些癌症恶化。通过对EPO对原始红系祖细胞影响的研究，我们发现并初步表征了两个重要的新因子(及其相关途径)，它们选择性地调节应激性红细胞生成。首先，我们对EPO诱导的一种新的细胞内Spi2Aserpin的研究(通过一个新的KO模型)表明，在选定的应激条件下，红细胞对溶酶体损伤变得高度敏感，溶酶体执行者组织蛋白-这是由Spi2A所抑制的。如果不加以控制，组织蛋白酶相关的损伤和红细胞死亡会升级，ROS和铁是细胞毒性的辅助因素。我们还证明了组织蛋白酶B/L抑制剂可以表型复制SPI2A的细胞保护作用。我们已经发现(并开始了解)的第二个EPO共同诱导的红细胞生成调节因子是分子适配器和假激酶Trib3。通过建立TRIB3-KO小鼠模型，我们揭示了TRIB3在早期和晚期红细胞形成中的非冗余新的应激性红细胞生成的特定作用。我们现在将通过以下特定目标推进对这两个新的应激性红细胞生成因子和耦合途径的洞察：SA#1将首先定义Spi2A和相关的途径如何影响由β-地中海贫血和铁失衡引起的应激性红细胞生成(#1.1)。在机制水平上，将确定Spi2a缺乏对红细胞自噬的影响，并将定义氧化应激加Spi2a缺乏时红细胞溶酶体造成的分子损伤(#1.2)。通过LOF研究，SA#2将首先确定Trib3假性激酶对β-地中海贫血早期和晚期红细胞生成以及铁失衡的影响(#2.1)。目的探讨TRIB3‘S在早期和晚期红系细胞未折叠蛋白应答通路中的作用以及TRIB3’S的调控分子作用机制。SA#3将推进对原始人类红系祖细胞(包括地中海贫血)的研究，并将首先确定溶酶体妥协和组织蛋白酶抑制对红细胞细胞保护的影响(#3.1)。AIM 3.2将定义Trib3、LOF和GOF在红细胞生成应激中的作用。关于红细胞形成的新调节因子和受损红系祖细胞中用于抗贫血药物的新靶向途径，将获得实质性的见解。

DESCRIPTION (provided by applicant): Anemia as associated with end-stage renal disease, chemo- and radiation therapies, inflammation, myelodysplasia, iron dysregulation and hemoglobinopathies leads to chronic bouts of accelerated, yet stressed and often compromised erythropoiesis. A specific understanding of key regulators of such stress erythropoiesis is of basic importance for insight into new anemia therapies. rhEPO per se can be therapeutic, but has substantial hypertensive side-effects, evokes thrombolytic events, is a high-cost recombinant glycoprotein, and may worsen certain cancers. Via investigations of EPO's effects on primary erythroid progenitors, we have uncovered and initially characterized two important new factors (and associated pathways) that selectively regulate stress erythropoiesis. First, our studies of a novel EPO-induced intracellular Spi2A serpin (via a new KO model) reveal that under select stress conditions, erythroblasts become highly sensitive to lysosome damage to the extent of leaching lysosomal executioner cathepsins- which are inhibited by Spi2A. If unchecked, cathepsin-associated damage and erythroblast death escalate, with ROS and iron as cytotoxic co-factors. We also demonstrate that a Cathepsin B/L inhibitor can phenocopy Spi2A's cytoprotective effects. A second EPO co-induced erythropoietic regulator we have discovered (and are beginning to understand) is the molecular adaptor and pseudokinase, Trib3. By developing a Trib3-KO mouse model, we reveal non-redundant novel stress erythropoiesis specific roles for Trib3 during both early, and late- stage erythroid cell formation We will now advance insight into these two new stress erythropoiesis factors, and coupled pathways, via the following SPECIFIC AIMS: SA#1 will first define how Spi2A and linked pathways impact on stress erythropoiesis due to β-thalassemia and iron imbalance (#1.1). At a mechanistic level, consequences of Spi2A- deficiency on erythroblast autophagy will be determined and molecular damage incurred by erythroblast lysosomes during oxidative stress plus Spi2A-deficiency will be defined (#1.2). Via LOF studies, SA#2 first will define effects exerted by Trib3 pseudokinase on early- and late-stage erythropoiesis during β-thalassemia, and iron imbalance (#2.1). Aim 2.2 will determine Trib3's roles in unfolded protein response pathways together with Trib3's regulatory molecular action mechanisms in early- and late- stage erythroid cells. SA#3 will advance studies to primary human erythroid progenitors (including thalassemia), and will first determine effects of lysosomal compromise, and cathepsin inhibition on erythroblast cytoprotection (#3.1). Aim 3.2 will define effects of Trib3 LOF and GOF during erythropoietic stress. Substantial insight will be gained concerning new regulators of red cell formation and novel target pathways within compromised erythroid progenitors for anti- anemia agents.