Novel regulators of stress erythropoiesis

描述（由申请人提供）：终末期肾脏疾病、化疗和放疗、炎症、骨髓发育不良、铁调节失调和血红蛋白病相关的贫血导致慢性加速发作，但应激和经常受损的红细胞生成。对这种应激性红细胞生成的关键调节因子的具体了解对于了解新的贫血疗法具有重要的基础意义。rhEPO本身可以治疗，但有严重的高血压副作用，引起溶栓事件，是一种高成本的重组糖蛋白，并可能使某些癌症恶化。通过对EPO对原发性红细胞祖细胞的影响的研究，我们发现并初步描述了两个重要的新因子（和相关途径），它们选择性地调节应激性红细胞生成。首先，我们对一种新的epo诱导的细胞内Spi2A丝氨酸的研究（通过一个新的KO模型）表明，在特定的应激条件下，红母细胞对溶酶体损伤变得高度敏感，其程度达到溶酶体刽子手组织蛋白酶的浸出程度，而溶酶体刽子手组织蛋白酶被Spi2A抑制。如果不加以控制，组织蛋白酶相关损伤和红母细胞死亡升级，ROS和铁作为细胞毒性辅助因子。我们还证明了一种组织蛋白酶B/L抑制剂可以表型化Spi2A的细胞保护作用。我们已经发现（并开始了解）的第二个EPO共诱导的红细胞生成调节因子是分子接头和假激酶Trib3。通过开发Trib3- ko小鼠模型，我们揭示了Trib3在早期和晚期红细胞形成过程中非冗多的新型应激性红细胞生成特异性作用。现在，我们将通过以下具体目的深入了解这两种新的应激性红细胞生成因子及其偶联途径：SA#1将首先定义Spi2A和相关途径如何影响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-缺乏对红母细胞自噬的影响，并定义氧化应激加Spi2A-缺乏时红母细胞溶酶体引起的分子损伤（#1.2）。通过LOF研究，SA#2将首先确定Trib3假激酶对β-地中海贫血和铁失衡期间早期和晚期红细胞生成的影响（#2.1）。Aim 2.2将确定Trib3在未折叠蛋白反应途径中的作用，以及Trib3在早期和晚期红细胞中的调节分子作用机制。SA#3将推进对人类原发性红系祖细胞（包括地中海贫血）的研究，并将首先确定溶酶体损伤和组织蛋白酶抑制对红母细胞保护的影响（#3.1）。目的3.2将定义Trib3 LOF和GOF在红细胞生成应激中的作用。将获得有关红细胞形成的新调控因子和受损红细胞祖细胞内抗贫血药物的新靶标途径的实质性见解。

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.