Understand and Improve Iron Distribution and Erythropoiesis in Beta-Thalassemia

描述（由申请人提供）：本申请描述了一项为期五年的指导培训计划，旨在培养一名独立的临床科学家，其学术医学生涯将专注于进一步了解人类红细胞的发育。人类-地中海贫血是一种与贫血、脾肿大和红细胞生成功能低下相关的疾病；这种疾病中的铁超载是由于输血和hepcidin缺乏引起的铁吸收增加。我们的实验室先前证明，转铁蛋白治疗β -地中海贫血小鼠可改善疾病表型，逆转贫血、脾肿大、无效红细胞生成和hepcidin表达增加。令人惊讶的是，虽然有更多的红细胞在循环中，但它们的大小和血红蛋白含量在治疗小鼠中减少了。我们假设补充转铁蛋白导致更多的缺铁红细胞生成，减少-地中海贫血中血红素、α -珠蛋白和β -珠蛋白合成之间的不平衡。我为这个奖项提出的研究目标是检验这个假设。我计划两个具体目标来评估这一点：1)确定当体内和体外有额外的转铁蛋白可用时，红细胞前体的铁摄取是否会改变；2)确定在体内和体外正常和β -地中海贫血细胞中添加转铁蛋白是否会改变血红蛋白成分的调节和产生以及铁和血红素的输出。这些研究将验证我的假设，即外源性转铁蛋白限制铁进入红细胞前体并保护早期红细胞免受血红素毒性的影响，将提供对β -地中海贫血表型生理学的见解，并将进一步加深我们对正常和紊乱红细胞的理解。我将通过以下方法实现这些具体目标：1)表征小鼠红细胞前体的铁动力学，分析这些细胞中转铁蛋白受体的体内表达，并评估细胞系和培养的人类细胞的胞质铁摄取；2)评估血红素和珠蛋白的表达水平和调控因子，测定细胞质中原卟啉锌和游离血红素的水平，表征红细胞前体分化过程中FLVCR和FPN-1B、血红素和铁输出蛋白分别对小鼠骨髓和脾脏样本以及培养的人样本的影响。这些研究的重点是基本的红细胞生理学，并可能为红细胞发育受到干扰的人类疾病提供见解。最后，了解转铁蛋白在-地中海贫血小鼠中的作用机制的进展可能会促进其治疗这种疾病的发展。

DESCRIPTION (provided by applicant): This application describes a five-year mentored training program to develop an independent clinician-scientist whose career in academic medicine will focus on furthering our understanding of human red blood cell development. Beta-thalassemia in humans is a disease associated with anemia, splenomegaly, and ineffective erythropoiesis; iron overload in this disease results from transfusion and increased iron absorption from hepcidin deficiency. Our lab previously demonstrated that treatment with transferrin in mice with beta- thalassemia improves the disease phenotype with reversal of anemia, splenomegaly, and ineffective erythropoiesis and an increase in hepcidin expression. Surprisingly, although there are more red cells in circulation, their size and hemoglobin content is reduced in treated mice. We hypothesize that supplemental transferrin results in more iron deficient erythropoiesis, reducing the imbalance between heme, alpha-globin, and beta-globin synthesis in beta-thalassemia. My goal in the studies proposed for this award is to test this hypothesis. I plan two specific aims to evaluate this: 1) to determine if iron uptake by erythroid precursors is altered when additional transferrin is available in vivo and in vitro; and 2) to determine if the regulation and production of hemoglobin components as well as iron and heme export is altered when supplemental transferrin is added in normal and beta-thalassemic cells in vivo and in vitro. These studies will test my hypothesis that exogenous transferrin restricts iron entry into erythroid precursors and protects early erythroid cells from heme toxicity, will provide insight into the physiology responsible for the phenotype in beta- thalassemia, and will further our understanding of normal and disordered erythropoiesis. I will accomplish these specific aims by the following methods: 1) characterize the ferrokinetics of erythroid precursors in mice given additional transferrin, analyze the expression of transferrin receptor in these cells in vivo, and evaluate cytosolic iron uptake in cell lines and human cells in culture; 2) evaluate expression levels and regulators of heme and globin production, measure levels of zinc protoporphyrin and free heme in the cytosol, and characterize the effect on FLVCR and FPN-1B, heme and iron exporters, respectively, during erythroid precursor differentiation in mouse bone marrow and spleen samples as well as in human samples in culture. These studies focus on basic red blood cell physiology and may lend insight into human diseases where red blood cell development is disturbed. Lastly, progress in understanding mechanisms of transferrin efficacy in beta-thalassemic mice may enable its therapeutic development for patients with this disease. PUBLIC HEALTH RELEVANCE: Our laboratory previously demonstrated that using the main iron carrying molecule, transferrin, to treat mice with the disease, beta-thalassemia reverses many of the abnormalities present in this disease. We will systematically analyze the mechanisms underlying this effect in order to create a foundation for further testing of transferrin. The treatment for Beta-thalassemia is suboptimal, but has remained unchanged for the past fifty years. Ultimately, our goal is to provide an alternative treatment for these patients that could possibly be effective for other diseases associated with anemia and iron overload.