Regulatory Role of Transferrin in Erythropoiesis and Iron Metabolism

描述(由申请人提供)：铁超载是贫血发病和死亡的主要原因，其特征是无效的红细胞生成，包括地中海贫血。近年来，在阐明铁代谢的分子参与者方面取得了前所未有的进展，为治疗提供了新的潜在途径。特别是，肝脏激素海普西丁已被确定为铁吸收和分布的中央调节因子。铁负荷性红细胞增多性贫血的特征是尽管铁负荷，但海普西丁水平过低。我们的研究表明，外源性转铁蛋白可以改善地中海贫血小鼠的贫血，减少循环中的非转铁蛋白结合铁，并增加海普西丁的表达，这表明转铁蛋白是红细胞生成和铁代谢之间相互作用的重要调节因素。我们建议进一步探讨转铁蛋白通过其在红血球和肝细胞中的作用来调节海普西丁表达的机制。最近研究发现，HFe和转铁蛋白受体2(TfR2)分子参与了海普西丁对肝脏的调节，在红系前体细胞中也具有重要的功能。然而，转铁蛋白对红系HFe和TfR2表达和信号的影响，以及HFe和TfR2如何调节红细胞生成在很大程度上仍不清楚。初步数据表明，外源性转铁蛋白(Tf)增加了单铁Tf的相对浓度，减少了铁进入红系前体细胞。我们提出了一个模型，在这个模型中，增加的单铁转铁蛋白改变了HFe、TfR1和TfR2介导的铁摄取和信号传递，在红系前体和肝细胞中。为了验证这一模型，我们提出了三个具体的目标：1.比较在HFe存在或不存在的情况下，TfR1和TfR2与单铁和二铁转铁蛋白结合的动力学和对铁的吸收。2.检测有效和无效红细胞生成过程中转铁蛋白浓度变化对红系前体细胞的影响。3.检测转铁蛋白浓度变化对肝细胞肝癌蛋白表达的影响。转基因小鼠和细胞系将作为实验系统来检验以下假设：HFe调节单铁转铁蛋白与二铁转铁蛋白竞争与转铁蛋白受体结合的能力(目标1)。Tf结合通过调节HFe、TfR1和TfR2的表达和信号来影响红系分化(Aim 2A)。Tf通过TfR1/HFe介导的红系细胞铁状态和小鼠铁动力学的变化来调节红细胞生成(Aim 2B)。单铁-转铁蛋白通过TfR2信号调节肝细胞中海普西丁的表达(目标3A)。增加的转铁蛋白减轻非转铁蛋白结合铁对海普西丁表达的影响(目标3B)。给予TF减轻红细胞生成障碍小鼠血清中的一种海普西丁下调因子(AIM 3C)。这些研究将阐明铁代谢和红细胞生成之间的相互作用的生理学，并可能为开发新的治疗方案来治疗并发贫血和铁超载疾病的患者提供基础。

DESCRIPTION (provided by applicant): Iron overload is the principal cause of morbidity and mortality in anemias characterized by ineffective erythropoiesis, including ¿-thalassemias. In recent years, unprecedented progress in elucidating the molecular participants in iron metabolism has provided novel potential therapeutic approaches. In particular, the hepatic hormone hepcidin has been identified as a central regulator of iron absorption and distribution. The iron loading dyserythropoietic anemias are characterized by inappropriately low hepcidin levels despite iron loading. We have shown that exogenous transferrin ameliorates anemia, reduces circulating non-transferrin bound iron, and increases hepcidin expression in ¿-thalassemic mice, suggesting that transferrin is an important regulator of the cross-talk between erythropoiesis and iron metabolism. We propose to further explore mechanisms by which transferrin regulates hepcidin expression via its roles in the erythron and in the hepatocyte. Recently HFE and transferrin receptor 2 (TfR2), molecules known to participate in the hepatic regulation of hepcidin, have been found to be also functionally important in erythroid precursors. However, the effects of transferrin on erythroid HFE and TfR2 expression and signaling, and how HFE and TfR2 modulate erythropoiesis remain largely uncharacterized. Preliminary data demonstrate that exogenous transferrin (Tf) increases the relative concentration of monoferric Tf and decreases iron entry into erythroid precursors. We propose a model in which increased monoferric transferrin alters HFE, TfR1, and TfR2 mediated iron uptake and signaling, in erythroid precursors and in hepatocytes. To test this model, we propose three specific aims: 1. Compare the binding kinetics and uptake of iron from monoferric and diferric transferrin via TfR1 and TfR2 in the presence or absence of HFE. 2. Examine the effects of changes in transferrin concentration on erythroid precursors during effective and ineffective erythropoiesis. 3. Examine the effects of changes in transferrin concentration on hepatocellular hepcidin expression. Genetically modified mice and cell lines will serve as experimental systems to test the following hypotheses: HFE modulates the ability of monoferric transferrin to compete with diferric transferrin for binding to transferrin receptors (Aim 1). Tf binding affects erythroid differentiaton through modulation of HFE, TfR1, and TfR2 expression and signaling (Aim 2A). Tf regulates erythropoiesis via TfR1/HFE-mediated changes in erythroid cellular iron status and mouse ferrokinetics (Aim 2B). Monoferric-Tf modulates hepcidin expression in hepatocytes via signaling through TfR2 (Aim 3A). Increased Tf mitigates the effects of non-transferrin bound iron on hepcidin expression (Aim 3B). Administration of Tf attenuates a hepcidin down-regulatory factor in serum of mice with dyserythropoiesis (Aim 3C). These studies will clarify the physiology of the cross-talk between iron metabolism and erythropoiesis, and potentially provide the basis for the development of novel therapeutic alternatives for patients with diseases of concurrent anemia and iron overload.