Regulation of mammalian iron homeostasis by iron and transferrin

描述(由申请人提供)： 由于铁(Fe)相关的疾病在世界范围内普遍存在，了解铁的动态平衡对于了解人类健康至关重要。铁的动态平衡在很大程度上受到海普西丁的调节，海普西丁是一种肝源性多肽，可以抑制饮食中铁的吸收和巨噬细胞铁的循环。红细胞生成驱动力增加和铁超载分别抑制和刺激了海普西丁的表达。我目前的工作集中在确定海普西丁表达的可溶性调节因子，我的工作涉及到血清中丰富的金属结合蛋白转铁蛋白(Tf)和必需的Tf结合金属锰(Mn)对海普西丁表达的调节。我的第一个目标，将在指导阶段完成，是确定转铁蛋白在体内对海普西丁表达的影响。我将构建表达金属亲和力改变的Tf变异体的转基因小鼠，将它们培育在Tf缺乏的背景上，并评估骨髓切除前后的Hepsidin表达，以最大限度地减少红细胞生成驱动对Hepsidin表达的抑制。第二个目标将在指导阶段启动，以确定细胞内锰和铁稳态之间的共性和差异，以此作为进一步剖析铁稳态的手段。我将分析已知金属转运蛋白水平改变的细胞系中54Mn和55Fe的转运，并确定改变的Mn和Fe水平对54Mn和55Fe的转运和掺入Tf的影响。第三个目标，也是独立阶段的重点，是确定细胞金属动态平衡所需的新因素。由于高锰对许多细胞系有毒害作用，我将利用shRNA和cDNA文库改变细胞的基因表达，筛选耐高锰的细胞，并通过54Mn和55Fe转运法鉴定感兴趣的突变系。从前两个目标，我将熟练研究铁的运输和分布；从后两个目标，我将建立一个独立的重点，有别于我导师的，铁在血液疾病中的作用。这一建议反映了我研究的总体趋势--研究哺乳动物的金属动态平衡及其与人类健康和疾病的关系--并将导致一个金属动态平衡的范例，从中可以设计出金属相关疾病的新治疗方法。 与公共健康相关：这项研究的目标是确定体内的铁是如何刺激肝脏产生一种限制肠道铁吸收的激素的。这项研究的结果应该会为贫血等铁相关疾病提供新的治疗选择。

DESCRIPTION (provided by applicant): As iron (Fe)-related disorders are prevalent world-wide, understanding Fe homeostasis is critical to understanding human health. Fe homeostasis is largely regulated by hepcidin, a liver-derived peptide that inhibits dietary Fe absorption and macrophage Fe recycling. Hepcidin expression is inhibited and stimulated respectively by increased erythropoietic drive and Fe overload. Focused on identifying soluble regulators of hepcidin expression, my current work has implicated transferrin (TF), the abundant serum metal-binding protein, and manganese (Mn), an essential TF-bound metal, in the regulation of hepcidin expression. My first objective, to be completed during the mentored phase, is to determine the effect of TF on hepcidin expression in vivo. I will construct transgenic mice expressing TF variants with altered metal affinity, breed them onto a TF-deficient background and evaluate hepcidin expression before and after bone marrow ablation to minimize the inhibition of hepcidin expression by erythropoietic drive. The second objective, to be initiated during the mentored phase, is to identify commonalities and divergences between cellular Mn and Fe homeostasis as a means to further dissect Fe homeostasis. I will assay 54Mn and 55Fe transport in cell lines with altered levels of known metal transporters and determine the effect of altered Mn and Fe levels on 54Mn and 55Fe transport and incorporation into TF. The third objective, the focus of the independent phase, is to identify novel factors required for cellular metal homeostasis. As high Mn levels are toxic to many cell lines, I will alter cellular gene expression with shRNA and cDNA libraries, select for cells resistant to high Mn levels and identify mutant lines of interest by 54Mn and 55Fe transport assay. From the first two objectives, I will achieve proficiency in the study of Fe transport and distribution; from the last two objectives, I will establish an independent focus divergent from my mentor's, the role of iron in hematologic disorders. This proposal reflects the overall trend in my research-the study of mammalian metal homeostasis and its relation to human health and disease-and will lead to a paradigm of metal homeostasis from which novel treatments for metal-related disorders can be designed. PUBLIC HEALTH RELEVANCE: The goal of this study is to determine how iron in the body stimulates the liver to produce a hormone that limits iron absorption from the gut. The results of this research should offer novel treatment options for iron-related disorders such as anemia.