Molecular Mechanisms of Intestinal Metal Ion Transport During Iron Deficiency

描述（由申请人提供）：由于没有活跃的排泄系统存在，膳食铁在十二指肠的吸收决定了全身铁水平。因此，这一过程必须严格控制，以避免组织铁积累（如遗传性血色素沉着症）或缺乏（如慢性炎症性贫血）的不良后果。在过去的十年里，我们一直在研究铁在肠上皮细胞（IECs）间运输的分子方面，长期目标是开发药物或饮食治疗来调节人体铁的吸收。虽然已经确定了铁的进口商和出口商，但关于铁在国际经济中心之间的运输和出口进入流通（这是限制速度的步骤）的具体细节的知识缺乏。我们做了新的观察，铜相关的过程被激活的铁剥夺啮齿动物。在肠细胞中，铜转运atp酶（Atp7a）和铜结合蛋白（金属硫蛋白）在细胞内铜水平升高的情况下上调。这些观察结果为体内铜水平（铁缺乏时增加，铁过载时减少）与肠道铁运输控制之间的关系提供了机制见解。基于这些发现，确定涉及铁通量控制的铜特异性机制是势在必行的。从肠细胞输出的亚铁（Fe2+）在功能上与铁（Fe3+）与组织液中的转铁蛋白（Tf）结合所必需的氧化步骤相耦合。一种膜结合的多铜氧化铁酶（FOX） Hephaestin （Heph）可能介导这一步骤。然而，Heph KO小鼠存活，肠道铁转运仅部分减弱，表明存在其他FOXs。我们最近发现肠细胞有两种不同的新型胞浆氧化铁酶（FOXs），一种是未被发现的可溶形式的Heph (sHeph)，另一种称为cytoFOX。我们假设胞质FOXs参与跨iec的铁的胞吞作用。对Heph KO小鼠的研究表明，这两种蛋白都有助于胞质FOX活性（sHeph ~35-40%; cytoFOX ~60-65%）。另一种循环FOX，铜蓝蛋白（Cp）也可能参与iec的铁输出。目前的建议将通过测试肠细胞中的新型多铜FOXs （sHeph和cytoFOX）和血液中的Cp在控制iec的铁输出中发挥重要作用的中心假设来阐明肠道铁运输的具体机制细节。我们还将确定Cu递送这些蛋白质的生物合成机制，可能涉及肠道Atp7a。本应用程序中概述的综合方法，使用独特的哺乳动物铁转运体内和体外模型，

DESCRIPTION (provided by applicant): Absorption of dietary iron in the doudenum determines overall body iron levels as no active excretory systems exist. As such, this process must be tightly controlled to avoid the adverse consequences of tissue iron accumulation (e.g. in hereditary hemochromatosis) or deficiency (e.g. in anemia of chronic inflammation). We have been investigating molecular aspects of iron transport across intestinal epithelial cells (IECs) fo the past decade, with a long-term goal of developing drugs or dietary treatments to modulate iron absorption in humans. Although iron importers and exporters have been identified, a paucity of knowledge exists regarding the specific details of iron movement across IECs and export into the circulation (which is the rate-limiting step). We made the novel observation that copper-related processes are activated by iron deprivation of rodents. In enterocytes, a copper transporting ATPase (Atp7a) and a copper-binding protein (metallothionein) were upregulated in the setting of increased intracellular copper levels. These observations provided mechanistic insight into the relationship between body copper levels (which increase during iron deficiency & decrease in iron overload) and control of intestinal iron transport. Based upon these findings, identification of copper-specific mechanisms involved in control of iron flux was an imperative. Ferrous iron (Fe2+) export from enterocytes is functionally coupled to an oxidation step which is required for iron (Fe3+) binding to transferrin (Tf) in the interstitial fluid. A membrane-bound, multi-copper ferroxidase (FOX), Hephaestin (Heph), may mediate this step. However, Heph KO mice are viable and intestinal iron transport is only partially attenuated, suggesting that other FOXs exist. We recently discovered that enterocytes have two, distinct novel cyosolic ferroxidases (FOXs), one being an undiscovered, soluble form of Heph (sHeph) and the other termed cytoFOX. We postulate that cytosolic FOXs participate in transcytosis of iron across IECs. Studies in Heph KO mice demonstrated that both proteins contribute to cytosolic FOX activity (sHeph ~35-40%; cytoFOX ~60-65%). Another, circulating FOX, ceruloplasmin (Cp), may also participate in Fe export from IECs. The current proposal will elucidate specific, mechanistic details of intestinal Fe transport by testing the central hypothesis that novel multi-copper FOXs (sHeph and cytoFOX) in enterocytes and Cp in blood play integral roles in control of iron export from IECs. We will also define the mechanism(s) of Cu delivery for the biosynthesis of these proteins, likely involving intestinal Atp7a. The integrative approach outlined in this application, using unique in vivo and in vitro models of mammalian iron transport, will first decipher (in Aim 1) the role of Atp7a in delivering copper to sHeph to mediate iron efflx and in potentiating hepatic copper loading. Aim 2 will define the role(s) of novel soluble FOXs in the transcytotic iron pathway, while Aim 3 will clarify the role of Cp in iron absorption and will determine the mechanism of enhanced production of holo-Cp during iron deficiency. Overall, this project will advance the field of iron biology by revealing new mechanistic details of iron transport and may provide opportunities to develop molecular approaches to modulate iron absorption.