Molecular Mechanisms of Intestinal Metal Ion Transport During Iron-Deficiency

描述（由申请人提供）：铁稳态的整体控制发生在近端小肠上皮的运输步骤，在那里吸收被精确调节以匹配体内铁的损失。重要的是，肠道铁转运的紊乱与人类几种重要的疾病状态有关，包括慢性病贫血和血色素沉着症。缺铁时，大鼠肠道铜转运增强，这可能是一种生理反应，与膳食铜在全身铁稳态各方面的作用有关。有趣的是，门克斯铜atp酶（Atpya）和二价金属转运蛋白i （Dmti）在不同出生年龄的缺铁大鼠十二指肠粘膜中被强烈诱导，后者可以运输铁和铜。因此，本研究的总体目标是：(1)确定Dmti和Atpya在缺铁过程中诱导铜转运的作用；(2)揭示缺铁过程中诱导Dmti和Atpya的分子机制；(3)确定铜对肠内跨上皮铁转运的分子机制的影响。这将通过利用细胞培养和啮齿动物肠道铁运输模型来完成。特异性AIM i将验证Dmti和Atpya的诱导是铁剥夺期间铜转运增加的原因。铁和铜转运研究和siRNA敲低将在我们的肠上皮体外模型IEC-6细胞中进行。一旦确定了在缺铁过程中参与铜转运的转运体，我们将在体内缺铁模型中进行补充研究，包括野生型、缺铁大鼠、贝尔格莱德大鼠（即dmti缺乏）和Atpya敲除小鼠。特异性AIM 2将验证Dmti和Atpya在缺铁期间受到不同分子机制调节的假设。我们将研究铁依赖性的Dmti转录后调控（由3' IRE介导）和IEC-6细胞中Atpya的转录调控。最后，具体的AIM 3将验证铁缺乏期间铜转运增加的假设，以增强肠道铁吸收的铜依赖方面。为了实现这一目标，铁转运研究将在从缺铁大鼠中分离的膜囊中进行，并测定肠细胞中的hephaestin活性和血清中的铜蓝蛋白活性。总的来说，这些研究将允许进一步定义在缺铁状态下参与促进肠道铁吸收的铜依赖过程。详细了解这些关系是至关重要的，因为肠道铁转运控制着全身铁稳态。此外，这项拟议的研究是新颖的，因为研究解决铁缺乏期间肠细胞和肝脏铜水平增加的影响迄今尚未报道。

DESCRIPTION (provided by applicant): The overall control of iron homeostasis occurs at the transport step in the epithelium of the proximal small bowel, where absorption is precisely regulated to match body iron losses. Importantly, perturbations in intestinal iron transport are associated with several important disease states in humans, including anemia of chronic disease and hemochromatosis. Intestinal copper transport is enhanced in rats during iron-deficiency, and this is likely a physiological response related to the role of dietary copper in various aspects of overall body iron homeostasis. Interestingly, the Menkes copper ATPase (Atpya) is strongly induced in the duodenal mucosa of iron-deprived rats at different postnatal ages along with Divalent Metal Transporter i (Dmti), which can transport iron and copper. Thus, the overall goals of this proposal are i) to determine the roles that Dmti and Atpya play in the induction of copper transport during iron-deprivation, 2) to decipher the molecular mechanisms of induction of Dmti and Atpya during iron-deprivation and 3) to determine the effect that copper has on molecular mechanisms of trans-epithelial iron transport in the intestine. This will be accomplished by utilizing cell culture and rodent models of intestinal iron transport. Specific AIM i will test the hypothesis that induction of Dmti and Atpya is responsible for increased transepithelial copper transport seen during iron-deprivation. Iron and copper transport studies and siRNA knockdowns will be performed in our in vitro model of the intestinal epithelium, the IEC-6 cells. Once the transporter(s) involved in the induction of copper transport during iron-deficiency have been identified, we will perform complementary studies in in vivo models of iron-deficiency, including wild-type, iron-deficient rats, Belgrade (i.e. Dmti-deficient) rats and Atpya knockout mice. Specific AIM 2, will test the hypothesis that Dmti and Atpya are regulated by distinct molecular mechanisms during iron-deficiency. Iron-dependent, post- transcriptional regulation of Dmti will be examined (mediated by the 3' IRE) and transcriptional regulation of Atpya in IEC-6 cells will be studied. Finally, specific AIM 3 will test the hypothesis that increased copper transport during iron deficiency functions to enhance copper-dependent aspects of intestinal iron absorption. To accomplish this goal, iron transport studies will be performed in membrane vesicles isolated from iron-deficient rats deprived of dietary copper, and hephaestin activity in enterocytes and ceruloplasmin activity in serum will be determined. Overall, these studies will allow further definition of the copper- dependent processes that are involved in enhancing intestinal iron absorption during states of iron- deficiency. A detailed understanding of these relationships is critical, as intestinal iron transport controls overall body iron homeostasis. Moreover, this proposed investigation is novel, as studies addressing the impact of increased enterocyte and liver copper levels during iron-deficiency have not been reported to date.