Molecular Mechanisms of Intestinal Metal Ion Transport During Iron-Deficiency

铁稳态的总体控制发生在近端小血管上皮细胞的运输步骤。 肠道，那里的吸收受到精确的调节，以匹配身体的铁损失。重要的是，微扰在 肠道铁转运与人类的几种重要疾病状态有关，包括 慢性病和血色素沉着症。缺铁时大鼠肠道铜转运增强， 这可能是一种生理反应，与饮食中铜在总体上不同方面的作用有关 体内铁平衡。有趣的是，门克斯铜ATPase(Atpya)在十二指肠中被强烈诱导 不同日龄缺铁大鼠粘膜与二价金属转运体I(DMTI)的关系 它可以运输铁和铜。因此，这项建议的总体目标是：一)确定角色 DMTI和ATPYA在缺铁诱导铜运输中的作用，2)破译 缺铁诱导DMTI和ATPIA的分子机制及3)测定 铜对肠道跨上皮铁转运分子机制的影响。这将是 通过利用细胞培养和肠道铁转运的啮齿动物模型来实现。特定的Aimi将 验证DMTI和ATPIA的诱导导致跨上皮铜升高的假说 缺铁期间所见的运输。铁和铜的运输研究和siRNA击倒将是 在我们的肠上皮细胞体外模型中进行，IEC-6细胞。曾经的传送者(S) 在缺铁期间参与铜的诱导运输的研究已经确定，我们将进行 在缺铁动物模型中的补充研究，包括贝尔格莱德野生型缺铁大鼠 (即DMTI缺陷)大鼠和Atpya基因敲除小鼠。规范AIM 2，将检验DMTI和 在缺铁过程中，ATPIA受到不同的分子机制的调节。 将研究DMTI的转录调控(由3‘IRE介导)和转录调控 将对Atpya在IEC-6细胞中的作用进行研究。最后，SpeciicAIM 3将检验铜含量增加的假设 缺铁时的转运功能增强肠道铁对铜的依赖 吸收。为了实现这一目标，将在分离的膜小泡中进行铁转运研究。 缺铁大鼠缺铜后肠道细胞和铜蓝蛋白的活性 将测定血清中的活性。总体而言，这些研究将进一步定义铜- 依赖的过程，参与促进肠道铁吸收在状态下- 缺乏症。对这些关系的详细了解是至关重要的，因为肠道铁运输是控制的。 全身铁平衡。此外，这项拟议的调查是新颖的，因为研究涉及 到目前为止，还没有关于缺铁期间肠细胞和肝脏铜水平升高的影响的报道。

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, includinganemia 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 ofthis proposal are i) to determine the roles that Dmti and Atpyaplay in the induction of copper transport duringiron-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 AIMi 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 ofthe intestinal epithelium, the IEC-6 cells. Oncethe transporter(s) involved in the induction of copper transport during iron-deficiency have been identified,we willperform complementary studies in in vivomodels of iron-deficiency, includingwild-type, iron-deficientrats, Belgrade (i.e. Dmti-deficient)rats and Atpya knockout mice. SpecificAIM 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, specificAIM 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 ofiron- deficiency. A detailed understandingof 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.