Molecular Mechanisms of Intestinal Metal Ion Transport During Iron-Deficiency

铁稳态的总体控制发生在近端小动脉上皮的运输步骤中。 肠，在那里吸收被精确地调节以匹配身体铁的损失。重要的是， 肠道铁转运与人类的几种重要疾病状态有关，包括 慢性疾病和血色病。缺铁时大鼠肠道铜转运增强， 这可能是一种生理反应，与膳食铜在整体免疫系统各方面的作用有关。 体内铁平衡有趣的是，门克斯铜ATP酶（Atpya）在十二指肠中被强烈诱导， 沿着生后不同日龄缺铁大鼠胃粘膜二价金属转运蛋白I（Dmti）的变化， 可以运输铁和铜。因此，本提案的总体目标是：i）确定 Dmti和Atpya在缺铁诱导铜转运中的作用，2） 铁剥夺过程中Dmti和Atpya诱导的分子机制和3）确定 铜对肠中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类动物肠道铁转运模型来实现。具体的AIMi将 检验Dmti和Atpya的诱导导致跨上皮铜增加的假设 在缺铁期间看到的运输。铁和铜转运研究和siRNA敲除将在 在我们的肠上皮细胞体外模型IEC-6细胞中进行。一旦运输机 在缺铁诱导铜转运的参与已经确定，我们将进行 补充研究在体内模型的铁缺乏，包括野生型，铁缺乏，贝尔格莱德 (i.e. Dmti缺陷）大鼠和Atpya敲除小鼠。SpecificAIM 2将检验Dmti和 在缺铁期间，Atpya由不同的分子机制调节。 将检查Dmti的转录调节（由3' IRE介导），并且将检测Dmti的转录调节（由3' IRE介导）。 Atpya在IEC-6细胞中的作用将被研究。最后，specificAIM 3将测试增加铜的假设， 铁缺乏期间的转运功能，以增强肠铁的铜依赖方面 吸收为了实现这一目标，将在分离的膜囊泡中进行铁转运研究 从缺铁大鼠剥夺膳食铜，和hephestin活性的肠细胞和铜蓝蛋白 将测定血清中的活性。总的来说，这些研究将允许进一步定义铜- 依赖的过程，参与增强肠铁吸收的状态ofiron， 缺陷详细了解这些关系是至关重要的，因为肠道铁转运控制着 全身铁平衡。此外，这项拟议的调查是新颖的，因为研究解决了 到目前为止，还没有关于缺铁期间肠上皮细胞和肝脏铜水平增加的影响的报道。

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.