Mechanism/Regulation of Intestinal Thiamin Uptake

项目总结： 是次续期申请的长远目标是继续调查 水在肠道吸收过程中所涉及的细胞和分子机制 可溶性维生素B1(硫胺素)及其调节。我们还旨在研究 感染肠道常见病原体--肠源性大肠埃希氏菌 硫胺素吸收过程。 硫胺素是正常细胞功能所必需的，它的缺乏(代表一种 严重的营养问题)导致各种临床异常，包括 心血管和神经疾病。人类(和其他哺乳动物)不能合成 因此，硫胺素必须通过肠道吸收来获得维生素。当前的研究 资助期的特点是肠道硫胺素摄取过程的许多方面。这些 包括人类硫胺素基因5‘-调节区的特征 转运蛋白1和2(hTHTR-1和hTHTR-2)在体外和体内都表明， 硫胺素缺乏时肠道硫胺素摄取过程适应性上调 转录调节机制(S)，这个过程也经历了分化-和 依赖发育的调节。我们还描述了所涉及的机制 硫胺素转运体在上皮细胞中的膜靶向和细胞内转运。 使用基因特异性siRNA方法，我们实验室的最新研究表明 HTHTR 1和hTHTR 2均参与人肠上皮细胞对硫胺素的摄取。 2体外培养细胞株。关于这些硫胺素转运体在硫胺素中的作用尚不清楚 在天然肠道中摄取。因此，在新的初步研究中，我们开发了SLC19a3-/- 基因敲除小鼠模型，并显示肠道硫胺素摄取显著受损 与野生型小鼠相比，我们还建立了SLC19a2/-基因敲除小鼠群体 我们的实验室。此外，我们还使用细菌双杂交系统对一名人类 并已鉴定出两个可能与hTHTR-1相互作用的蛋白质 (Tetraspanin和E-cadherin-1)。此外，我们还研究了 致病性大肠埃希菌(EPEC)对硫胺素的摄取受到显著抑制。 根据我们以前和新的初步发现，我们在这项建议中的目标是：1)进一步 用slc19a2-/-和slc19a3-/-基因敲除研究肠道硫胺素吸收过程 建立小鼠模型，探讨THTR-1和THTR-2在跨上皮和跨上皮细胞中的作用。 天然肠道中的膜转运事件，2)识别顺式调节元件和 反式作用核因子参与硫胺素摄取适应性上调 缺乏，3)鉴定人肠道中与hTHTR-1和hTHTR-2相互作用的蛋白质 并了解它们的生物学/生理作用，以及4)确定 EPEC抑制肠道硫胺素摄取的细胞和分子机制 进程。 这些研究的结果应该继续提供新的和有价值的信息 关于肠道硫胺素摄取的细胞和分子机制 进程及其监管，以及对这一进程产生负面影响的因素。这 应该最终帮助我们设计有效的策略来优化硫胺素身体 在与硫胺素缺乏和次优水平相关的条件下的动态平衡，以及 将可能对该营养参数产生负面影响的外源因素的影响降至最低。

Project Summary: The long-term objective of this renewal application is to continue our investigations into the cellular and molecular mechanisms involved in the intestinal absorption process of the water- soluble vitamin B1 (thiamin) and their regulation. We also aim at examining the effect of infection with enteropathogenic E. coli (EPEC), a common intestinal pathogen, on the intestinal thiamin absorption process. Thiamin is essential for normal cellular functions and its deficiency (which represents a significant nutritional problem) leads to a variety of clinical abnormalities including cardiovascular and neurological disorders. Humans (and other mammals) cannot synthesize thiamin, and thus, must obtain the vitamin via intestinal absorption. Studies during the current funding period have characterized many aspects of the intestinal thiamin uptake process. These include characterization of the 5' -regulatory regions of the genes of the human thiamin transporters 1 & 2 (hTHTR-1 & hTHTR-2) both in vitro and in vivo, demonstration that the intestinal thiamin uptake process is adaptively up-regulated in thiamin deficiency via transcriptionally-mediated mechanism(s) and that the process also undergoes differentiation- and developmental- dependent regulation. We have also characterized the mechanisms involved in membrane targeting and intracellular trafficking of the thiamin transporters in epithelial cells. Using gene specific siRNA approaches, recent studies from our laboratory have shown that both the hTHTR 1 & 2 are involved in thiamin uptake by a human intestinal epithelial Caco- 2 cell line in vitro. Nothing is known about the role of these thiamin transporters in thiamin uptake in the native intestine. Thus, in new preliminary studies we have developed an Slc19a3-/- knockout mouse model and showed significant impairment in intestinal thiamin uptake compared to wild-type mice; we have also established a colony of Slc19a2-/- knockout mice in our laboratory. In addition, we have used the bacterial two-hybrid system to screen a human intestinal cDNA library and have identified two putative proteins that can interact with hTHTR-1 (tetraspanin and E-cadherin-1). Furthermore, we have examined the effect of the enteropathogenic Escherichia coli (EPEC) and found significant inhibition in thiamin uptake. Based on our previous and new preliminary findings, our aims in this proposal are: 1) To further characterize the intestinal thiamin absorption process using Slc19a2 -/-and Slc19a3 -/- knockout mouse models and to determine the role of THTR-1 and THTR-2 in trans-epithelial and trans- membrane transport events in the native intestine, 2) To identify the cis-regulatory elements and trans-acting nuclear factors involved in the adaptive up-regulation of thiamin uptake in thiamin deficiency, 3) To identify proteins that interact with hTHTR-1 and hTHTR-2 in human intestinal epithelial cells and to understand their biological/physiological roles, and 4) to determine the cellular and molecular mechanisms involved in EPEC inhibition of the intestinal thiamin uptake process. Results of these studies should continue to provide novel and valuable information regarding the cellular and molecular mechanisms involved in the intestinal thiamin uptake process and their regulation as well as of the factors that negatively impact the process. This should ultimately assist us in the designing of effective strategies to optimize thiamin body homeostasis in conditions associated with thiamin deficiency and sub-optimal levels, and in minimizing the effect of exogenous factors that may negatively impact this nutritional parameter.