Human Intestinal Biotin Uptake: Molecular/Cell Mechanisms

描述（由申请人提供）：本次更新申请的长期目标是更好地了解维生素H（生物素）在人体肠道中的吸收过程，特别关注相关转运系统的分子调控，即，人钠依赖性多种维生素转运蛋白（hSMVT），以及涉及hSMVT蛋白靶向和细胞内运输至人肠上皮细胞顶膜的机制。生物素是水溶性维生素家族的一员，对正常细胞功能、生长和发育至关重要。生物素缺乏导致严重的临床异常，包括神经系统疾病、生长迟缓和皮肤异常。人类和其他哺乳动物不能合成生物素，因此必须通过肠道吸收从外源来源获得维生素。因此，肠道在决定和调节正常生物素体稳态中起着核心作用。本实验室在过去几年的研究中克隆了大鼠肠道生物素SMVT系统，鉴定了其基因的5'调控区，并研究了个体发育对肠道生物素摄取过程的影响。然而，目前对人类肠道生物素摄取过程的分子调控以及生物素缺乏和细胞分化如何调控该过程知之甚少。关于肠上皮细胞中hSMVT蛋白的细胞生物学，关于其靶向顶膜和细胞内运输所涉及的分子机制，也一无所知。在最近的初步研究中，我们已经获得的证据表明，存在两个启动子，驱动hSMVT基因的转录，并证明人肠道生物素摄取过程中的生物素缺乏和细胞分化过程中的调节（通过转录机制）。我们还建立了使用活的人肠上皮Caco-2细胞的共聚焦成像来研究hSMVT靶向顶端膜和细胞内运输的机制。在本申请中，我们提出继续这些研究并描述三个具体目的：I）继续在体外人肠上皮细胞中hSMVT基因的5'调控区的表征，并在转基因小鼠中体内确认所鉴定的启动子的活性和生理相关性。II）继续表征在生物素缺乏和细胞分化期间参与人肠上皮细胞生物素摄取调节的分子机制。III）研究涉及hSMVT蛋白靶向人肠上皮细胞顶膜的机制，并确定涉及其细胞内运输的机制。这些研究的结果应提供新的和有价值的信息，在人肠道生物素吸收过程的调节和hSMVT系统的细胞生物学的分子/细胞机制。这将最终帮助我们设计有效的策略，以优化生物素体内稳态的条件下与生物素缺乏和次优水平。

DESCRIPTION (provided by applicant): The long-term objectives of this renewal application are to develop a better understanding of the absorption process of vitamin H (biotin) in the human intestine, with specific focus on the molecular regulation of the involved transport system, i.e., the human sodium-dependent multivitamin transporter (hSMVT), as well as the mechanisms involved in the targeting and intracellular trafficking of the hSMVT protein to the apical membrane of human intestinal epithelial cells. Biotin, a member of the water-soluble family of vitamins, is essential for normal cellular functions, growth, and development. Biotin deficiency leads to serious clinical abnormalities that include neurological disorders, growth retardation, and dermal abnormalities. Humans and other mammals cannot synthesize biotin, and thus, must obtain the vitamin from exogenous sources via absorption in the intestine. Therefore, the intestine plays a central role in determining and regulating normal biotin body homeostasis. Studies from our laboratory over the past several years have cloned the rat intestinal biotin SMVT system, characterized the 5' regulatory region of its gene, and examined the effect of ontogeny on intestinal biotin uptake process. Much less, however, is currently known about the molecular regulation of the biotin uptake process in the human intestine, and how biotin deficiency and cell differentiation regulates the process. Nothing is also known about the cell biology of the hSMVT protein in intestinal epithelial cells with regards to the molecular mechanisms involved in its targeting to the apical membrane and intracellular trafficking. In recent preliminary studies, we have obtained evidence showing the existence of two promoters that drive the transcription of the hSMVT gene, and demonstrating that the human intestinal biotin uptake process is regulated (via transcriptional mechanisms) in biotin deficiency and during cell differentiation. We have also established the use of confocal imaging of living human intestinal epithelial Caco-2 cells to study the mechanisms involved in hSMVT targeting to the apical membrane and intracellular trafficking. We propose in this application to continue these investigations and describe three specific aims: I) To continue the characterization of the 5' regulatory region of the hSMVT gene in vitro in human intestinal epithelial cells, and to confirm activity and physiological relevance of the identified promoters in vivo in transgenic mice. II) To continue the characterization of the molecular mechanism(s) involved in the regulation of biotin uptake by human intestinal epithelial cells in biotin deficiency and during cell differentiation. Ill) To study the mechanisms involved in the targeting of the hSMVT protein to the apical membrane of the human intestinal epithelial cells, and to determine the mechanisms involved in its intracellular trafficking. Results of these studies should provide novel and valuable information regarding the molecular/cellular mechanisms involved in the regulation of the human intestinal biotin absorption process and the cell biology of the hSMVT system. This should ultimately assist us in the design of effective strategies to optimize biotin body homeostasis in conditions associated with biotin deficiency and sub-optimal levels.