Human Intestinal Biotin Uptake: Molecular/Cell Mechanisms

描述(由申请人提供)：这项续期申请的长期目标是更好地了解维生素H(生物素)在人体肠道的吸收过程，特别是有关转运系统的分子调控，即人钠依赖多维生素转运体(HSMVT)，以及hSMVT蛋白靶向和细胞内转运至人肠道上皮细胞顶膜的机制。生物素是水溶性维生素家族的一员，对正常的细胞功能、生长和发育是必不可少的。生物素缺乏会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部来源获得维生素。因此，肠道在决定和调节正常生物素体内动态平衡方面起着核心作用。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，目前对人类肠道中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这一过程的了解要少得多。HSMVT蛋白在肠上皮细胞中的细胞生物学也不是很清楚，涉及其靶向顶膜和细胞内转运的分子机制。在最近的初步研究中，我们获得的证据表明，存在两个驱动hSMVT基因转录的启动子，并证明在生物素缺乏和细胞分化过程中，人类肠道生物素摄取过程受到(通过转录机制)调节。我们还建立了利用活体人肠上皮Caco-2细胞的共聚焦成像来研究hSMVT靶向顶膜和细胞内转运的机制。在本申请中，我们建议继续这些研究，并描述三个具体目标：i)继续在体外人类肠上皮细胞中鉴定hSMVT基因的5‘调控区，并在转基因小鼠中证实所识别的启动子在体内的活性和生理意义。Ii)继续描述在生物素缺乏和细胞分化过程中调节人肠道上皮细胞摄取生物素的分子机制(S)。研究hSMVT蛋白靶向人肠上皮细胞顶膜的机制，并确定其在细胞内转运的机制。这些研究的结果将为调节人类肠道生物素吸收过程的分子/细胞机制和hSMVT系统的细胞生物学提供新的和有价值的信息。这最终应该有助于我们设计有效的策略，在与生物素缺乏和次优水平相关的条件下优化生物素体内的动态平衡。

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.