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.
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