The processes of intestinal growth and differentiation are essential to the maintenance of normal gut function. Derangements in gut mucosal growth and differentiation result in diarrhea, malabsorption, and altered barrier function. Thyroid hormone (T3) is one of the most potent regulators of intestinal epithelial growth and differentiation, but its mechanism of action in the GI tract is largely unknown. This research proposal is designed to elucidate the molecular mechanisms by which T3 exerts its profound effects upon GI mucosal structure and function. Both in vivo and in vitro studies have demonstrated that T3 has two major effects upon the small intestinal epithelium (1) induction of crypt cell hyperplasia and (2) alteration in the pattern of enterocyte gene expression, e.g., increased intestinal alkaline phosphatase (IAP) expression. The effects of T3 on target tissues are generally mediated through binding to a nuclear receptor protein which interacts directly with DNA cis-elements (TRE) within specific T3-responsive genes. Multiple forms of the T3 receptor (TR) are encoded by either the alpha or beta cerbA genes, including a non-hormone binding variant, c-erbA alpha-2, which is thought to inhibit T3 action. Since cellular T3-responsiveness is dependent upon the relative abundance of the various TRs, we will use in situ hybridization and crypt-villus separation techniques to determine the cell type-specific patterns of TR expression within the small intestinal mucosa. They have established TR-transfected HT-29 cells (HT-29TR) as an excellent in vitro model to study the T3 effects on intestinal epithelial cells. HT-29TR cells will be treated with T3 to examine the nature of the growth response, e.g., 3H thymidine incorporation, proto-oncogene induction, and gel shift assays to identify a nuclear complex with the c-fox serum response element. Direct comparisons will be made with the well characterized crypt cell growth factor, EGF. Sodium butyrate-treated HT-29TR cells will then be used as a model of villus enterocytes and the T3 effects upon IAP gene expression examined. Transient transfection experiments with IAP reporter gene constructs will enable them to identify the important DNA cis-regulatory elements. Finally, DNAse 1 footprinting and gel shift assays will be performed to more precisely define the relevant DNA-protein interactions underlying T3-regulated gene expression. The proposed studies will enhance the understanding of the role that T3 plays in regards to intestinal mucosal structure and function, and should have broader implications for the biology of development and neoplasia. It is hoped that the studies will identify potential therapeutic targets which could be used in the future to maintain gut integrity during critical illness.
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