Goblet cells are abundant throughout the gastrointestinal tract and secrete products which form a continuous viscoelastic coat on the mucosal surface. Trefoil peptides, a family of structurally distinct small peptides specifically expressed in a regionalized fashion at all levels of the gastrointestinal tract, have been identified as a previously unappreciated major product of goblet cell populations. Trefoil factors are critical constituents at the mucosal-lumenal interface contributing to preservation of mucosal integrity, and protecting against a variety of noxious insults in in vivo model systems. Furthermore, preliminary studies suggest that expression of the representative trefoil ITF (Intestinal Trefoil Factor) is directly dependent on transcriptional factors which are associated with commitment of a pluripotent intestinal epithelial stem cell to the goblet cell lineage. However, insight into the molecular basis of trefoil peptide expression in a cell- and tissue-specific manner and the dimensions of their functional activity in sustaining epithelial integrity remains incomplete. The main goals of the studies in this proposal are the delineation of the mechanisms through which these abundant peptides sustain mucosal integrity, and the elucidation of the molecular regulation of the genes encoding the trefoil in order to define both the molecular basis of goblet cell specific gene expression and commitment to goblet cell differentiation. Two major specific aims are planned. (I) Determination of function and mechanisms of action of trefoil peptides. Preliminary studies have suggested that trefoil peptides are mediated through receptors and utilize distinct intracellular signalling pathways. The trefoil peptide receptor will be cloned and its mechanism of action characterized, while the intracellular processes that mediate its mitogenic effects will be defined. (II) Determination of the molecular basis of goblet-cell specific expression of ITF. Studies have identified a novel cis regulatory element in the ITF promoter which confers goblet cell expression of reporter genes in vivo. A nuclear factor which binds this element will be characterized through transient transfection and transgenic approaches. Collectively, these studies should provide insights into the trefoil family of proteins a newly recognized dimension of mucosal biology as well as the molecular basis of intestinal and goblet cell function, most importantly including the molecular basis of stem cell commitment to the goblet cell lineage. These insights promise to field new perspective on mechanisms of mucosal destruction, repair and function in inflammatory bowel disease and other forms of injury in the gastrointestinal tract.
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