Neurotensin (NT), an important regulatory peptide, affects numerous aspects of intestinal function, including motility, secretion and gut mucosal growth. In addition, the expression of the gene encoding NT and neuromedin N (designated NT/N) is specifically localized to the adult small bowel in a distinctive spatial distribution. This small bowel- specific pattern of expression suggests that the NT/N gene will provide an important molecular model to better delineate the complex mechanisms leading to gut differentiation. The major focus of this project is to define the molecular mechanisms that regulate NT/N gene expression. Our preliminary findings, using gut-derived cell lines, demonstrate that the NT/N gene is a downstream target for activation by ras documenting, for the first time, that the ras signaling pathway affects a cellular gene which is normally expressed only in highly-differentiated cells of the gut mucosa. Expression of the NT/N gene is mainly dependent on intact cis- elements located in the proximal 120 nucleotides of the NT/N promoter and include a crucial, multifunctional AP-1/CRE site that binds c-Jun, CREB and ATF proteins and a purine-rich region that binds the apolipoprotein AI regulatory protein-1 (ARP-1). Furthermore, we demonstrate that c-jun activates NT/N transcription while the CRE binding protein, ATF- 1, represses c-jun-mediated NT/N activation. Based on our findings, the central hypothesis of this proposal is that NT/N gene expression in the GI tract is regulated by a complicated interaction of both ubiquitous and tissue-specific transcription binding proteins in conjunction with the ras regulatory pathway to either activate or repress NT/N expression in a cell-specific manner. To examine this hypothesis, we have planned experiments with the following SPECIFIC AIMS: l) We will determine the mechanisms for activation of NT/N transcription by cellular ras. To accomplish this goal, we will perform a series of transfection experiments using gut-derived cell lines to dissect the ras signaling pathway culminating in NT/N expression that occurs with differentiation. Also, normal small intestine will be assessed for concomitant changes in ras expression. 2) We will examine the role of AP-1 and CREB/ATF proteins in NT/N activation and repression. To accomplish this goal, we will delineate the proteins binding to the proximal AP-1/CRE site. 3) We will further define the role of ARP-1 in NT/ N gene activation. To accomplish this goal, a series of functional promoter analyses and protein binding experiments will be performed. The long-term goal of this proposal is to define the molecular mechanisms that regulate NT/N expression. An in-depth analysis of the mechanisms that lead to the activation or repression of the NT/N gene will result in a better understanding of how NT regulates different aspects of gut function and will reveal possible insights into the role of NT in gut growth. Furthermore, the NT/N gene will provide a useful molecular paradigm to better define the complex differentiation pathway leading to small bowel and colonic phenotypes during normal gut development.
Showing the most recent 10 out of 132 publications
