Aging is characterized by an increase in the proliferative activity of gut mocosa which may contribute to development of pathological processes in the gut including cancer. Neurotensin (NT), a trophic gut peptide, stimulates gut mocosal growth in young and aged rats. In addition, NT mRNA levels increase with aging. The central hypothesis of this proposal is that NT gene expression in the gut is regulated in a temporal and tissue-specific fashion by mechanisms which involve transcriptional and post-transcriptional regulation; increases in NT levels may play a role in the increased proliferative activity found in gut mucosa with aging. To test the central hypothesis, we will employ the following specific aims: 1) We will further characterize the developmental regulation of NT in the gut. We will determine whether NT mRNA levels reflect actual NT peptide content by radioimmunoassay. We will next qualitatively determine the distribution of NT mRNA and peptide in gut mucosa. We will determine whether changes in NT mRNA levels in the jejunum and ileum are mediated by alterations in transcription and, if so, whether these changes occur by activation of a single or different promoters; 2) We will examine the increases in gut NT expression with aging. We will repeat our nuclear run-on studies to determine whether the increases of NT mRNA are the result of changes in transcription. If the increases in NT are not mediated by changes in transcription then we will determine the site (nucleus vs. cytoplasm) of increased NT mRNA. Finally, we will determine whether the increases of NT mRNA are associated with concomitant increases of NT peptide content and release; 3) We will delineate the sequences (cis-elements) essential for basal and tissue-specific expression of NT. We will then more precisely map the regulatory sequences of the NT gene using a series of mutant constructs. We will determine whether this 5' region of NT can confer activation to a minimal heterologous promoter. Next we will determine possible DNA-binding domains of the rat NT promoter. The long-term goal of this proposal is to determine whether NT plays a role in the increased proliferative activity that is found in gut mucosa with aging. The finding of an age-dependent increase of a growth factor that stimulates gut mucosal proliferation would be of potential clinical importance that may lead to a better understanding of the molecular mechanisms regulating the hyperproliferative changes that occur in the gut with age.
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