THE NEUROTENSIN GENE IS A DOWNSTREAM TARGET FOR RAS ACTIVATION

Ras regulates novel patterns of gene expression and the differentiatio n of various eukaryotic cell types. Stable transfection of Ha-ras into the human colon cancer line CaCo2 results in the morphologic differen tiation to a small bowel phenotype. The purpose of our study was to de termine whether the Ras regulatory pathway plays a role in the express ion of the neurotensin gene (NT/N), a terminally differentiated endocr ine product specifically localized in the gastrointestinal tract to th e adult small bowel, We found that CaCo2-ras cells, but not parental C aCo2, express high levels of the human NT/N gene and, moreover, that t his increase in gene expression is regulated at the level of transcrip tion. Transfection experiments using NT/N-CAT mutation constructs iden tify the proximal 200 bp of NT/N flanking sequence as sufficient for m aximal Ras-mediated NT/N reporter gene induction. Furthermore, a proxi mal AP-1/CRE moth is crucial for this Ras-mediated NT/N activation. Wi ld-type Ha-ras induces NT/N gene expression, albeit at lower levels th an activated Ras; a dominant-negative Raf blocks this NT/N induction, suggesting that Raf lies downstream of Ras in this pathway. In additio n, postconfluent cultures of CaCo2 cells, which are differentiated to a small bowel phenotype, express the NT/N gene by 6 d after reaching c onfluency; this increase of NT/N expression is associated with concomi tant increases of cellular p21(ras) protein. We conclude that Ras (bot h wild-type and activated) enhances expression of the NT/N gene in the gut-derived CaCo2 cell line, suggesting an important role for the Ras signaling pathway in NT/N gene transcription. Our results underscore the possibility that tissue-specific genes (such as NT/N) expressed in distinct subpopulations of the gut may be subject to Ras regulation, Finally, we speculate that the NT/N gene and the CaCo2 and CaCo2-ras c ell systems will provide unique models to further define the cellular mechanisms leading to mammalian intestinal differentiation.