The neurotensin gene is a downstream target for Ras activation.
Ras regulates novel patterns of gene expression and the differentiation of various eukaryotic cell types. Stable transfection of Ha-ras into the human colon cancer line CaCo2 results in the morphologic differentiation to a small bowel phenotype. The purpose of our study was to determine whether the Ras regulatory pathway plays a role in the expression of the neurotensin gene (NT/N), a terminally differentiated endocrine product specifically localized in the gastrointestinal tract to the adult small bowel. We found that CaCo2-ras cells, but not parental CaCo2, express high levels of the human NT/N gene and, moreover, that this increase in gene expression is regulated at the level of transcription. Transfection experiments using NT/N-CAT mutation constructs identify the proximal 200 bp of NT/N flanking sequence as sufficient for maximal Ras-mediated NT/N reporter gene induction. Furthermore, a proximal AP-1/CRE motif is crucial for this Ras-mediated NT/N activation. Wild-type Ha-ras induces NT/N gene expression, albeit at lower levels than activated Ras; a dominant-negative Raf blocks this NT/N induction, suggesting that Raf lies down-stream of Ras in this pathway. In addition, postconfluent cultures of CaCo2 cells, which are differentiated to a small bowel phenotype, express the NT/N gene by 6 d after reaching confluency; this increase of NT/N expression is associated with concomitant increases of cellular p21ras protein. We conclude that Ras (both 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 cell systems will provide unique models to further define the cellular mechanisms leading to mammalian intestinal differentiation.
Evers
B M
BM
Department of Surgery, University of Texas Medical Branch, Galveston 77555, USA.
Zhou
Z
Z
Celano
P
P
Li
J
J
D002454:Cell Differentiation; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D011905:Genes, ras; D006801:Humans; D066298:In Vitro Techniques; D007422:Intestines / Q000166:cytology / Q000502:physiology; D009496:Neurotensin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D009496:Neurotensin; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)