Background. Neurotensin, an important gut hormone, binds its receptor (NTR) to stimulate proliferation of intestinal cells; the molecular mechanisms remain largely undefined. Mitogen-activated protein kinases (MAPKs) translocate to the nucleus and induce transcription factors (eg, c- Fos) in response to certain trophic agents. The purpose of this study was (1) to define the signaling mechanisms regulating neurotensin and (2) to determine whether the AP-1 transcription factor c-Fos is induced. Methods. Expression of the NTR gene was determined in the human colon cancer cell lines KM12C, KML4A, and KM20 by Northern blot analysis and ribonuclease- protection experiments. To assess whether NTR was functionally coupled to the Ca2+-signaling pathway, intracellular Ca2+ ([Ca2+](i)) mobilization was assessed by fura-2 spectrofluorometry. To determine whether the MAPK signaling pathway was activated in KM20 cells by neurotensin, Western blots for the Phosphorylated forms of MAPK (p42 and p44) and in vitro kinase assays were performed. In addition, Western blots were performed to assess levels of c-Fos after neurotensin treatment. Results. The NTR gene was expressed in the KM20 cell line but not in KM12C or KM12LA. The NTR in KM20 cells was functionally coupled to the Ca2+-signaling pathway as noted by a rapid (30 seconds) mobilization of [Ca2+](i) after addition of neurotensin; the neurotensin-mediated response was blocked by the NTR antagonist SR48692. Both p42(MAPK) and p44(MAPK) were stimulated by neurotensin ~3 to 6 minutes after treatment. Increased levels of c-Fos were demonstrated, with peak levels occurring 2 hours after addition of neurotensin. Conclusions. Our results demonstrate that neurotensin binds to its endogenous NTR in KM20 cells with stimulation of the Ca2+- and MAPK-signaling pathways and an increase in the levels of the AP-1 protein c-Fos. Delineating the signal transduction mechanisms regulating the cellular effects of neurotensin will provide important insights into the molecular pathways responsible for gut hormone- mediated proliferation.
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