Abstract
Neurotensin (NT), a neuropeptide released in the gastrointestinal tract in response to several stimuli, is involved in the pathophysiology of colonic inflammation. However, the molecular mechanism(s) mediating this proinflammatory response remains unclear. We found that NCM460, non-transformed human colonocytes, express a functional high affinity NT receptor that mediates NT-induced Erk activation. By using NCM460 cells stably transfected with NTR1, we show that NTR1 activation leads to interleukin (IL)-8 secretion that is mediated via both NF-kappaB- and Erk-dependent pathways. In addition, NT-stimulated NF-kappaB activation is dependent on intracellular calcium release. NT-stimulated Erk activity requires Ras activation because overexpression of the dominant negative Ras mutant Ras-17N almost completely inhibits the Erk activation. Furthermore, NT directly stimulates Ras-GTP formation as shown by a Ras-GTP pull-down assay. By using reporter gene constructs containing targeted substitutions in the IL-8 promoter, we show that the NF-kappaB, AP-1, and to a lesser degree the C/EBP sites in the IL-8 promoter region are required for IL-8 gene expression induced by NT. In summary, our results demonstrate that NT stimulates calcium-dependent NF-kappaB and Ras-dependent Erk pathways that mediate the release of IL-8 from non-transformed human colonocytes. We speculate that these NT-related proinflammatory pathways are important in the pathophysiology of colonic inflammation.
Highlights
Neurotensin (NT),1 a 13-amino acid neuropeptide originally isolated by Carraway and Leeman [1], is highly expressed in the gastrointestinal tract [2]
We first determined whether NCM460 cells express NTR1 mRNA by RT-PCR, and we examined the functionality of this receptor
Our results show that the NCM460 cells express NTR1 as indicated by an expected 1315-bp PCR product that corresponds to the full-length coding region of human NTR1 cDNA
Summary
NT was purchased from Phoenix Pharmaceuticals (Belmont, CA). [3,11,-tyrosyl-3,5-3H]NT was from PerkinElmer Life Sciences. Construction and Functionality of a Puromycin-resistant Retroviral Vector—To create stably transfected cell lines that selectively grow in medium containing puromycin, we constructed a highly efficient puromycin-resistant expression retroviral vector named pCMBP from the vector pCMMP The retroviral promoter from pCMMP was removed with the restriction enzymes Sbf and PmlI (the fragment named Sbf-PmlI), and ligated into the pBabe-puro vector which was first digested with BamHI, endblunted with Klenow, and digested with PstI. The retroviruses expressing -galactosidase were prepared following the procedure described below and used to infect NIH3T3 cells to measure the titer of produced viruses. The NTR1 fragment was removed from pCR2.1 by EcoRI digestion and ligated into the puromycin-expressing retroviral vector pCMBP (pCMBP-NTR1) described above. Preparation of Retroviruses and Infection of NCM460 Cells—293T cells Selected cells were pooled (named NCM460-NTR1 cells) and used for this study
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