title: Metalloproteinase-dependent transforming growth factor-alpha release mediates neurotensin-stimulated MAP kinase activation in human colonic epithelial cells.
abstract: Expression of the neuropeptide neurotensin (NT) and its high affinity receptor (NTR1) is increased during the course of Clostridium difficile toxin A-induced acute colitis, and NTR1 antagonism attenuates the severity of toxin A-induced inflammation. We recently demonstrated in non-transformed human colonic epithelial NCM460 cells that NT treatment caused activation of a Ras-mediated MAP kinase pathway that significantly contributes to NT-induced interleukin-8 (IL-8) secretion. Here we used NCM460 cells, which normally express low levels of NTR1, and NCM460 cells stably transfected with NTR1 to identify the upstream signaling molecules involved in NT-NTR1-mediated MAP kinase activation. We found that inhibition of the epidermal growth factor receptor (EGFR) by either an EGFR neutralizing antibody or by its specific inhibitor AG1478 (0.2 microm) blocked NT-induced MAP kinase activation. Moreover, NT stimulated tyrosine phosphorylation of the EGFR, and pretreatment with a broad spectrum metalloproteinase inhibitor batimastat reduced NT-induced MAP kinase activation. Using neutralizing antibodies against the EGFR ligands EGF, heparin-binding-EGF, transforming growth factor-alpha (TGFalpha), or amphiregulin we have shown that only the anti-TGFalpha antibody significantly decreases NT-induced phosphorylation of EGFR and MAP kinases. Furthermore, inhibition of the EGF receptor by AG1478 significantly reduced NT-induced IL-8 promoter activity and IL-8 secretion. This is the first report demonstrating that NT binding to NTR1 transactivates the EGFR and that this response is linked to NT-mediated proinflammatory signaling. Our findings indicate that matrix metalloproteinase-mediated release of TGFalpha and subsequent EGFR transactivation triggers a NT-mediated MAP kinase pathway that leads to IL-8 gene expression in human colonic epithelial cells.
authors.affiliation: Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02468, USA.
authors.forename: Dezheng
authors.initials: D
authors.lastname: Zhao
authors.forename: Yanai
authors.initials: Y
authors.lastname: Zhan
authors.forename: Hon Wai
authors.initials: HW
authors.lastname: Koon
authors.forename: Huiyan
authors.initials: H
authors.lastname: Zeng
authors.forename: Sarah
authors.initials: S
authors.lastname: Keates
authors.forename: Mary P
authors.initials: MP
authors.lastname: Moyer
authors.forename: Charalabos
authors.initials: C
authors.lastname: Pothoulakis
mesh_terms: D002460:Cell Line; D003106:Colon; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D007700:Kinetics; D045726:Metalloproteases / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009496:Neurotensin / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D014409:Tumor Necrosis Factor-alpha; D009496:Neurotensin; D066246:ErbB Receptors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D045726:Metalloproteases