Signal Transduction Pathways Mediating Neurotensin-stimulated Interleukin-8 Expression in Human Colonocytes

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-κB- and Erk-dependent pathways. In addition, NT-stimulated NF-κB 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-κB, 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-κB 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. 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-κB- and Erk-dependent pathways. In addition, NT-stimulated NF-κB 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-κB, 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-κB 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. neurotensin guanine nucleotide-binding protein-coupled receptor extracellular signal-regulated protein kinase 1,2-bis(o-aminophenooxy)ethane-N,N,N′,N′-tetraacetic acid, acetoxymethyl ester phorbol 12-myristate acetate pertussis toxin interleukin neurotensin receptor epidermal growth factor reverse transcriptase-polymerase chain reaction base pair fetal bovine serum phosphate-buffered saline enzyme-linked immunosorbent assay electrophoretic mobility shift assay(s) mitogen-activated protein Neurotensin (NT),1 a 13-amino acid neuropeptide originally isolated by Carraway and Leeman (1Carraway R. Leeman S.E. J. Biol. Chem. 1973; 248: 6854-6861Abstract Full Text PDF PubMed Google Scholar), is highly expressed in the gastrointestinal tract (2Carraway R. Leeman S.E. J. Biol. Chem. 1976; 251: 7045-7052Abstract Full Text PDF PubMed Google Scholar). 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Invest. 1999; 103: 843-849Crossref PubMed Scopus (120) Google Scholar) demonstrate that NT is a proinflammatory peptide in the colon because blockage of the NT-NTR1 interaction with SR 48692 inhibited colonic secretion and inflammation mediated by Clostridium difficile toxin A. We also showed that, compared with normal colonic epithelial cells, there was a dramatic up-regulation of NTR1 during human colonic inflammation (15Castagliuolo I. Valenick L. Pasha A. Sturniolo G.C. D'Incá R.D. Fiocchi C. Pothoulakis C. Gastroenterology. 1999; 116: 799Google Scholar) as well as in Clostridium difficile toxin A-mediated colitis (14Castagliuolo I. Wang C.C. Valenick L. Pasha A. Nikulasson S. Carraway R.E. Pothoulakis C. J. Clin. Invest. 1999; 103: 843-849Crossref PubMed Scopus (120) Google Scholar). NT exerts its proinflammatory effects by interacting with several cell types, including mast cells (13Castagliuolo I. Leeman S.E. Bartolak-Suki E. Nikulasson S. Qiu B. Carraway R.E. Pothoulakis C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12611-12615Crossref PubMed Scopus (69) Google Scholar, 14Castagliuolo I. Wang C.C. Valenick L. Pasha A. Nikulasson S. Carraway R.E. Pothoulakis C. J. Clin. Invest. 1999; 103: 843-849Crossref PubMed Scopus (120) Google Scholar, 16Pothoulakis C. Castagliuolo I. Leeman S.E. Ann. N.Y. Acad. Sci. 1998; 840: 635-648Crossref PubMed Scopus (56) Google Scholar, 17Miller L.A. Cochrane D.E. Carraway R.E. Feldberg R.S. Br. J. Pharmacol. 1995; 114: 1466-1470Crossref PubMed Scopus (40) Google Scholar), leukocytes (14Castagliuolo I. Wang C.C. Valenick L. Pasha A. Nikulasson S. Carraway R.E. Pothoulakis C. J. Clin. Invest. 1999; 103: 843-849Crossref PubMed Scopus (120) Google Scholar, 18Goldman R. Bar-Shavit Z. Romeo D. FEBS Lett. 1983; 159: 63-67Crossref PubMed Scopus (38) Google Scholar), endothelial cells (19Schaeffer P. Laplace M.C. Savi P. Pflieger A.M. Gully D. Herbert J.M. J. Biol. 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However, the importance of the sites in IL-8 gene expression on the cell and the In this we NCM460, a non-transformed human colonic epithelial cell to the molecular by which NT exerts its proinflammatory We found that NCM460 cells express a functional NTR1 of NCM460 cells transfected with the NTR1 to NT stimulates IL-8 secretion as well as gene in a and We also demonstrate that NF-κB and Erk activation are required for NT-induced IL-8 gene Our results that NT-stimulated NF-κB DNA binding activity is we show for the that NT stimulates Ras-GTP formation and that NT-induced Erk activation is NT was from was from and from and from and and from DNA from NCM460 cells and from for of protein from stably transfected cell lines that in containing we a highly expression from the by C. and by R. of The promoter from was with the and and the which was with with and with the of this we also a gene the by a that, was with to the and was isolated from by this with and The was with the and the The the described and to cells to the of cell was isolated from NCM460 cells by the as described previously P. N. Biochem. PubMed Scopus Google Scholar). Two of with to the of was for of human NTR1 using DNA under the by of and and The on the of human NTR1 N. P. S. P. X. M. Gully D. Le Fur G. P. D. FEBS Lett. PubMed Scopus Google Scholar). The containing was and the reverse containing a was was using the and the of the DNA was by DNA The NTR1 was from by and the described containing NTR1 using the described cells by A. a of cells in containing of for and the was with The by C. and in a of and to using to the the and and the of NCM460 cells was as for and with containing of and of growth for in the of The cells in containing and for cells and for this NCM460 cells cells in in containing The cells with and with for The cells with of containing of was to the for the cells on and with in of for and the in the was to the of in The of receptors was from a by IL-8 protein in colonic epithelial by a enzyme-linked immunosorbent assay using IL-8 as described previously Pothoulakis C. S. M. Am. J. Physiol. Google Scholar). expressed as S.E. for with of a reporter containing to of the promoter region of human IL-8 gene has been described previously M. S. Castagliuolo I. S. A. Pothoulakis C. J. Clin. Invest. 2000; PubMed Scopus Google Scholar). IL-8 reporter constructs containing in NF-κB, AP-1, C/EBP sites by The on the described by N. X. J. Biol. Chem. PubMed Scopus Google Scholar). The NF-κB was the was and the C/EBP was The by DNA using specific for the expression and the IL-8 promoter activity in response to NT, cells in and transfected using with IL-8 constructs a DNA constructs as cells for by to NT for in cell using The activity was by IL-8 activity to from are as the activity from of with for DNA binding as described previously M. Res. PubMed Scopus Google Scholar). in with and of and for The cell with of for of of for by for with of for and for of with of of and of for was to the reaction and for on the was to the and for of the The and as well as normal from of specific protein to the was by the a for in The was for under to The NF-κB was from The was by DNA kinase and cells in with and with of bovine serum in for by and for in of bovine serum and and the in of containing and and for for and in of of the cell was with for with NT. was with the with and in containing a inhibitor for for of cell by and for blocked in in and with to Erk by Ras activity was using a Ras the cells stimulated with NT with and with containing a inhibitor for for The with for and for The with for and the with The with and of the to using a the of Ras to of of cell also to using the Ras expressed as S.E. using the of with for Our recent (14Castagliuolo I. Wang C.C. Valenick L. Pasha A. Nikulasson S. Carraway R.E. Pothoulakis C. J. Clin. Invest. 1999; 103: 843-849Crossref PubMed Scopus (120) Google Scholar) that NTR1 mediates colonic inflammation. NTR1 mediates release of the proinflammatory we the non-transformed human colonic epithelial cell NCM460 L.A. L.R. In Biol. 1996; PubMed Scopus Google Scholar). We NCM460 cells express NTR1 by and we the of this Our results show that the NCM460 cells express NTR1 as by that to the region of human NTR1 not NCM460 cells to NT to for NT induced Erk a of with the specific NTR1 antagonist SR 48692 a of completely inhibited NT Erk activation, indicating that NTR1 mediates this response NT of to IL-8 secretion not suggesting that the of the receptor may not high to mediate this we to NTR1 in NCM460 cells to expression of NTR1 results in IL-8 secretion in response to NT. that, the NTR1 by from from NCM460 cells was the NTR1 and to stably transfected NCM460 cell lines NTR1 as described under cells express of NT we a binding assay using The results not that compared with the NCM460 cell that expressed NT receptors cells receptors of cells to NT stimulated IL-8 secretion with a NT IL-8 secretion induced by NT was and and a was However, NT of not IL-8 secretion in the NCM460 cells We also NT stimulates IL-8 gene in cells transfected with a reporter containing the human IL-8 The results showed that NT stimulated IL-8 activity by in cells in the NCM460 cells these results demonstrate that NT stimulates IL-8 secretion as well as IL-8 gene in the of NF-κB activation for NT-induced IL-8 we NT stimulated translocation and DNA binding activity of NF-κB in NT DNA binding of a protein that NF-κB and as shown by The of the was by the with a that not NF-κB as well as by of the binding We of NF-κB activation NT-induced IL-8 of cells with the NF-κB inhibitor inhibited NT-induced NF-κB DNA binding activity and IL-8 secretion not the of NF-κB in NT-induced IL-8 we endogenous NF-κB Our show that NT-induced IL-8 secretion was by in cells overexpressing human as compared with the overexpression of inhibits NT-induced IL-8 gene cells transfected with the IL-8 reporter with Our that with the NT-induced IL-8 gene the NF-κB for NT-induced IL-8 we IL-8 promoter that targeted in the The results showed that the NF-κB mutant not to NT the IL-8 promoter was by NT NF-κB activation is a for NT-induced IL-8 gene and IL-8 protein release. NT stimulated intracellular calcium release in cells a which to of the cells with completely inhibited NT-induced the and NT-induced IL-8 secretion the of was mediated NF-κB, cells with and with NT and NF-κB DNA binding activity was by Our results showed that with NT-stimulated NF-κB DNA binding indicating that the of intracellular calcium on NT-induced IL-8 secretion NF-κB activation We also found that of with a of inhibitor of the calcium-dependent inhibited NT-induced IL-8 secretion by and not results that the NF-κB activation in response to NT mediated and We Erk-dependent mediates NT-induced IL-8 cells with a specific kinase and with NT for to IL-8 release and IL-8 inhibited both NT-induced IL-8 release and IL-8 promoter activity suggesting that Erk activation is involved in NT-induced IL-8 Ras is of NT-induced Erk activation, we NT directly activates Ras using a Ras-GTP pull-down assay D. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). cells with NT for for and the of Ras-GTP in the cell We found that NT Ras-GTP from to NT stimulated Ras-GTP demonstrate for the that binding of NT to NTR1 activates It is that Erk activation Ras-dependent T. T. T. T. M. F. K. K. M. J. 1999; PubMed Scopus Google Scholar, A. A. S. I. Biol. 2000; 20: PubMed Scopus Google Scholar). However, Ras is involved in the NT signaling is not this we a dominant negative Ras mutant Ras-17N to events of Ras activation. cells with the and to NT a phorbol ester that activates Erk in a Y. S. S. A. K. S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), which is to Erk in a Ras-dependent We found that the dominant negative Ras mutant blocked Erk activation induced by NT as by on Erk activation NT-induced activation of the Erk is intracellular calcium is involved in Erk activation in cells J.M. Res. 1996; PubMed Scopus Google Scholar), we the that calcium in NT-induced Erk activation. with for cells with NT for The that on NT-induced Erk activation not We also the of PTX on NT-induced Erk activation. cells with PTX and with acid NT for The results that PTX almost completely inhibited Erk activation on NT-induced Erk activation. our results demonstrate that NT-stimulated Erk activation is Ras-dependent not intracellular calcium and Ras activation is required for NT-induced IL-8 secretion and IL-8 gene cells with and to NT for of the dominant negative Ras inhibited NT-induced IL-8 protein release the Ras-17N mutant also inhibited NT-induced IL-8 cells transfected with the Ras-17N with IL-8 and to NT for shown in overexpression of Ras-17N inhibited NT-stimulated IL-8 promoter We have previously (14Castagliuolo I. Wang C.C. Valenick L. Pasha A. Nikulasson S. Carraway R.E. Pothoulakis C. J. Clin. Invest. 1999; 103: 843-849Crossref PubMed Scopus (120) Google Scholar, I. Valenick L. Pasha A. Sturniolo G.C. D'Incá R.D. Fiocchi C. Pothoulakis C. Gastroenterology. 1999; 116: 799Google Scholar) that NTR1 are in colonic inflammation and for expression of NTR1 receptors on colonic epithelial that NCM460 human colonic epithelial cells express the high affinity NT receptor that mediates Erk activation in response to NT. of NCM460 cells overexpressing NTR1 to NT secretion of the IL-8 in a and NT-induced IL-8 secretion and gene dependent on NF-κB and Erk activation. In addition, NF-κB activation was mediated by intracellular calcium release. We also for the that NT stimulates Ras activity which is required for NT-induced Erk activation and that both calcium and Ras activation are involved in IL-8 release in response to NT. Our studies demonstrate that the of NF-κB activation and IL-8 secretion in response to NT in is calcium-dependent NF-κB activation is required for IL-8 expression induced by the neuropeptide in human K. M. J. K. J. Immunol. 159: Google Scholar). The by which intracellular calcium release leads to activation of NF-κB activity NT have not been elucidated. of cells to stimuli as cytokines triggers a signaling and of to the activation, and translocation of NF-κB Immunol. 1996; PubMed Scopus Google Scholar). The of intracellular calcium on may mediated by a as a S. M. L.A. J. PubMed Scopus Google Scholar, K. A. T. FEBS Lett. 1998; PubMed Scopus (50) Google Scholar). In addition, the calcium-dependent protein may with protein kinase C to NF-κB by a S. M. L.A. J. PubMed Scopus Google Scholar, B. J. Immunol. 1995; Google Scholar, A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Our that partially inhibited IL-8 secretion that calcium-dependent pathways may involved in NF-κB activation NT Our results that Erk is required for NT-induced IL-8 promoter activity and IL-8 a proinflammatory for in the NTR1 signaling results also a for in IL-8 the inhibitor of and the inhibitor of inhibited IL-8 release induced by C. difficile toxin in human M. S. Castagliuolo I. S. A. Pothoulakis C. J. Clin. Invest. 2000; PubMed Scopus Google Scholar) as well as by in gastric epithelial cells S. M. R.M. J. Immunol. 1999; Google Scholar). Although both kinase and NF-κB are required for IL-8 gene expression in response to stimuli, involvement of kinase pathways in NF-κB activation to dependent on the Erk and kinase activation not to with NF-κB S. M. R.M. J. Immunol. 1999; Google Scholar). in the kinase inhibitor IL-8 in response to translocation of NF-κB M. T. S. N. M. N. T. FEBS Lett. 2000; PubMed Scopus Google Scholar). a was shown to required for gene expression and mucin in epithelial cells M. J.H. J. Y. C. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). In addition, our results demonstrate that Erk is also involved in NT-induced gene expression as inhibited In to the NF-κB the IL-8 promoter binding sites for the and Our also that the and sites involved in NT-induced IL-8 promoter activity not these a is to the transcriptional activity of as well as expression of and the of the transcriptional factor M.R. J. Biol. 2000; PubMed Scopus Google Scholar, J. 1996; PubMed Scopus Google Scholar). IL-8 expression in human both NF-κB and pathways. Although NT-stimulated Erk activation in Chinese hamster ovary cells NTR1 was partially inhibited by PTX and completely blocked by the protein kinase C inhibitor GF 109203X (25Poinot-Chazel C. Portier M. Bouaboula M. Vita N. Pecceu F. Gully D. Monroe J.G. Maffrand J.P. Le Fur G. Casellas P. Biochem. J. 1996; 320: 145-151Crossref PubMed Scopus (66) Google Scholar), the that leads to Erk activation is not Our results demonstrate that a dominant negative Ras mutant Ras-17N completely inhibited NT-induced Erk and NT-induced IL-8 NT stimulated Ras-GTP our this is the to show that NT activates Ras and that NT-induced Erk activation is The NT stimulates Ras-dependent Erk activation in a protein and remains to elucidated. It is that Ras-dependent Erk activation by mediated is in which the released from the kinase to activation of the Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, P. N. Nature. PubMed Scopus Google Scholar, Y. S. F. K. 1999; PubMed Scopus Google Scholar). to Ras-dependent Erk activation is via the calcium-dependent kinase which is by and I. G. S. J. Nature. 1996; PubMed Scopus Google Scholar). However, the of PTX to NT-induced Erk activation in our that these pathways are to mediate NT-induced Ras-dependent Erk activation. In addition, of the receptor has also been shown to mediate Erk activation induced by as the receptors for acid, C. A. Nature. 1996; PubMed Scopus Google Scholar), and neuropeptide I. Valenick L. J. Pothoulakis C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The that the of the receptor is involved in NT-induced Erk activation remains to elucidated. In summary, our that NT expression of IL-8 via NF-κB and Ras-dependent Erk activation our of the molecular by which this neuropeptide mediates its in the gastrointestinal We C. R. and for of in this We to for the antagonist