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• W2000960883 abstract "Substance P (SP) participates in acute intestinal inflammation via binding to the G-protein-coupled neurokinin-1 receptor (NK-1R) and release of proinflammatory cytokines from colonic epithelial cells. SP also stimulates cell proliferation, a critical event in tissue healing during chronic colitis, via transactivation of the epidermal growth factor (EGF) receptor (EGFR) and activation of mitogen-activated protein kinase (MAPK). Here we examined the mechanism by which SP induces EGFR and MAPK activation. We used non-transformed human NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) as well as untransfected U373 MG cells expressing high levels of endogenous NK-1R. Exposure of both cell lines to SP (10–7m) stimulated EGFR activation (1 min) followed by extracellular signal-regulated protein kinase (ERK1/2) activation (2–5 min). SP-induced ERK1/2 activation was blocked by pretreatment with the metalloproteinase inhibitor Batimastat/GM6001, the EGFR phosphorylation inhibitor AG1478, and the tumor necrosis factor-α-converting enzyme (TACE) inhibitor TAPI-1. Pretreatment with antibodies against potential EGFR ligands suggested that transforming growth factor-α (TGFα), but not the other EGFR ligands EGF, heparin-binding EGF, or amphiregulin, mediates SP-induced EGFR transactivation. SP stimulated TGFα release into the extracellular space that was measurable within 2 min, and this release was inhibited by metalloproteinase inhibitors and the TACE inhibitor TAPI-1. SP also induced MAPK-mediated cell proliferation that was inhibited by TACE, matrix metalloproteinase (MMP), EGFR, and MEK1 inhibitors. Thus, in human colonocytes, NK-1R-induced EGFR and MAPK activation and cell proliferation involve matrix metalloproteinases (most likely TACE) and the release of TGFα. These signaling mechanisms may be involved in the protective effects of NK-1R in chronic colitis. Substance P (SP) participates in acute intestinal inflammation via binding to the G-protein-coupled neurokinin-1 receptor (NK-1R) and release of proinflammatory cytokines from colonic epithelial cells. SP also stimulates cell proliferation, a critical event in tissue healing during chronic colitis, via transactivation of the epidermal growth factor (EGF) receptor (EGFR) and activation of mitogen-activated protein kinase (MAPK). Here we examined the mechanism by which SP induces EGFR and MAPK activation. We used non-transformed human NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) as well as untransfected U373 MG cells expressing high levels of endogenous NK-1R. Exposure of both cell lines to SP (10–7m) stimulated EGFR activation (1 min) followed by extracellular signal-regulated protein kinase (ERK1/2) activation (2–5 min). SP-induced ERK1/2 activation was blocked by pretreatment with the metalloproteinase inhibitor Batimastat/GM6001, the EGFR phosphorylation inhibitor AG1478, and the tumor necrosis factor-α-converting enzyme (TACE) inhibitor TAPI-1. Pretreatment with antibodies against potential EGFR ligands suggested that transforming growth factor-α (TGFα), but not the other EGFR ligands EGF, heparin-binding EGF, or amphiregulin, mediates SP-induced EGFR transactivation. SP stimulated TGFα release into the extracellular space that was measurable within 2 min, and this release was inhibited by metalloproteinase inhibitors and the TACE inhibitor TAPI-1. SP also induced MAPK-mediated cell proliferation that was inhibited by TACE, matrix metalloproteinase (MMP), EGFR, and MEK1 inhibitors. Thus, in human colonocytes, NK-1R-induced EGFR and MAPK activation and cell proliferation involve matrix metalloproteinases (most likely TACE) and the release of TGFα. These signaling mechanisms may be involved in the protective effects of NK-1R in chronic colitis. Substance P (SP), 1The abbreviations used are: SP, substance P; NK-1R, neurokinin-1 receptor; IL, interleukin; TNFα, tumor necrosis factor-α; MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; EGFR, EGF receptor; TACE, TNFα-converting enzyme; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MMP, matrix metalloproteinase; ERK, extracellular signal-regulated kinase; HB, heparin-binding; TGFα, transforming growth factor-α; ELISA, enzyme-linked immunosorbent assay; ADAM, adisintegrin and metalloproteinase.1The abbreviations used are: SP, substance P; NK-1R, neurokinin-1 receptor; IL, interleukin; TNFα, tumor necrosis factor-α; MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; EGFR, EGF receptor; TACE, TNFα-converting enzyme; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MMP, matrix metalloproteinase; ERK, extracellular signal-regulated kinase; HB, heparin-binding; TGFα, transforming growth factor-α; ELISA, enzyme-linked immunosorbent assay; ADAM, adisintegrin and metalloproteinase. an 11-amino acid neuropeptide belonging to the tachykinin family, is localized in the central nervous system (1Mantyh P.W. J. Clin. Psychiatry. 2002; 63: 6-10PubMed Google Scholar), enteric nerves (2Costa M. Furness J.B. Franco R. Llewellyn-Smith I. Murphy R. Beardsley A.M. CIBA Found. Symp. 1982; : 129-144PubMed Google Scholar), sensory neurons (3Maggi C.A. Arch. Int. Pharmacodyn. Ther. 1990; 303: 157-166PubMed Google Scholar), and immune cells, such as macrophages (4Bost K.L. Adv. Exp. Med. Biol. 1995; 373: 219-223Crossref PubMed Scopus (17) Google Scholar). SP has been associated with several intestinal functions, including motility (5Holzer P. Holzer-Petsche U. Pharmacol. Ther. 1997; 73: 173-217Crossref PubMed Scopus (311) Google Scholar), mucosal permeability (6Pothoulakis C. Castagliuolo I. Lamont J.T. Jaffer A. O'Keane J.C. Snider R.M. Leeman S.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 947-951Crossref PubMed Scopus (255) Google Scholar), and chloride secretion (7Riegler M. Castagliuolo I. So P.T. Lotz M. Wang C. Wlk M. Sogukoglu T. Cosentini E. Bischof G. Hamilton G. Teleky B. Wenzl E. Matthews J.B. Pothoulakis C. Am. J. Physiol. 1999; 276: G1473-G1483PubMed Google Scholar), via binding to its high affinity neurokinin-1 receptor (NK-1R) expressed on several intestinal cell types. SP and the NK-1R play a pivotal role in the pathophysiology of intestinal inflammation. Thus, SP and NK-1R expression is increased in the colon of patients with inflammatory bowel disease (8Goldin E. Karmeli F. Selinger Z. Rachmilewitz D. Dig. Dis. Sci. 1989; 34: 754-757Crossref PubMed Scopus (142) Google Scholar), in Clostridium difficile-associated colitis (9Mantyh C.R. Maggio J.E. Mantyh P.W. Vigna S.R. Pappas T.N. Dig. Dis. Sci. 1996; 41: 614-620Crossref PubMed Scopus (44) Google Scholar), and in the ileum of rats injected with C. difficile toxin A (10Castagliuolo I. Keates A.C. Qiu B. Kelly C.P. Nikulasson S. Leeman S.E. Pothoulakis C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4788-4793Crossref PubMed Scopus (143) Google Scholar, 11Pothoulakis C. Castagliuolo I. Leeman S.E. Wang C.C. Li H. Hoffman B.J. Mezey E. Am. J. Physiol. 1998; 275: G68-G75Crossref PubMed Google Scholar). Pharmacologic antagonism of NK-1R (6Pothoulakis C. Castagliuolo I. Lamont J.T. Jaffer A. O'Keane J.C. Snider R.M. Leeman S.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 947-951Crossref PubMed Scopus (255) Google Scholar, 12Mantyh C.R. Pappas T.N. Lapp J.A. Washington M.K. Neville L.M. Ghilardi J.R. Rogers S.D. Mantyh P.W. Vigna S.R. Gastroenterology. 1996; 111: 1272-1280Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 13Stucchi A.F. Shofer S. Leeman S. Materne O. Beer E. McClung J. Shebani K. Moore F. O'Brien M. Becker J.M. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G1298-G1306Crossref PubMed Google Scholar) or genetic deletion of this receptor (14Castagliuolo I. Riegler M. Pasha A. Nikulasson S. Lu B. Gerard C. Gerard N.P. Pothoulakis C. J. Clin. Investig. 1998; 101: 1547-1550Crossref PubMed Scopus (160) Google Scholar) results in reduced intestinal inflammation, whereas deletion of neutral endopeptidases, which degrade SP, enhances acute gut inflammation (15Kirkwood K.S. Bunnett N.W. Maa J. Castagliolo I. Liu B. Gerard N. Zacks J. Pothoulakis C. Grady E.F. Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 281: G544-G551Crossref PubMed Google Scholar). Along these lines SP acts as a proinflammatory peptide in vitro and induces secretion of interleukin-1 (IL-1), IL-6, IL-8, and tumor necrosis factor-α (TNFα) (10Castagliuolo I. Keates A.C. Qiu B. Kelly C.P. Nikulasson S. Leeman S.E. Pothoulakis C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4788-4793Crossref PubMed Scopus (143) Google Scholar, 16Derocq J.M. Segui M. Blazy C. Emonds-Alt X. Le Fur G. Brelire J.C. Casellas P. FEBS Lett. 1996; 399: 321-325Crossref PubMed Scopus (36) Google Scholar, 17Fiebich B.L. Schleicher S. Butcher R.D. Craig A. Lieb K. J. Immunol. 2000; 165: 5606-5611Crossref PubMed Scopus (118) Google Scholar, 18Laurenzi M.A. Persson M.A. Dalsgaard C.J. Haegerstrand A. Scand. J. Immunol. 1990; 31: 529-533Crossref PubMed Scopus (133) Google Scholar, 19Lieb K. Fiebich B.L. Busse-Grawitz M. Hull M. Berger M. Bauer J. J. Neuroimmunol. 1996; 67: 77-81Abstract Full Text PDF PubMed Google Scholar, 20Zhao D. Kuhnt-Moore S. Zeng H. Pan A. Wu J.S. Simeonidis S. Moyer M.P. Pothoulakis C. Biochem. J. 2002; 368: 665-672Crossref PubMed Scopus (67) Google Scholar) via activation of the transcription factor NF-κB in target cells (21Azzolina A. Bongiovanni A. Lampiasi N. Biochim. Biophys. Acta. 2003; 1643: 75-83Crossref PubMed Scopus (185) Google Scholar, 22Lieb K. Fiebich B.L. Berger M. Bauer J. Schulze-Osthoff K. J. Immunol. 1997; 159: 4952-4958PubMed Google Scholar). However, studies with NK-1R-deficient mice also indicated that although NK-1R deletion protects mice from the acute phase of experimental colitis, all aspects of colonic inflammation were enhanced at later stages of colitis, indicating that SP may play a protective role in chronic colonic inflammation (28Al Sarraj A. Thiel G. Neurosci. Lett. 2002; 332: 111-114Crossref PubMed Scopus (25) Google Scholar). Ligand binding to NK-1R, a G-protein-coupled receptor, leads to phosphoinositide hydrolysis (23Rollandy I. Dreux C. Imhoff V. Rossignol B. Neuropeptides. 1989; 13: 175-185Crossref PubMed Scopus (18) Google Scholar), calcium mobilization (24Pradier L. Heuillet E. Hubert J.P. Laville M. Le Guern S. Doble A. J. Neurochem. 1993; 61: 1850-1858Crossref PubMed Scopus (18) Google Scholar), and mitogen-activated protein kinase (MAPK) activation (25Luo W. Sharif T.R. Sharif M. Cancer Res. 1996; 56: 4983-4991PubMed Google Scholar, 26DeFea K.A. Vaughn Z.D. O'Bryan E.M. Nishijima D. Dery O. Bunnett N.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11086-11091Crossref PubMed Scopus (353) Google Scholar). We reported previously that SP stimulation leads to activation of MAPK and increased DNA synthesis in U373 MG astrocytoma cells by a mechanism involving transactivation of the epidermal growth factor receptor (EGFR) (27Castagliuolo I. Valenick L. Liu J. Pothoulakis C. J. Biol. Chem. 2000; 275: 26545-26550Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Al-Sarrai and Thiel (28Al Sarraj A. Thiel G. Neurosci. Lett. 2002; 332: 111-114Crossref PubMed Scopus (25) Google Scholar) also reported that SP via NK-1R causes transactivation of the EGFR in human glioma cells. However, whether SP transactivates EGFR in human colonocytes is not known. Moreover, the signaling mechanism(s) by which SP-NK-1R interactions lead to EGFR and MAPK activation and cell proliferation has not been identified. In this current study, we sought to identify the mechanisms of SP-induced activation of EGFR and MAPK and SP-induced cell proliferation in non-transformed human colonic epithelial cells stably transfected with NK-1R, as well as in U373 MG astrocytoma cells expressing high levels of endogenous NK-1R. Cell Culture—Non-transformed human colonic epithelial NCM460 cells stably expressing NK-1R (NCM460-NK-1R) were made by a retroviral approach using the protocol described by us previously (20Zhao D. Kuhnt-Moore S. Zeng H. Pan A. Wu J.S. Simeonidis S. Moyer M.P. Pothoulakis C. Biochem. J. 2002; 368: 665-672Crossref PubMed Scopus (67) Google Scholar). These cells possess characteristics similar to native colonocytes and have been used by us to study NK-1R signaling (20Zhao D. Kuhnt-Moore S. Zeng H. Pan A. Wu J.S. Simeonidis S. Moyer M.P. Pothoulakis C. Biochem. J. 2002; 368: 665-672Crossref PubMed Scopus (67) Google Scholar). NCM460 and NCM460-NK-1R cells were cultured in M3D medium (INCELL Corporation, San Antonio, CA) containing 10% fetal calf serum (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) solution. U373 MG astrocytoma cells expressing high levels of endogenous NK-1R (27Castagliuolo I. Valenick L. Liu J. Pothoulakis C. J. Biol. Chem. 2000; 275: 26545-26550Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) were obtained from ATCC (Manassas, VA) and cultured in minimum essential medium containing 10% fetal calf serum, 1% penicillin/streptomycin solution, 25 mm sodium bicarbonate, and 10 mm sodium pyruvate (Sigma). Cell Signaling Experiments—NCM460, NCM460-NK-1R, and U373 MG cells seeded in 12-well plates at a density of 0.2 × 106 cells/well were incubated overnight in serum-free M3D (NCM460 and NCM460-NK-1R cells) or minimal essential medium (U373 MG cells) containing 1% penicillin/streptomycin. Cells were then pretreated with the EGFR inhibitor AG1478 (0.4 μm) (Calbiochem, La Jolla, CA), the metalloproteinase inhibitors Batimastat (3 μg/ml) (BB94, British Biotech, Oxford, UK), GM6001 (8 μm) (Calbiochem), the TNFα-converting enzyme (TACE) inhibitor TAPI-1 (8 μm) (Calbiochem), the MAPK kinase (MEK1) inhibitor PD98059 (25 μm) (Calbiochem), or Me2SO for 30 min and then stimulated with SP, vehicle (control), or in some experiments EGF (20 ng/ml) (positive control for EGFR activation) for the time points indicated in Fig. 1. In some experiments, NCM460-NK-1R or U373 MG cells were treated with either 8 μm MMP2/9 inhibitor or 8 μm CL-82198 (Calbiochem), and SP (10–7m)-induced ERK1/2 phosphorylation was determined by Western blot analysis. To determine the role of EGFR and its ligands in SP-induced EGFR and MAPK activation, serum-starved NCM460-NK-1R cells and U373 MG cells were pretreated with either control IgG (20 μg/ml) or antibodies against EGFR (20 μg/ml), EGF (20 μg/ml), HB-EGF (20 μg/ml), TGFα (20 μg/ml), or amphiregulin (20 μg/ml) (R&D Systems Inc., Minnesota, MN) for 1 h, followed by incubation with SP (10–7m) for 15 min. Western blot analyses of cell lysates were used to determine the levels of ERK1/2, phospho-ERK1/2, and/or phospho-EGFR as described below. Western Blot Analysis—SP-treated cells were lysed in 1× lysis buffer (62.5 mm Tris-HCl, 2% SDS, 10% glycerol, 0.01% bromphenol blue, and 1% 2-mercaptoethanol). Equal amounts of cell extracts were fractionated by 10% SDS-PAGE, and proteins were transferred onto nitrocellulose membranes (Bio-Rad) at 400 mA for 2 h at 4 °C. Membranes were blocked in 5% nonfat dried milk in TBST (50 mm Tris, pH 7.5, 0.15 m NaCl, 0.05% Tween 20) and then incubated with antibodies directed against phospho-EGFR (Santa Cruz Biotechnology, Santa Cruz, CA), ERK1/2, and phospho-ERK1/2 (Cell Signaling, Beverly, MA). Membranes were washed with TBST and incubated with horseradish peroxidase-labeled secondary antibodies for 1 h. The peroxidase signal was detected by Supersignal chemiluminescent substrate (Pierce), and the image of the signal was exposed to x-ray film (Fujifilm, Tokyo, Japan). In some experiments, Western blot bands were quantified by densitometry and Scion image analysis software (Scion Corporation, Frederick, MD) with normalization of the phospho-ERK1/2 or phospho-EGFR phosphorylated bands to the corresponding band of control signal (ERK1/2) from the same samples. TGFα ELISA—TGFα protein levels were determined by double ligand ELISA using goat anti-human TGFα (R&D Systems Inc.) according to the manufacturer's instructions (minimal detection limit of 0.5 pg/ml). Results were expressed as means ± S.E. (pg/ml). Cell Proliferation Assay—NCM460-NK-1R cells were seeded on 96-well plates (1 × 106 cells/plate). Serum-starved NCM460-NK-1R cells in 100 μl/well medium were pretreated with PD98059 (25 μm), AG1478 (2 μm), GM6001 (20 μm), TAPI-1 (20 μm), or vehicle (Me2SO) for 30 min and then exposed to SP (10–7m), EGF (20 ng/ml), or TGFα (20 ng/ml). After 24 h, 20 μl of CellTiter AQueous One solution (MTS tetrazolium compound) (Promega) were added into each well and incubated for 1 h. The color changes resulting from the increased cell number were detected by reading absorbance at 490 nm using a 96-well plate reader. Statistical Analysis—ELISA results were analyzed using the Prism professional statistics software program (GraphPad Software Inc., San Diego, CA). Analyses of variance (ANOVA) were used for intergroup comparison. SP Stimulates Phosphorylation of the EGFR and ERK in a Time- and Dose-dependent Manner—SP-NK-1R interactions participate in colonic mucosa healing during colitis presumably by stimulating EGFR-mediated colonic fibroblast proliferation (29Castagliuolo I. Morteau O. Keates A.C. Valenick L. Wang C.C. Zacks J. Lu B. Gerard N.P. Pothoulakis C. Br. J. Pharmacol. 2002; 136: 271-279Crossref PubMed Scopus (54) Google Scholar). To study the mechanism of SP-induced EGFR and ERK1/2 activation in colonic epithelial cells, we used non-transformed human colonic epithelial NCM460 cells, either non-transfected or stably transfected with NK-1R (NCM460-NK-1R). NCM460-NK-1R cells were used previously to study the signaling mechanisms by which substance P induces IL-8 expression (20Zhao D. Kuhnt-Moore S. Zeng H. Pan A. Wu J.S. Simeonidis S. Moyer M.P. Pothoulakis C. Biochem. J. 2002; 368: 665-672Crossref PubMed Scopus (67) Google Scholar). Using a semiquantitative reverse transcription-PCR for human NK-1R mRNA (30Simeonidis S. Castagliuolo I. Pan A. Liu J. Wang C.C. Mykoniatis A. Pasha A. Valenick L. Sougioultzis S. Zhao D. Pothoulakis C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2957-2962Crossref PubMed Scopus (80) Google Scholar), we found that NCM460 cells express NK-1R as shown by the presence of a 324-bp DNA fragment that corresponded to the full-length human NK-1R cDNA (data not shown). We also verified the authenticity of the PCR product by sequencing analysis (data not shown). To examine whether SP causes EGFR transactivation and ERK1/2 phosphorylation, NCM460-NK-1R and NCM460 cells were treated with SP (10–7m) for the indicated time points. Cells were then lysed, and equal amounts of cell proteins were subjected to Western blot analyses using antibodies directed against tyrosine-phosphorylated EGFR or dual phospho-ERK1/2. In non-transfected NCM460 cells, SP did not induce significant EGFR or ERK1/2 phosphorylation (data not shown), indicating low levels of expression of NK-1R in these cells as indicated previously (20Zhao D. Kuhnt-Moore S. Zeng H. Pan A. Wu J.S. Simeonidis S. Moyer M.P. Pothoulakis C. Biochem. J. 2002; 368: 665-672Crossref PubMed Scopus (67) Google Scholar). However, in NCM460-NK-1R cells, SP induced a transient increase in tyrosine phosphorylation of EGFR that was detectable as early as 1 min after SP exposure, peaked at 5 min, and returned to control levels at 45 min (Fig. 1A). SP also induced phosphorylation of ERK1/2 that was evident at 2 min and with maximal intensity at 5–10 min (Fig. 1A). This result is consistent with the notion that the ERK1/2 pathway is downstream of EGFR (27Castagliuolo I. Valenick L. Liu J. Pothoulakis C. J. Biol. Chem. 2000; 275: 26545-26550Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). To confirm these responses in cells expressing high levels of endogenous NK-1R we used U373 MG cells. Our results show that SP induced ERK1/2 phosphorylation as early as 1 min, with maximal intensity at 5–10 min (Fig. 1B). The phospho-ERK signal was predominantly an ERK2 signal because it was correlated to a higher expression level of the ERK2 p42 subunit (Fig. 1B). As expected, exposure of both cell types to EGF (20 ng/ml) induced ERK1/2 and EGFR phosphorylation in a similar fashion as in NK-1R-transfected and non-transfected U373 MG cells (Fig. 1), as well as in non-transfected NCM460 cells (data not shown), indicating intact EGFR and ERK1/2 pathways in all cell lines. Next, NCM460-NK-1R and U373 MG cells were treated with different concentrations of SP for 15 min, and phosphorylation of ERK1/2 was measured. As shown in Fig. 2, SP, at concentrations ranging between 10–6 and 10–9m, stimulated ERK1/2 phosphorylation in a dose-dependent manner, whereas 10–10m SP had no significant effect. EGFR- and Metalloproteinase-dependent Pathways Are Involved in SP-induced MAPK Activation—To confirm the role of EGFR in SP-induced ERK1/2 phosphorylation in human colonocytes, we pretreated NCM460-NK-1R cells and U373 MG cells with either a neutralizing antibody directed against EGFR or a control IgG (20 μg/ml) for 30 min and then exposed the cells to SP (10–7m) for 15 min. Our results showed that pretreatment with anti-EGFR neutralizing antibody, but not control IgG, significantly inhibited SP-induced ERK1/2 phosphorylation (Fig. 3), indicating that SP induces ERK1/2 phosphorylation via the EGFR pathway. It is known that EGF receptor transactivation by several G-protein-coupled receptors involves cleavage of precursors of EGF receptor ligands by metalloproteinases (31Buteau J. Foisy S. Joly E. Prentki M. Diabetes. 2003; 52: 124-132Crossref PubMed Scopus (322) Google Scholar, 32Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1499) Google Scholar, 33Schafer B. Gschwind A. Ullrich A. Oncogene. 2004; 23: 991-999Crossref PubMed Scopus (222) Google Scholar). To determine whether SP-induced EGF receptor phosphorylation requires metalloproteinase activity, NCM460-NK-1R and U373 MG cells were pretreated with the broad metalloproteinase inhibitors Batimastat (BB94, 3 μg/ml) and GM6001 (8 μm), the EGFR inhibitor AG1478 (0.4 μm), or a TACE inhibitor (TAPI-1, 8 μm) for 30 min and then stimulated with SP (10–7m) for various time points. We found that BB94 and GM6001 inhibited SP-induced ERK1/2 phosphorylation (Fig. 4). In addition, the EGFR inhibitor AG1478 (0.4 μm) and the MEK1 inhibitor PD98059 (25 μm) also completely inhibited SP-mediated ERK1/2 phosphorylation (Fig. 4). The TACE inhibitor TAPI-1 also blocked SP-induced ERK1/2 phosphorylation. Preliminary Western blot experiments showed that TACE is highly expressed in NCM460-NK-1R cells (data not shown). SP Stimulates TGFα Release, Which Activates EGFR—Our experiments described above (Figs. 3 and 4) indicate that SP-induced EGFR phosphorylation involves an extracellular ligand for the EGFR. It is well established that EGF (34Modjtahedi H. Komurasaki T. Toyoda H. Dean C. Int. J. Cancer. 1998; 75: 310-316Crossref PubMed Scopus (31) Google Scholar), HB-EGF (35Wallasch C. Crabtree J.E. Bevec D. Robinson P.A. Wagner H. Ullrich A. Biochem. Biophys. Res. Commun. 2002; 295: 695-701Crossref PubMed Scopus (104) Google Scholar), TGFα (36Borrell-Pages M. Rojo F. Albanell J. Baselga J. Arribas J. EMBO J. 2003; 22: 1114-1124Crossref PubMed Scopus (239) Google Scholar), and amphiregulin (37Johnson G.R. Kannan B. Shoyab M. Stromberg K. J. Biol. Chem. 1993; 268: 2924-2931Abstract Full Text PDF PubMed Google Scholar) represent the four ligands that bind and activate the EGFR. To identify the EGFR ligand(s) participating in SP-NK-1R signaling, NCM460-NK-1R cells were exposed to antibodies directed against EGF, HB-EGF, TGFα, and amphiregulin or control antibodies for 1 h prior to SP (10–7m) stimulation. After 15 min, cell lysates were processed for Western blot analysis using an antibody directed against phosphorylated EGFR. Our results showed that SP-induced EGFR and ERK1/2 phosphorylation was significantly inhibited by anti-TGFα neutralizing antibodies, but not by control IgG nor by antibodies directed against the other EGFR ligands, in both NCM460-NK-1R (Fig. 5A) and U373 MG (Fig. 5B) cells. Densitometric analysis of the results from NCM460-NK-1R cells showed that compared with control IgG, the TGFα neutralizing antibody significantly reduced SP-induced phospho-EGFR and phospho-ERK1/2 activation by 63 and 60%, respectively (Fig. 5C). However, neutralizing antibodies against EGF, HB-EGF, and amphiregulin had no inhibitory effect in SP-induced phospho-EGFR and phospho-ERK1/2 activation (Fig. 5, A and B). To confirm and further explore the results with the TGFα neutralizing antibody (Fig. 5C) we examined whether SP directly stimulated TGFα release from its membrane-bound form into the extracellular space. To do this, NCM460-NK-1R (Fig. 6A) and U373 MG (Fig. 6B) cells were treated with SP for the indicated time intervals, and TGFα levels were measured in the conditioned media by ELISA. Compared with non-stimulated cells, SP induced a prompt release of TGFα into the media, which reached the maximal level within 2 min and remained elevated after 10 min (Fig. 6). As expected (38Ramesh G. Levine A.E. Int. J. Cancer. 1995; 62: 492-497Crossref PubMed Scopus (6) Google Scholar), phorbol 12-myristate 13-acetate at 1 μm also stimulated increased TGFα levels in both cell types (∼3-fold, data not shown). Moreover, the MMP inhibitors Batimastat and GM6001 and the TACE inhibitor TAPI-1 prevented SP-induced TGFα release (Fig. 6). TGFα release in GM6001-pretreated U373 MG cells without SP stimulation was below the detection limit of the assay (Fig. 6B). Thus, SP-induced release of TGFα is metalloproteinase-dependent. SP Stimulates Colonic Epithelial Cell Proliferation via MMP-, EGFR-, and MAPK-dependent Pathways—Although SP was known to induce DNA synthesis in U373 MG astrocytoma cells (27Castagliuolo I. Valenick L. Liu J. Pothoulakis C. J. Biol. Chem. 2000; 275: 26545-26550Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), a similar mitogenic SP response has not been investigated in normal colonic epithelial cells. Using a MTS-based cell proliferation assay, SP (10–7m) was found to significantly increase cell proliferation by ∼18% within 24 h of exposure (Fig. 7A, **, p < 0.01), which was almost completely abolished by inhibition of MEK1 with 25 μm PD98059 (Fig. 7A, *, p < 0.05). PD98059 pretreatment without addition of SP did not significantly affect cell viability. Exposure to EGF (20 ng/ml) and TGFα (20 ng/ml), serving as positive controls, also stimulated a similar PD98059-sensitive mitogenic response (Fig. 7A, ***, p < 0.001). Inhibition of EGFR, MMPs, and TACE by AG1478, GM6001, and TAPI-1, respectively, also blocked SP-induced mitogenesis to basal level (Fig. 7B, *, p < 0.05). Previous studies with NK-1R-deficient mice indicated that the NK-1R plays an important protective role in the healing phase of experimental colitis (28Al Sarraj A. Thiel G. Neurosci. Lett. 2002; 332: 111-114Crossref PubMed Scopus (25) Google Scholar) and presented evidence in mouse colonic fibroblasts that activation of EGFR may participate in this response in a NK-1R-dependent manner (28Al Sarraj A. Thiel G. Neurosci. Lett. 2002; 332: 111-114Crossref PubMed Scopus (25) Google Scholar). We have also shown that SP can stimulate proliferation of human astrocytoma cells via an EGFR-dependent MAPK pathway (27Castagliuolo I. Valenick L. Liu J. Pothoulakis C. J. Biol. Chem. 2000; 275: 26545-26550Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). However, whether similar NK-1R-dependent signaling events can take place in human colonocytes, an important cell target for the development of colitis, is not known. Most importantly, the upstream NK-1R-dependent signaling pathways leading to EGFR and MAPK activation have not been studied. We now report the new finding that in non-transformed human colonic epithelial cells, SP induces EGFR and MAPK activation via a metalloproteinase-dependent pathway. Our results suggest that NK-1R engagement by SP leads to activation of metalloproteinases that specifically cleave proTGFα, which is plasma membrane-bound. Active TGFα is then released into the medium that subsequently binds and activates the EGFR receptor, leading to ERK1/2 signaling. Although SP has been demonstrated to induce cell proliferation in U373 MG astrocytoma cells (27Castagliuolo I. Valenick L. Liu J. Pothoulakis C. J. Biol. Chem. 2000; 275: 26545-26550Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) and transformed colonic epithelial Caco2 cells (39Goode T. O'Connor T. Hopkins A. Moriarty D. O'Sullivan G.C. Collins J.K. O'Donoghue D. Baird A.W. O'Connell J. Shanahan F. J. Cell. Physiol. 2003; 197: 30-41Crossref PubMed Scopus (42) Google Scholar), we now present evidence that SP induces cell proliferation in non-transformed colonic epithelial cells and that this proliferation is linked to EGFR and MAPK activation following NK-1R engagement. Our data demonstrating that these responses can be reproduced in another cell line expressing high levels of endogenous NK-1R (U373 MG cells) provide physiological relevance to the results obtained with NK-1R-transfected NCM460 cells. Our results demonstrate that SP-induced EGFR transactivation can be blocked by the matrix metalloproteinase inhibitors Batimastat and GM6001, indicating that membrane metalloproteinases are involved in this SP-dependent signaling event. Several metalloproteinases are available in cells for shedding of EGFR ligands, and different metalloproteinases are capable of shedding more than one EGFR ligand. For example, HB-EGF can be cleaved by a variety of metalloproteinases such as MMP3 (40Suzuki M. Raab G. Moses M.A. Fernandez C.A. Klagsbrun M. J. Biol. Chem. 1997; 272: 31730-31737Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar), MMP7 (41Yu W.H. Woessner Jr., J.F. McNeish J.D. Stamenkovic I. Genes Dev. 2002; 16: 307-323Crossref PubMed Scopus (383) Google Scholar), ADAM9 (42Izumi Y. Hirata M. Hasuwa H. Iwamoto R. Umata T. Miyado K. Tamai Y. Kurisaki T. Sehara-Fujisawa A. Ohno S. Mekada E. EMBO J. 1998; 17: 7260-7272Crossref PubMed Scopus (474) Google Scholar), ADAM10 (43Lemjabbar H. Basbaum C. Nat. Med. 2002; 8: 41-46Crossref PubMed Scopus (299) Google Scholar), ADAM12 (44Asakura M. Kitakaze M. Takashima S. Liao Y. Ishikura F. Yoshinaka T. Ohmoto H. Node K. Yoshino K. Ishiguro H. Asanuma H. Sanada S. Matsumura Y. Takeda H. Beppu S. Tada M. Hori M. Higashiyama S. Nat. Med. 2002; 8: 35-40Crossref PubMed Scopus (640) Google Scholar), as well as the TNFα-converting enzyme (TACE or ADAM17) (45Sunnarborg S.W. Hinkle C.L. Stevenson M. Russell W.E. Raska C.S. Peschon J.J. Castner B.J. Gerhart M.J. Paxton R.J. Black R.A. Lee D.C. J. Biol. Chem. 2002; 277: 12838-12845Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Our results with neutralizing antibodies demonstrate that among several EGFR ligands, only a TGFα antibody inhibited SP-induced EGFR phosphorylation. Together with our finding that SP exposure of NCM460-NK-1R colonocytes leads to a rapid release of mature TGFα into the medium, our results strongly suggest that TGFα may be the only EGF receptor ligand responsible for SP-induced EGFR activation in these cells. The specific metalloproteinase(s) responsible for cleaving proTGFα to mature TGFα in response to SP appears to be TACE, which was originally known to cleave TNFα but then was found to be the only matrix metalloproteinase to efficiently shed proTGFα, leading to the release of its mature form (46Hinkle C.L. Mohan M.J. Lin P. Yeung N. Rasmussen F. Milla M.E. Moss M.L. Biochemistry. 2003; 42: 2127-2136Crossref PubMed Scopus (50) Google Scholar). We report here that the TACE inhibitor, TAPI-1, diminished SP-induced TGFα release and subsequent ERK1/2 phosphorylation in NCM460-NK-1R cells, indicating that SP-induced TGFα release and MAPK activation were mediated by TACE. Although we did not have the opportunity to examine the participation of all metalloproteinases in SP-induced TGFα-related EGFR and MAPK signaling, experiments with the MMP2/9 inhibitor (8 μm), which blocks both MMP2 and MMP9, and with CL-82198 (8 μm), an inhibitor of MMP13, did not affect SP-induced ERK1/2 phosphorylation in either NCM460-NK-1R or U373 MG cells (data not shown). Thus, MMP2, MMP9, and MMP13 are not likely to mediate SP-induced MAPK activation. Results in this study and prior studies indicate that SP-induced MAPK activation requires the presence of a functional EGFR kinase domain (27Castagliuolo I. Valenick L. Liu J. Pothoulakis C. J. Biol. Chem. 2000; 275: 26545-26550Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Moreover, studies with G-protein-coupled receptors that transactivate EGFR indicate that the EGFR signaling pathway is linked to Ras activation (47Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar). Our results indicating that NK-1R engagement leads to transactivation of the EGF receptor in colonocytes are in line with our prior finding demonstrating that NK-1R stimulation leads to activation of Ras (20Zhao D. Kuhnt-Moore S. Zeng H. Pan A. Wu J.S. Simeonidis S. Moyer M.P. Pothoulakis C. Biochem. J. 2002; 368: 665-672Crossref PubMed Scopus (67) Google Scholar). Our finding that EGF receptor transactivation is responsible for SP-induced ERK1/2 activation is also consistent with results indicating that Ras may be involved in SP-induced MAPK stimulation and cell proliferation (48Yang C.M. Hsiao L.D. Chien C.S. Lin C.C. Luo S.F. Wang C.C. Cell. Signalling. 2002; 14: 913-923Crossref PubMed Scopus (32) Google Scholar). Previous results with non-intestinal cells also demonstrated that the proliferative and antiapoptotic effects of SP required the formation of a scaffolding complex comprising internalized receptor, β-arrestin, src, and ERK1/2. The relationship of the SP-mediated EGFR-dependent ERK1/2 activation observed in our study to the NK-1R-related scaffolding complex reported by DeFea et al. (26DeFea K.A. Vaughn Z.D. O'Bryan E.M. Nishijima D. Dery O. Bunnett N.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11086-11091Crossref PubMed Scopus (353) Google Scholar) remains to be elucidated. SP failed to induce MAPK activation or EGFR transactivation in non-transfected NCM460 colonocytes. On the other hand, we were able to detect NK-1R mRNA in these cells. These results are consistent with low levels of expression of NK-1R in several cell types, including colonic epithelial cells. For example, Goode et al. (39Goode T. O'Connor T. Hopkins A. Moriarty D. O'Sullivan G.C. Collins J.K. O'Donoghue D. Baird A.W. O'Connell J. Shanahan F. J. Cell. Physiol. 2003; 197: 30-41Crossref PubMed Scopus (42) Google Scholar) indicated that although several colonic adenocarcinoma cell lines were negative for NK-1R mRNA and protein, NK-1R could be induced upon exposure to a mixture of proinflammatory cytokines. Moreover, SP was also able to induce a mitotic response only in cytokine-exposed and not in untreated colonic epithelial SW620 cells (39Goode T. O'Connor T. Hopkins A. Moriarty D. O'Sullivan G.C. Collins J.K. O'Donoghue D. Baird A.W. O'Connell J. Shanahan F. J. Cell. Physiol. 2003; 197: 30-41Crossref PubMed Scopus (42) Google Scholar). Studies from our laboratory also indicate that the proinflammatory cytokines IL-1β and TNFα can induce expression of the human NK-1R in monocytic THP-1 cells at the protein and mRNA level (30Simeonidis S. Castagliuolo I. Pan A. Liu J. Wang C.C. Mykoniatis A. Pasha A. Valenick L. Sougioultzis S. Zhao D. Pothoulakis C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2957-2962Crossref PubMed Scopus (80) Google Scholar). Along these lines expression of functional NK-1R is only evident in lamina propria macrophages isolated from inflamed, but not from normal, intestine (10Castagliuolo I. Keates A.C. Qiu B. Kelly C.P. Nikulasson S. Leeman S.E. Pothoulakis C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4788-4793Crossref PubMed Scopus (143) Google Scholar). Based on these considerations, our results using NK-1R-transfected NCM460 cells may be pathophysiologically relevant taking also into account that increased levels of NK-1R expression have been noted in several forms of intestinal inflammation in animals and humans, including C. difficile colitis and inflammatory bowel disease (8Goldin E. Karmeli F. Selinger Z. Rachmilewitz D. Dig. Dis. Sci. 1989; 34: 754-757Crossref PubMed Scopus (142) Google Scholar, 9Mantyh C.R. Maggio J.E. Mantyh P.W. Vigna S.R. Pappas T.N. Dig. Dis. Sci. 1996; 41: 614-620Crossref PubMed Scopus (44) Google Scholar, 10Castagliuolo I. Keates A.C. Qiu B. Kelly C.P. Nikulasson S. Leeman S.E. Pothoulakis C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4788-4793Crossref PubMed Scopus (143) Google Scholar, 11Pothoulakis C. Castagliuolo I. Leeman S.E. Wang C.C. Li H. Hoffman B.J. Mezey E. Am. J. Physiol. 1998; 275: G68-G75Crossref PubMed Google Scholar, 49Renzi D. Pellegrini B. Tonelli F. Surrenti C. Calabro A. Am. J. Pathol. 2000; 157: 1511-1522Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 50Weinstock J.V. Blum A. Metwali A. Elliott D. Bunnett N. Arsenescu R. J. Immunol. 2003; 171: 3762-3767Crossref PubMed Scopus (56) Google Scholar). In summary, we present evidence indicating that the mechanism of SP-NK-1R-mediated EGFR transactivation in colonic epithelial cells involves activation of TAPI-1-sensitive matrix metalloproteinases (most likely TACE) able to cleave the membrane-bound precursor proTGFα, leading to shedding of its mature form to the extracellular space. TGFα in turn engages the EGFR, causing its activation and leading to MAPK signaling, which induces colonic epithelial and astrocytoma cell proliferation. This novel pathway may also participate in SP-related cell proliferation in various cell types, as well as in mucosal healing during colonic inflammation. Lastly, these NK-1R signaling events may also be important in the growing evidence for involvement of SP and NK-1R in the development and progression of tumor growth of various etiologies, including pancreatic cancer (51Friess H. Zhu Z. Liard V. Shi X. Shrikhande S.V. Wang L. Lieb K. Korc M. Palma C. Zimmermann A. Reubi J.C. Buchler M.W. Lab. Investig. 2003; 83: 731-742Crossref PubMed Scopus (107) Google Scholar), human gliomas (52Palma C. Nardelli F. Manzini S. Maggi C.A. Br. J. Cancer. 1999; 79: 236-243Crossref PubMed Scopus (67) Google Scholar), and breast cancer (53Singh D. Joshi D.D. Hameed M. Qian J. Gascon P. Maloof P.B. Mosenthal A. Rameshwar P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 388-393Crossref PubMed Scopus (152) Google Scholar), as well as colon dysplasia and carcinoma (54Frucht H. Gazdar A.F. Park J.A. Oie H. Jensen R.T. Cancer Res. 1992; 52: 1114-1122PubMed Google Scholar, 55Sitohy B. El Salhy M. Acta Oncol. 2002; 41: 543-549Crossref PubMed Scopus (20) Google Scholar)." @default.
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• W2000960883 title "Metalloproteinases and Transforming Growth Factor-α Mediate Substance P-induced Mitogen-activated Protein Kinase Activation and Proliferation in Human Colonocytes" @default.
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