Neurotensin stimulates IL-8 expression in human colonic epithelial cells through Rho GTPase-mediated NF-kappa B pathways

Dezheng Zhao1, Sabina Kuhnt-Moore1, Huiyan Zeng2, Jack S. Wu1, Mary P. Moyer3, and Charalabos Pothoulakis1

1 Division of Gastroenterology, 2 Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02468; and 3 INCELL Corporation, San Antonio, Texas 78249


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Neurotensin (NT), a neuropeptide highly expressed in the gastrointestinal tract, participates in the pathophysiology of intestinal inflammation. We recently showed that NT stimulates interleukin-8 (IL-8) expression in NCM460 nontransformed human colonic epithelial cells via both mitogen-activating protein kinase (MAPK)- and NF-kappa B-dependent pathways. However, the molecular mechanism by which NT induces expression of proinflammatory cytokines such as IL-8 has not been investigated. In this study we show that inhibition of endogenous Rho family proteins (RhoA, Rac1, and Cdc42) by their respective dominant negative mutants inhibits NT-induced IL-8 protein production and promoter activity. Western blot experiments demonstrated that NT strongly activated RhoA, Rac1, and Cdc42. Overexpression of the dominant negative mutants of RhoA, Rac1, and Cdc42 significantly inhibited NT-induced NF-kappa B-dependent reporter gene expression and NF-kappa B DNA binding activity. NT also stimulated p38 MAPK phosphorylation, and overexpression of dominant negative mutants of RhoA, Rac1, and Cdc42 did not significantly alter p38 and ERK1/2 phosphorylation in response to NT. Together, our findings indicate that NT-stimulated IL-8 expression is mediated via a Rho-dependent NF-kappa B-mediated pathway.

neuropeptide; inflammation; signal transduction; gene regulation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NEUROTENSIN (NT), a 13-amino acid neuropeptide, is localized in the central nervous system and in peripheral tissues, most notably the gastrointestinal tract (5, 6). NT increases motility and secretion of small bowel, colon, and stomach (35, 41, 43) and stimulates proliferation of intestinal epithelial cells in vivo and in vitro (16, 28, 44-46). NT mediates several intestinal effects by binding to a high-affinity cell surface G protein-coupled receptor (GPCR) with seven membrane-spanning domains, named NT receptor 1 (NTR1). NT-NTR1 interaction participates in stress-mediated colonic responses such as mucin and prostaglandin E2 secretion and mast cell degranulation (8). NT via NTR1 also plays a proinflammatory role in acute colonic inflammation. Thus NT and NTR1 expression are elevated in the colonic mucosa of rats exposed to Clostridium difficile toxin A (10), and administration of the NTR1 antagonist SR-48692 inhibits colonic secretion and inflammation in response to toxin A (10). Along these lines, we recently reported the presence of NTR1 in nontransformed human colonic epithelial NCM460 cells (47). We also found that in NCM460 cells overexpressing NTR1, NT stimulates release of the potent neutrophil chemoattractant interleukin-8 (IL-8) (47), indicating that NT may mediate colonic inflammation by acting directly on human colonocytes.

NT stimulates the formation of inositol 1,4,5-trisphosphate and increases intracellular calcium (1, 3). NT activates ERK, a member of the mitogen-activating protein kinase (MAPK) family in colonic adenocarcinoma HT29 cells (33), colonic epithelial NCM460 cells (47), and pancreatic MIA PaCa-2 cells (13). In NCM460 cells transfected with NTR1 (NCM460-NTR1 cells), NT-stimulated ERK activation is Ras dependent, and a Ras-mediated signaling pathway is involved in NT-induced IL-8 expression (47). Experiments with NCM460-NTR1 cells indicate that the mechanism of NT-mediated stimulation of IL-8 gene expression involves a nuclear factor-kappa B (NF-kappa B)-dependent pathway (47). This is consistent with a prior study (9) demonstrating that NT can directly stimulate DNA binding activity of NF-kappa B in isolated human intestinal microvascular endothelial cells. NF-kappa B is a critical regulator for the expression of genes involved in inflammation of the gastrointestinal tract (2, 14, 37). It consists of homo- and heterodimers of Rel family proteins, such as p65 and p50, sequestered in an inactive form in the cytoplasm by Ikappa B inhibitory proteins. It is well established that the NF-kappa B pathway can be activated by the members of the Rho family proteins RhoA, Rac1, and Cdc42 (32). RhoA is important for GPCR signaling (36, 38), and involvement of Rho family proteins in inflammatory responses such as IL-1beta and IL-8 production has been previously demonstrated (21, 27, 31, 48). Evidence also indicates that the effect of RhoA in expression of inflammatory genes may involve NF-kappa B activation (27, 29, 31, 32). However, whether the Rho family of proteins participates in NT signaling is not known.

In this study we examined the hypothesis that Rho proteins participate in NT-NTR1 signaling as it relates to IL-8 secretion. Using NCM460 colonocytes, we have shown in this current study that inhibition of endogenous Rho family proteins (RhoA, Rac1, and Cdc42) by their respective dominant negative mutants significantly decreases NT-induced IL-8 promoter activity and IL-8 secretion. Our results indicate that NT strongly activates RhoA, Rac1, and Cdc42 and that overexpression of the dominant negative mutants of RhoA, Rac1, and Cdc42 significantly inhibits NT-induced NF-kappa B DNA binding activity and NF-kappa B-dependent reporter gene expression but not p38 and ERK1/2 MAPK phosphorylation. Together, these findings indicate that NT-stimulated IL-8 expression is mediated via a Rho-dependent NF-kappa B pathway.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Nontransformed human colonic epithelial cells NCM460 were obtained from INCELL (San Antonio, TX). NCM460-NTR1 cells were generated as previously described (47). M3D serum-free medium was purchased from INCELL, and Effectene transfection reagents were from Qiagen (Valencia, CA). Retroviral vectors expressing dominant negative mutants of RhoA, Rac1, and Cdc42 (RhoA-19N, Rac1-17N, and Cdc42-17N, respectively) have been previously described (48). The pMD-VSVG and pMD-gag-pol plasmids and the 293T cell line were gifts from Dr. Richard C. Mulligan (Children's Hospital, Harvard Medical School, Cambridge, MA). Bacteria expressing glutathione S-transferase (GST)-rhotekin Rho binding domain fusion protein (GST-TRBD) and GST-p21-activated kinase (PAK) binding domain fusion protein (GST-PBD) were kindly provided by Drs. Martin A. Schwartz (34) and Richard A. Cerione (Cornell University, Ithaca, NY), respectively. The monoclonal antibody directed against RhoA was from Santa Cruz Biotechnology (Santa Cruz, CA), and the monoclonal antibodies against Rac1 and Cdc42 were from Upstate Biotechnology (Lake Placid, NY). The enhanced chemiluminescent (ECL) detection reagents were from Pierce Biotechnology (Rockford, IL). NF-kappa B and serum responsive element (SRE)-driven luciferase reporter constructs were from BD Sciences Clontech (Palo Alto, CA). The IL-8 promoter construct was kindly provided by Dr. Andrew C. Keates (Beth Israel Deaconess Medical Center, Boston, MA). The NF-kappa B consensus oligonucleotide probe was purchased from Promega (Madison, WI), and [gamma -32P]ATP was from Perkin-Elmer (Boston, MA). T4 DNA kinase was from New England Biolabs (Beverly, MA), and the monoclonal antibodies against phospho-ERK1/2 and phospho-p38 were from Cell Signaling (Beverly, MA). Rabbit polyclonal antibodies against ERK2 and p38 were from Santa Cruz Biotechnology.

Preparation of retroviruses and cell infection. Retroviruses were prepared using a previously described procedure (47). Briefly, 293T cells were transiently transfected with the indicated retroviral expression vector, pMD-VSVG, and pMD-gag-pol at a ratio of 4:1:3 using Effectene transfection reagents. Sixteen hours after transfection, cells were incubated in fresh growth medium for 32 h. The virus-containing media were then filtered through a 0.45-µm disk filter and either used immediately or kept at -80°C. To infect NCM460-NTR1 monolayers, we used a method previously described by us (47). Briefly, 4 × 104 cells/cm2 were incubated (16-24 h) with 2 volumes of filtered virus-containing supernatants and 1 volume of fresh growth medium in the presence of 10 µg/ml Polybrene (Sigma). Cells were then washed twice with phosphate-buffered saline (PBS) and incubated in the presence of serum-free medium for 24 h before NT treatment.

IL-8 measurements. IL-8 protein levels were determined in cell-conditioned media by enzyme-linked immunosorbent assay using goat anti-human IL-8 (R&D Systems, Minneapolis, MI) as described previously (25). Results are expressed as means ± SE (in ng/ml).

Determination of RhoA, Rac1, and Cdc42 activation. The activity of RhoA, Rac1, and Cdc42 was determined as recently described by us (48). Briefly, equal amounts of cell lysates were prepared and incubated with equal amounts of freshly prepared Sepharose beads containing GST-TRBD (for RhoA-GTP) or GST-PBD (for Rac1-GTP and Cdc42-GTP) for 45 min on ice, and the beads were washed with AP buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 1% Triton X-100, 10 mM MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF). The beads were then boiled with 2× SDS sample buffer (100 mM Tris-Cl, pH 6.8, 4% SDS), and equal volumes of the samples were subjected to Western blot analysis by using a monoclonal antibody against RhoA, Rac1, and Cdc42, respectively. To control equal protein loading, we also subjected the same amounts of cell lysates to Western blot analysis using monoclonal antibodies against RhoA, Rac1, and Cdc42.

Luciferase reporter assay. Cells were seeded in 12-well plates (0.2 × 106 cells/well) overnight and transiently transfected with IL-8 promoter luciferase constructs, an SRE or NF-kappa B luciferase construct, a control luciferase plasmid (pRL-TK; Promega), or other DNA constructs as indicated using the Effectene transfection reagent (Qiagen). Transfected cells were serum-starved for 24 h, followed by exposure to NT for 4 h. Firefly and Renilla luciferase activities in cell extracts were measured using the Dual-Luciferase Reporter assay system (Promega). The relative luciferase activity was then calculated by normalizing IL-8 promoter-driven firefly luciferase activity to control Renilla luciferase activity.

Electrophoretic mobility shift assays. Nuclear NF-kappa B DNA binding activity was determined as previously described by us (47). Briefly, nuclear extracts were prepared, and equal amounts of protein were incubated with 32P-labeled NF-kappa B consensus oligonucleotide probe in the presence of poly(dI-dC). Binding of specific nuclear protein to the probe was determined by fractionating the nuclear proteins through a nondenaturing 6% polyacrylamide gel. The gel was dried and then exposed to X-ray autoradiography film.

ERK and p38 phosphorylation assay. Cells were washed twice with ice-cold PBS and then incubated in RIPA buffer containing a protease inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany) for 10 min. Cell lysates were centrifuged at 1,000 g for 10 min. Equal amounts of cell extracts were separated by SDS-PAGE (10%), and proteins were transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA) at 100 volts for 1 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 phosphospecific antibodies (0.2 µg/ml) to ERK1/2 or p38. Horseradish peroxidase-labeled antibodies were detected by SuperSignal chemiluminescent substrate (Pierce). To ensure equal protein loading, the blots were stripped and reprobed with polyclonal antibodies against ERK2 or p38, respectively.

Statistical analyses. Results are expressed as means ± SE. Data were analyzed using the SigmaStat professional statistics software program (Jandel Scientific Software, San Rafael, CA). ANOVA with protected t-tests were used for intergroup comparison.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of Rho family GTPases is required for NT-induced IL-8 gene expression. Our laboratory has demonstrated that NT-NTR1 receptor interaction plays an important role in intestinal inflammation (10) and that NT-stimulated IL-8 expression in NCM460-NTR1 cells involves ERK1/2 and NF-kappa B activation (47). Because the members of the Rho family GTPases RhoA, Rac1, and Cdc42 were shown to mediate NF-kappa B activation and NF-kappa B-dependent gene expression (32), we sought to determine whether the Rho family of GTPases is involved in NT-induced IL-8 gene expression. For this purpose, we used a retroviral expression system developed by Dr. Richard Mulligan that could produce viruses at a titer of 109-1010 cfu/ml by transient transfection of human kidney carcinoma 293T cells. This titer of retroviruses was shown to infect human colonic epithelial cells NCM460 at an ~100% efficiency (47). To examine the effect of overexpression of dominant negative forms of Rho GTPases on NT-induced IL-8 protein production, we infected NCM460-NTR1 cells with equal amounts of LacZ-, RhoA-19N-, Rac1-17N-, or Cdc42-17N-expressing retroviruses. The infected cells were incubated with serum-free M3D media and treated with NT (10-7 M), and IL-8 secretion was measured in the conditioned media. The data show that, as expected, NT stimulated IL-8 release in control, LacZ-infected cells. However, overexpression of RhoA-19N, Rac1-17N, or Cdc42-17N significantly inhibited NT-induced IL-8 secretion (Fig. 1A). Next, we examined whether overexpression of RhoA-19N, Rac1-17N, or Cdc42-17N affects NT-induced IL-8 promoter activity. Cells were cotransfected with equal amounts of LacZ-, RhoA-19N-, Rac1-17N-, or Cdc42-17N-expressing constructs together with IL-8 promoter reporter construct plus an internal control DNA. The transfected cells were rendered quiescent and then treated with NT (10-7 M). Cell extracts were used to determine luciferase reporter activity. The results show that overexpression of RhoA-19N, Rac1-17N, or Cdc42-17N significantly inhibited NT-induced IL-8 promoter activity (Fig. 1B), indicating that RhoA, Rac1, and Cdc42 are involved in NT-induced IL-8 gene expression.


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Fig. 1.   Neurotensin (NT)-induced IL-8 expression requires activation of Rho family GTPases. A: NCM460-NTR1 cells were infected with equal amounts of retroviruses expressing LacZ, RhoA-19N, Rac1-17N, or Cdc42-17N. The infected cells were serum-starved and treated with NT (10-7 M) for 4 h. IL-8 secretion in the conditioned media was determined by ELISA. Data are expressed as means ± SE (n = 4). *P < 0.005, RhoA-19N-, Rac1-17N-, or Cdc42-17N-expressing cells with NT stimulation vs. LacZ-expressing cells. B: NCM460-NTR1 cells were transiently transfected with IL-8 promoter reporter construct together with LacZ-, RhoA-19N-, Rac1-17N-, or Cdc42-17N-expressing plasmid. The transfected cells were serum-starved and treated with NT (10-7 M) for 4 h. Cell extracts were prepared to measure IL-8 promoter activity. Values are expressed as means ± SE (relative luciferase activity; n = 3). The results are representative of at least 3 separate experiments.

NT rapidly activates RhoA. On the basis of these results (Fig. 1), we speculated that NT might directly stimulate the activity of RhoA, Rac1, and Cdc42 measured as levels of their GTP-bound forms. We first determined whether NT activates RhoA in NCM460-NTR1 cells, measured by using the pull-down assay described in MATERIALS AND METHODS. Cells were treated with NT (10-7 M) for the indicated time intervals (Fig. 2), and cell extracts were prepared and incubated with RhoA-binding domain-containing GST-fusion protein conjugated to Sepharose beads. The bound protein was eluted and subjected to Western blot analysis by using a monoclonal antibody against RhoA. Equal amounts of cell extracts were also subjected to Western blot analysis by using a monoclonal antibody against RhoA to ensure equal protein loading. We found that NT strongly stimulated RhoA-GTP loading as early as 20 s and that this stimulation was evident 10 min after NT treatment (Fig. 2).


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Fig. 2.   NT stimulates formation of GTP-bound RhoA-GTP. Quiescent NCM460-NTR1 cells were treated with NT (10-7 M) for the indicated times. Equal amounts of cell extracts were used to determine the levels of GTP-bound RhoA as described in MATERIALS AND METHODS. The results are representative of 3 independent experiments.

NT rapidly activates Rac1 and Cdc42. Because Rac1 and Cdc42 GTPases also play a role in NT-induced IL-8 expression (Fig. 1), we determined whether NT activates these proteins, using the pull-down assay described in MATERIALS AND METHODS. Briefly, cells were treated with NT (10-7 M) for the indicated times, and equal amounts of cell extracts were incubated with Rac1/Cdc42-binding domain-containing GST-fusion protein conjugated to Sepharose beads. The bound protein was eluted and subjected to Western blot analysis by using a monoclonal antibody against Rac1 and Cdc42. Our results showed that NT rapidly stimulated the formation of Rac1-GTP (Fig. 3A) and Cdc42-GTP (Fig. 3B) as early as 1 min after exposure.


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Fig. 3.   NT stimulates formation of GTP-bound Rac1 and Cdc42. Quiescent NCM460-NTR1 cells were treated with NT (10-7 M) for the indicated times. Cell extracts were used to determine the levels of GTP-bound Rac1 (A) and Cdc42 (B) as described in MATERIALS AND METHODS. The results are representative of 3 independent experiments.

NT stimulates Rho-dependent SRE-driven gene expression. Stimulation of SRE-mediated gene expression is a commonly used parameter of Rho-dependent signaling, and Rho GTPases exert this effect primarily through the transcription factor SRF (serum response factor) (20). Here we determined whether NT stimulates SRE-driven luciferase activity and examined the involvement of RhoA, Rac1, and Cdc42 in this response. NCM460-NTR1 cells were transiently transfected with SRE-luciferase construct together with equal amounts of LacZ-, RhoA-19N-, Rac1-17N-, or Cdc42-17N-expressing plasmids. The transfected cells were then treated with NT (10-7 M), and luciferase reporter activity was measured. We found that NT increased SRE-driven luciferase activity ~15-fold and that cotransfection with RhoA-19N, Rac1-17N, or Cdc42-17N significantly reduced NT-induced luciferase activity (Fig. 4). These results provide further evidence that NT signaling involves activation of the Rho family of proteins.


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Fig. 4.   NT-induced serum responsive element (SRE)-luciferase activity requires activation of RhoA, Rac1, and Cdc42. NCM460-NTR1 cells were transiently transfected with a SRE-luciferase reporter construct together with LacZ (control)-, RhoA-19N-, Rac1-17N-, or Cdc42-17N-expressing plasmid. The transfected cells were serum-starved and treated with NT (10-7 M) for 4 h. Cell extracts were prepared to measure luciferase activity. Values are expressed as means ± SE (relative luciferase activity; n = 3).

NT-induced NF-kappa B-dependent gene expression involves RhoA, Rac1, and Cdc42. Because NT-stimulated IL-8 expression in NCM460-NTR1 cells requires NF-kappa B activation (47), we examined whether the effect of the dominant negative mutants for Rho-19N, Rac1-17N, or Cdc42-17N on NT-induced IL-8 expression is mediated through the NF-kappa B pathway. To do this, we transfected cells with LacZ-, Rho-19N-, Rac1-17N-, or Cdc42-17N-expressing constructs together with a NF-kappa B promoter reporter construct and an internal control plasmid. The transfected cells were serum-starved and treated with NT (10-7 M), and cell extracts were used to measure NF-kappa B promoter activity. The results show that overexpression of Rho-19N, Rac1-17N, or Cdc42-17N significantly inhibited NT-induced NF-kappa B-dependent reporter gene expression (Fig. 5).


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Fig. 5.   NT-induced NF-kappa B-dependent gene expression requires activation of RhoA, Rac1, and Cdc42. NCM460-NTR1 cells were transiently transfected with a NF-kappa B promoter-driven luciferase reporter construct together with LacZ (control)-, RhoA-19N-, Rac1-17N-, or Cdc42-17N-expressing plasmid. The transfected cells were serum-starved and treated with NT (10-7 M) for 4 h. Cell extracts were prepared to measure NF-kappa B promoter activity. Values are expressed as means ± SE (relative luciferase activity; n = 3). *P < 0.01, RhoA-19N-, Rac1-17N-, or Cdc42-17N-expressing cells with NT stimulation vs. control LacZ-expressing cells.

NT-induced NF-kappa B DNA binding requires RhoA, Rac1, and Cdc42. Previous studies indicated that constitutive active mutants of RhoA, Rac1, and Cdc42 are able to stimulate DNA binding activity of NF-kappa B (32). Here we examined whether dominant negative mutants of RhoA, Rac1, and Cdc42 inhibit NT-induced NF-kappa B-dependent reporter gene expression (Fig. 5) by reducing NF-kappa B DNA binding activity. NCM460-NTR1 cells were infected with equal amounts of LacZ-, RhoA-19N-, Rac1-17N-, or Cdc42-17N-expressing retroviruses. The infected cells were incubated with serum-free media and then treated with NT (10-7 M) for 30 min. Nuclear extracts were prepared, and NF-kappa B DNA binding activity was determined as previously described (47). The data indicate that overexpression of RhoA-19N, Rac1-17N, or Cdc42-17N significantly reduced NT-induced DNA binding activity of NF-kappa B (Fig. 6). These results suggest that the three members of Rho family GTPases mediate NT-induced IL-8 expression and NF-kappa B-dependent reporter gene transcription, at least in part, by activating NF-kappa B DNA binding.


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Fig. 6.   Overexpression of RhoA-19N, Rac1-17N or Cdc42 attenuates NF-kappa B binding activity in response to NT. NCM460-NTR1 cells were incubated with retroviruses expressing LacZ (control), RhoA-19N, Rac1-17N, or Cdc42-17N for 16 h. The infected cells were serum-starved and treated with NT (10-7 M) or PBS for 30 min. Nuclear extracts were prepared, and NF-kappa B DNA binding activity was determined by electrophoretic mobility shift assay as described previously (47). In the last two lanes, an excess amount of cold NF-kappa B oligonucleotide was added to the reaction mixtures to demonstrate the specificity of NF-kappa B binding.

Rho GTPases are not involved in NT-induced activation of the MAPKs ERK1/2 and p38. It is well established that NT activates the MAPK ERK1/2 in various cell types, including NCM460 cells (13, 33, 47). Rho GTPases mediate MAPK activation (12, 39, 40), whereas ERK1/2 and p38 MAPK regulate NF-kappa B activation in response to specific stimuli (7, 24, 30, 42). Here we determined whether Rho GTPases are involved in activation of the MAPKs ERK1/2 and p38 in response to NT. We first examined whether NT activates p38 in parental NCM460 cells, in which NT is known to activate ERK1/2 (47). Our data indicate that NT rapidly but transiently stimulates p38 phosphorylation in NCM460 cells (Fig. 7A). In contrast, NT-induced p38 phosphorylation in NCM460-NTR1 cells was also rapid, but more persistent (Fig. 7B). The results suggest that strong and prolonged p38 activation in response to NT requires a high level of cell surface NTR1 expression. To examine whether Rho GTPases are involved in NT-induced ERK and p38 activation, we infected NCM460-NTR1 cells with viruses expressing LacZ, RhoA-19N, Rac1-17N, or Cdc42-17N for 16 h. The infected cells were incubated with serum-free media and then treated with NT for 5 min. Cell extracts were subjected to Western blot analysis, using monoclonal antibodies against phospho-ERK1/2 and phospho-p38. The results indicate that dominant negative mutants of RhoA, Rac1, and Cdc42 did not affect NT-induced ERK1/2 and p38 phosphorylation (Fig. 8).


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Fig. 7.   NT stimulates p38 MAPK phosphorylation. A: quiescent NCM460 cells were treated with NT (100 nM) or TNF-alpha (20 ng/ml) for the indicated times. B: quiescent NCM460-NTR1 cells were treated with NT (50 nM) for the indicated times. C: NCM460-NTR1 cells were treated with different concentrations of NT for 15 min. Equal amounts of cell extracts were subjected to Western blot analysis, using a monoclonal antibody against phospho-p38.



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Fig. 8.   Overexpression of RhoA-19N, Rac1-17N, or Cdc42-17N has little effect on NT-induced activation of ERK1/2 and p38. NCM460-NTR1 cells were infected with equal amounts of retroviruses expressing LacZ, RhoA-19N, Rac1-17N, or Cdc42-17N for 16 h as described in MATERIALS AND METHODS. The infected cells were washed with PBS and then treated with NT (10-7 M) for 5 min. Phosphorylation of ERK1/2 and p38 was determined as described in MATERIALS AND METHODS. The results are representative of 3 independent experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our laboratory has previously reported (10) that expression of NT and NTR1 is increased in the intestinal mucosa during inflammation and have presented functional evidence for a major requirement for NT receptors in the pathogenesis of acute inflammatory diarrhea. Studies from our laboratory also indicate that the proinflammatory effects of NT at the cellular level involve activation of the NF-kappa B/Ikappa B system. For example, nontransformed human colonic epithelial NCM460 cells express functional NTR1, and increased expression of this receptor leads to NF-kappa B activation and IL-8 gene expression following NT exposure (47). Primary human intestinal endothelial cells also express a functional NTR1, and incubation of these cells with NT results in increased NF-kappa B DNA binding activity (9). Using NCM460 cells overexpressing NTR1, we report here that NT rapidly activates Rho family GTPases RhoA, Rac1, and Cdc42 and that activation of these molecules is required for NT-induced IL-8 gene expression. Consistent with these results, our study also demonstrates that activation of RhoA, Rac1, and Cdc42 mediates NT-induced NF-kappa B as well as SRE-dependent gene expression.

The Rho family of proteins mediate many cellular responses stimulated by a variety of extracellular factors including growth factors, cytokines, and GPCR ligands (26, 36, 38). In addition to their roles in regulating actin cytoskeleton, focal adhesion complex formation, and cell mobility (15, 18, 19), Rho GTPases are also involved in NF-kappa B activation and expression of inflammation-related genes in response to many stimuli. For example, RhoA activation is required for IL-8 expression stimulated by LPS (21) and for IL-1beta expression induced by the GPCR ligands bradykinin (31) and the neutrophil chemotactic factor fMLP (22). Rho GTPases also mediate IL-8 gene expression in response to the peptide substance P in NCM460 colonocytes transfected with the substance P, neurokinin-1 receptor (48). Rho GTPases regulate proinflammatory cytokine gene expression by activating the NF-kappa B pathway (21, 22, 31, 48). In line with these findings, results presented in this current study indicate that NT-stimulated IL-8 gene expression involves RhoA-, Rac1-, and Cdc42-mediated NF-kappa B activation.

The molecular mechanism(s) whereby Rho family GTPases activate NF-kappa B has not been completely elucidated. It is well established that nuclear translocation and DNA binding of NF-kappa B heterodimers is primarily regulated through phosphorylation and degradation of the inhibitory proteins Ikappa B by Ikappa B kinases (IKKs) or other, not yet identified kinases. Thus, Cammarano and Minden (4) reported that Rac1 stimulates NF-kappa B via NF-kappa B-inducing kinase (NIK)-mediated IKKbeta activation, whereas, in contrast, RhoA and Cdc42 activate NF-kappa B via IKK-independent pathways. Consistent with these results, Naumann and colleagues (11, 17) reported that p21-activating kinase 1 (PAK1), the immediate downstream effector of Rac1 and Cdc42, binds and activates NIK, leading to degradation of Ikappa Balpha and increased NF-kappa B DNA binding in Helicobacter pylori-infected gastric epithelial cells. On the other hand, Rac1 regulates IL-1-induced NF-kappa B-dependent reporter gene expression without causing Ikappa Balpha degradation and nuclear translocation of NF-kappa B (23). Instead, it was suggested that Rac1 activates both p38 and p42/p44, leading to enhanced transactivation of gene expression by the p65 subunit of NF-kappa B following IL-1 exposure (23). These seemingly conflicting results suggest that the ability of Rho GTPases to activate NF-kappa B by inducing IKK activity might depend on a particular stimulus as well as a particular cell type. Our results indicate that both NT-induced NF-kappa B DNA binding activity and kappa B site-mediated reporter gene expression involve Rho GTPases. In addition, we present evidence that NT activates another member of the MAP kinase family, namely, the MAPK p38, and that Rho GTPases are not involved in NT-induced activation of ERK1/2 and p38. These results suggest that MAPKs are not involved in NT-induced, Rho GTPase-mediated NF-kappa B signaling, a notion consistent with our recent studies indicating that inhibition of ERK activation has no effect on NT-induced NF-kappa B activation (47).

In summary, our current finding that the neuropeptide NT and its GPCR NTR1 mediate gene expression of the proinflammatory cytokine IL-8 by a RhoA-dependent pathway further supports the notion that RhoA GTPases play an important role in GPCR signaling. In addition to RhoA, our present data indicate that another two members of the Rho family proteins, Rac1 and Cdc42, are also important for proinflammatory responses mediated by the GPCR NTR1. Together, the Rho family of proteins not only participates in cytoskeletal reorganization involved in cell proliferation and migration in response to many extracellular factors but also plays an important role in NF-kappa B-dependent proinflammatory gene expression. We speculate that NT-induced, Rho family-dependent, proinflammatory signaling may represent an important pathway participating in the pathogenesis of intestinal inflammation.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-47343, DK-60729, and T32-DK-07760.


    FOOTNOTES

Address for reprint requests and other correspondence: C. Pothoulakis, Beth Israel Deaconess Medical Center, Division of Gastroenterology, Dana 501, 330 Brookline Ave., Boston, MA 02215 (E-mail: cpothoul{at}caregroup.harvard.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 12, 2003;10.1152/ajpcell.00328.2002

Received 12 July 2002; accepted in final form 22 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Amar, S, Kitabgi P, and Vincent JP. Activation of phosphatidylinositol turnover by neurotensin receptors in the human colonic adenocarcinoma cell line HT29. FEBS Lett 201: 31-36, 1986[ISI][Medline].

2.   Ardite, E, Panes J, Miranda M, Salas A, Elizalde JI, Sans M, Arce Y, Bordas JM, Fernandez-Checa JC, and Pique JM. Effects of steroid treatment on activation of nuclear factor kappaB in patients with inflammatory bowel disease. Br J Pharmacol 124: 431-433, 1998[Abstract].

3.   Bozou, JC, Rochet N, Magnaldo I, Vincent JP, and Kitabgi P. Neurotensin stimulates inositol trisphosphate-mediated calcium mobilization but not protein kinase C activation in HT29 cells. Involvement of a G-protein. Biochem J 264: 871-878, 1989[ISI][Medline].

4.   Cammarano, MS, and Minden A. Dbl and the Rho GTPases activate NFkappa B by Ikappa B kinase (IKK)-dependent and IKK-independent pathways. J Biol Chem 276: 25876-25882, 2001[Abstract/Free Full Text].

5.   Carraway, R, and Leeman SE. Characterization of radioimmunoassayable neurotensin in the rat. Its differential distribution in the central nervous system, small intestine, and stomach. J Biol Chem 251: 7045-7052, 1976[Abstract].

6.   Carraway, R, and Leeman SE. The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami. J Biol Chem 248: 6854-6861, 1973[Abstract/Free Full Text].

7.   Carter, AB, Knudtson KL, Monick MM, and Hunninghake GW. The p38 mitogen-activated protein kinase is required for NF-kappa B-dependent gene expression. The role of TATA-binding protein (TBP). J Biol Chem 274: 30858-30863, 1999[Abstract/Free Full Text].

8.   Castagliuolo, I, Leeman SE, Bartolak-Suki E, Nikulasson S, Qiu B, Carraway RE, and Pothoulakis C. A neurotensin antagonist, SR 48692, inhibits colonic responses to immobilization stress in rats. Proc Natl Acad Sci USA 93: 12611-12615, 1996[Abstract/Free Full Text].

9.   Castagliuolo, I, Valenick L, Pasha A, Sturniolo GC, D'Inca RD, Fiocchi C, and Pothoulakis C. Neurotensin (NT) mediates inflammatory responses in human intestinal microvascular endothelial cells (HIMEC) (Abstract). Gastroenterology 116: A799, 1999.

10.   Castagliuolo, I, Wang CC, Valenick L, Pasha A, Nikulasson S, Carraway RE, and Pothoulakis C. Neurotensin is a proinflammatory neuropeptide in colonic inflammation. J Clin Invest 103: 843-849, 1999[Abstract/Free Full Text].

11.   Churin, Y, Kardalinou E, Meyer TF, and Naumann M. Pathogenicity island-dependent activation of Rho GTPases Rac1 and Cdc42 in Helicobacter pylori infection. Mol Microbiol 40: 815-823, 2001[ISI][Medline].

12.   Coso, OA, Chiariello M, Yu JC, Teramoto H, Crespo P, Xu N, Miki T, and Gutkind JS. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell 81: 1137-1146, 1995[ISI][Medline].

13.   Ehlers, RA, Zhang Y, Hellmich MR, and Evers BM. Neurotensin-mediated activation of MAPK pathways and AP-1 binding in the human pancreatic cancer cell line, MIA PaCa-2. Biochem Biophys Res Commun 269: 704-708, 2000[ISI][Medline].

14.   Ellis, RD, Goodlad JR, Limb GA, Powell JJ, Thompson RP, and Punchard NA. Activation of nuclear factor kappa B in Crohn's disease. Inflamm Res 47: 440-445, 1998[ISI][Medline].

15.   Etienne-Manneville, S, and Hall A. Rho GTPases in cell biology. Nature 420: 629-635, 2002[ISI][Medline].

16.   Evers, BM, Izukura M, Chung DH, Parekh D, Yoshinaga K, Greeley GH, Uchida T, Townsend CM, and Thompson JC. Neurotensin stimulates growth of colonic mucosa in young and aged rats. Gastroenterology 103: 86-91, 1992[ISI][Medline].

17.   Foryst-Ludwig, A, and Naumann M. p21-activated kinase 1 activates the nuclear factor kappa B (NF-kappa B)-inducing kinase-Ikappa B kinases NF-kappa B pathway and proinflammatory cytokines in Helicobacter pylori infection. J Biol Chem 275: 39779-39785, 2000[Abstract/Free Full Text].

18.   Hall, A. Rho GTPases and the actin cytoskeleton. Science 279: 509-514, 1998[Abstract/Free Full Text].

19.   Hall, A, and Nobes CD. Rho GTPases: molecular switches that control the organization and dynamics of the actin cytoskeleton. Philos Trans R Soc Lond B Biol Sci 355: 965-970, 2000[ISI][Medline].

20.   Hill, CS, Wynne J, and Treisman R. The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell 81: 1159-11570, 1995[ISI][Medline].

21.   Hippenstiel, S, Soeth S, Kellas B, Fuhrmann O, Seybold J, Krull M, Eichel-Streiber C, Goebeler M, Ludwig S, and Suttorp N. Rho proteins and the p38-MAPK pathway are important mediators for LPS-induced interleukin-8 expression in human endothelial cells. Blood 95: 3044-3051, 2000[Abstract/Free Full Text].

22.   Huang, S, Chen LY, Zuraw BL, Ye RD, and Pan ZK. Chemoattractant-stimulated NF-kappa B activation is dependent on the low molecular weight GTPase RhoA. J Biol Chem 276: 40977-40981, 2001[Abstract/Free Full Text].

23.   Jefferies, CA, and O'Neill LA. Rac1 regulates interleukin 1-induced nuclear factor kappa B activation in an inhibitory protein kappa Balpha -independent manner by enhancing the ability of the p65 subunit to transactivate gene expression. J Biol Chem 275: 3114-3120, 2000[Abstract/Free Full Text].

24.   Li, JD, Feng W, Gallup M, Kim JH, Gum J, Kim Y, and Basbaum C. Activation of NF-kappa B via a Src-dependent Ras-MAPK-pp90rsk pathway is required for Pseudomonas aeruginosa-induced mucin overproduction in epithelial cells. Proc Natl Acad Sci USA 95: 5718-5723, 1998[Abstract/Free Full Text].

25.   Linevsky, JK, Pothoulakis C, Keates S, Warny M, Keates AC, Lamont JT, and Kelly CP. IL-8 release and neutrophil activation by Clostridium difficile toxin-exposed human monocytes. Am J Physiol Gastrointest Liver Physiol 273: G1333-G1340, 1997[Abstract/Free Full Text].

26.   Mackay, DJ, and Hall A. Rho GTPases. J Biol Chem 273: 20685-20688, 1998[Free Full Text].

27.   Mainiero, F, Soriani A, Strippoli R, Jacobelli J, Gismondi A, Piccoli M, Frati L, and Santoni A. RAC1/P38 MAPK signaling pathway controls beta1 integrin-induced interleukin-8 production in human natural killer cells. Immunity 12: 7-16, 2000[ISI][Medline].

28.   Maoret, JJ, Anini Y, Rouyer-Fessard C, Gully D, and Laburthe M. Neurotensin and a non-peptide neurotensin receptor antagonist control human colon cancer cell growth in cell culture and in cells xenografted into nude mice. Int J Cancer 80: 448-454, 1999[ISI][Medline].

29.   Montaner, S, Perona R, Saniger L, and Lacal JC. Activation of serum response factor by RhoA is mediated by the nuclear factor-kappa B and C/EBP transcription factors. J Biol Chem 274: 8506-8515, 1999[Abstract/Free Full Text].

30.   Nemoto, S, DiDonato JA, and Lin A. Coordinate regulation of Ikappa B kinases by mitogen-activated protein kinase kinase kinase 1 and NF-kappa B-inducing kinase. Mol Cell Biol 18: 7336-7343, 1998[Abstract/Free Full Text].

31.   Pan, ZK, Ye RD, Christiansen SC, Jagels MA, Bokoch GM, and Zuraw BL. Role of the Rho GTPase in bradykinin-stimulated nuclear factor-kappa B activation and IL-1beta gene expression in cultured human epithelial cells. J Immunol 160: 3038-30345, 1998[Abstract/Free Full Text].

32.   Perona, R, Montaner S, Saniger L, Sanchez-Perez I, Bravo R, and Lacal JC. Activation of the nuclear factor-kappa B by Rho, CDC42, and Rac-1 proteins. Genes Dev 11: 463-475, 1997[Abstract].

33.   Poinot-Chazel, C, Portier M, Bouaboula M, Vita N, Pecceu F, Gully D, Monroe JG, Maffrand JP, Le Fur G, and Casellas P. Activation of mitogen-activated protein kinase couples neurotensin receptor stimulation to induction of the primary response gene Krox-24. Biochem J 320: 145-151, 1996[ISI][Medline].

34.   Ren, XD, Kiosses WB, and Schwartz MA. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 18: 578-585, 1999[Abstract/Free Full Text].

35.   Riegler, M, Castagliuolo I, Wang C, Wlk M, Sogukoglu T, Wenzl E, Matthews JB, and Pothoulakis C. Neurotensin stimulates Cl- secretion in human colonic mucosa in vitro: role of adenosine. Gastroenterology 119: 348-357, 2000[ISI][Medline].

36.   Sah, VP, Seasholtz TM, Sagi SA, and Brown JH. The role of Rho in G protein-coupled receptor signal transduction. Annu Rev Pharmacol Toxicol 40: 459-489, 2000[ISI][Medline].

37.   Schmid, RM, Adler G, and Liptay S. Activation of NFkappa B in inflammatory bowel disease. Gut 43: 587-588, 1998[Medline].

38.   Seasholtz, TM, Majumdar M, and Brown JH. Rho as a mediator of G protein-coupled receptor signaling. Mol Pharmacol 55: 949-956, 1999[Free Full Text].

39.   Teramoto, H, Coso OA, Miyata H, Igishi T, Miki T, and Gutkind JS. Signaling from the small GTP-binding proteins Rac1 and Cdc42 to the c-Jun N-terminal kinase/stress-activated protein kinase pathway. A role for mixed lineage kinase 3/protein-tyrosine kinase 1, a novel member of the mixed lineage kinase family. J Biol Chem 271: 27225-27228, 1996[Abstract/Free Full Text].

40.   Teramoto, H, Crespo P, Coso OA, Igishi T, Xu N, and Gutkind JS. The small GTP-binding protein rho activates c-Jun N-terminal kinases/stress-activated protein kinases in human kidney 293T cells. Evidence for a Pak-independent signaling pathway. J Biol Chem 271: 25731-25734, 1996[Abstract/Free Full Text].

41.   Thor, K, and Rosell S. Neurotensin increases colonic motility. Gastroenterology 90: 27-31, 1986[ISI][Medline].

42.   Vanden Berghe, W, Plaisance S, Boone E, De Bosscher K, Schmitz ML, Fiers W, and Haegeman G. p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways are required for nuclear factor-kappa B p65 transactivation mediated by tumor necrosis factor. J Biol Chem 273: 3285-3290, 1998[Abstract/Free Full Text].

43.   Walsh, JH. Gastrointestinal hormones. In: Physiology of the Gastrointestinal Tract (2nd ed.), edited by Johnson LR.. New York: Raven, 1987, p. 181-253.

44.   Wood, JG, Hoang HD, Bussjaeger LJ, and Solomon TE. Effect of neurotensin on pancreatic and gastric secretion and growth in rats. Pancreas 3: 332-339, 1988[ISI][Medline].

45.   Wood, JG, Hoang HD, Bussjaeger LJ, and Solomon TE. Neurotensin stimulates growth of small intestine in rats. Am J Physiol Gastrointest Liver Physiol 255: G813-G817, 1988[Abstract/Free Full Text].

46.   Yoshinaga, K, Evers BM, Izukura M, Parekh D, Uchida T, Townsend CM, and Thompson JC. Neurotensin stimulates growth of colon cancer. Surg Oncol 1: 127-134, 1992[Medline].

47.   Zhao, D, Keates AC, Kuhnt-Moore S, Moyer MP, Kelly CP, and Pothoulakis C. Signal transduction pathways mediating neurotensin-stimulated interleukin-8 expression in human colonocytes. J Biol Chem 276: 44464-44471, 2001[Abstract/Free Full Text].

48.   Zhao, D, Kuhnt-Moore S, Zeng H, Pan A, Wu JS, Simeonidis S, Moyer MP, and Pothoulakis C. Substance P-stimulated interleukin-8 expression in human colonic epithelial cells involves Rho family small GTPases. Biochem J 368: 665-672, 2002[ISI][Medline].


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