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
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ABSTRACT |
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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-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-
B-dependent reporter
gene expression and NF-
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-
B-mediated pathway.
neuropeptide; inflammation; signal transduction; gene regulation
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INTRODUCTION |
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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-B (NF-
B)-dependent pathway
(47). This is consistent with a prior study
(9) demonstrating that NT can directly stimulate DNA binding activity of NF-
B in isolated human intestinal microvascular endothelial cells. NF-
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 I
B inhibitory proteins. It is well established that the NF-
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-1
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-
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-B DNA binding activity and NF-
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-
B pathway.
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MATERIALS AND METHODS |
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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-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-
B consensus
oligonucleotide probe was purchased from Promega (Madison, WI), and
[
-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-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-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-
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.
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RESULTS |
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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-B activation (47). Because the members of the Rho
family GTPases RhoA, Rac1, and Cdc42 were shown to mediate NF-
B
activation and NF-
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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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 (107 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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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 (107
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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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 (107 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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NT-induced NF-B-dependent gene expression involves RhoA, Rac1,
and Cdc42.
Because NT-stimulated IL-8 expression in NCM460-NTR1 cells requires
NF-
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-
B pathway. To
do this, we transfected cells with LacZ-, Rho-19N-, Rac1-17N-, or
Cdc42-17N-expressing constructs together with a NF-
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-
B promoter activity. The
results show that overexpression of Rho-19N, Rac1-17N, or Cdc42-17N
significantly inhibited NT-induced NF-
B-dependent reporter gene
expression (Fig. 5).
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NT-induced NF-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-
B
(32). Here we examined whether dominant negative mutants of RhoA, Rac1, and Cdc42 inhibit NT-induced NF-
B-dependent reporter gene expression (Fig. 5) by reducing NF-
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-
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-
B (Fig. 6). These
results suggest that the three members of Rho family GTPases
mediate NT-induced IL-8 expression and NF-
B-dependent reporter gene
transcription, at least in part, by activating NF-
B DNA binding.
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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-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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DISCUSSION |
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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-B/I
B system. For example, nontransformed human colonic
epithelial NCM460 cells express functional NTR1, and increased
expression of this receptor leads to NF-
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-
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-
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-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-1
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-
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-
B activation.
The molecular mechanism(s) whereby Rho family GTPases activate NF-B
has not been completely elucidated. It is well established that nuclear
translocation and DNA binding of NF-
B heterodimers is primarily
regulated through phosphorylation and degradation of the inhibitory
proteins I
B by I
B kinases (IKKs) or other, not yet identified
kinases. Thus, Cammarano and Minden (4) reported that Rac1
stimulates NF-
B via NF-
B-inducing kinase (NIK)-mediated IKK
activation, whereas, in contrast, RhoA and Cdc42 activate NF-
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 I
B
and increased NF-
B
DNA binding in Helicobacter pylori-infected gastric
epithelial cells. On the other hand, Rac1 regulates IL-1-induced
NF-
B-dependent reporter gene expression without causing I
B
degradation and nuclear translocation of NF-
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-
B following IL-1 exposure (23). These seemingly conflicting results suggest that the ability of Rho GTPases
to activate NF-
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-
B DNA binding activity and
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-
B signaling, a notion consistent with our recent studies indicating that inhibition of ERK activation has no
effect on NT-induced NF-
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-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.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-47343, DK-60729, and T32-DK-07760.
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FOOTNOTES |
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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.
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