Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois, West Side Department of Veterans Affairs Medical Center, Chicago, Illinois 60612
Submitted 26 September 2002 ; accepted in final form 17 April 2003
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ABSTRACT |
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inflammation; enteropathogenic Escherichia coli; nuclear factor-B; protein kinase C
; I
B kinase; extracellular signal-regulated kinase
Activation of NF-B by microbial pathogens uses various upstream
cellular signals that result in inflammation. Helicobacter pylori
(21a) and Clostridium
difficile toxin A (56a)
utilize p38 to stimulate the inflammatory response; Salmonella
typhimurium utilizes intracellular Ca2+
(18); and Pseudomonas
aeruginosa (25), S.
typhimurium (20a), and
H. pylori (24) have
all been reported to engage ERK as a means of activating NF-
B. We
showed (47) that EPEC
activates the ERK pathway in host intestinal epithelial cells, which then
participates in I
B
phosphorylation and degradation and
subsequent IL-8 expression. Various upstream signal transduction molecules can
activate ERK, the best defined of which is the Ras-Raf1-MEK cascade
(56b). The ERK pathway,
however, can be alternatively activated by atypical PKCs (aPKCs)
(20,
26), and aPKCs have been shown
to activate NF-
B in a number of model systems
(6,
8,
12,
39,
53).
aPKCs (aPKC and -
/
) are unresponsive to calcium and
play important roles in controlling cell growth and survival
(6,
15,
35), most likely by activating
ERK and NF-
B pathways
(8,
9,
1214,
33,
34,
49). The mechanism of ERK
regulation by aPKC requires its interaction with and subsequent activation of
MEK, which can then activate ERK, or IKK
(6,
7,
3234,
36,
42). Additionally, PKC
can independently regulate NF-
B through interaction with and activation
of IKK (12,
17,
28,
53) or phosphorylation of the
p65 subunit of NF-
B (1,
23,
31).
EPEC has been shown to activate conventional PKCs in host cells
(3,
10), but their role in the
intestinal epithelial inflammatory response is not known. We reported
(47) that intracellular
calcium is not involved in EPEC-induced inflammation, suggesting that
calcium-dependent, conventional PKCs are not involved and thus implicating
either novel PKCs or aPKCs. Therefore, the aim of this study was to determine
whether PKC is activated by infection with EPEC and to investigate its
role in EPEC-associated inflammation. In this report, we show that EPEC
infection induces the translocation and activation of aPKC
in target
intestinal epithelial cells. PKC
inhibition with pharmacological
inhibitors, a specific inhibitory pseudosubstrate, or expression of a
dominant-negative PKC
significantly attenuated EPEC-induced
I
B
phosphorylation, demonstrating its involvement in the
proinflammatory signaling cascade. In contrast, PKC
inhibition had no
effect on EPEC activation of ERK, suggesting that these two signaling
molecules independently stimulate the inflammatory response. The mechanism by
which EPEC-activated PKC
regulates NF-
B includes activation of
IKK. Although the serine phosphorylation of p65 is significantly increased
after EPEC infection, inhibition of PKC
did not consistently attenuate
this response, suggesting that other signaling pathways participate in this
event. These studies show that epithelium-based signaling pathways evoked by
EPEC infection are complex and independently stimulate inflammation, thus
guaranteeing this protective response.
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MATERIALS AND METHODS |
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Bacterial strains and infection. Overnight cultures of EPEC strain E2948/69, grown in Luria-Bertani broth, were diluted (1:33) in serum- and antibiotic-free tissue culture medium containing 0.5% mannose and grown at 37°C to mid-log growth phase. Monolayers were infected as previously described (46) to yield a multiplicity of infection (MOI) of 100.
Treatment with inhibitors. To define the PKC isoforms involved in
activation of NF-B, cells were treated for 1 h before infection with
select inhibitors. Bisindolylmaleimide (BIM; Calbiochem, La Jolla, CA) at a
concentration of 50 µM inhibits many PKC isoforms, whereas Gö-6976
(Calbiochem) at a concentration of 10 µM inhibits only calcium-dependent
PKCs. Rottlerin (Calbiochem) in lower concentrations (36 µM)
selectively inhibits PKC
, whereas higher concentrations (3045
µM) also suppress calcium-dependent PKCs. At a concentration of 100 µM,
rottlerin also blocks PKC
. The myristoylated PKC
pseudosubstrate
(MYR-PKC
-PS; Biosource International, Camarillo, CA) suppresses only the
PKC
isoform. Intestinal epithelial cells were incubated with
MYR-PKC
-PS (20 µM) for 1 h before infection with EPEC.
Plasmids and cell transfection. The pcDNA3 and pPKC-KA
dominant negative form of PKC
inserted into pcDNA3 vector were kind
gifts from Dr. J. Moscat (Universidad Autonoma, Madrid, Spain). The PKC
mutant was generated by a substitution of lysine-275 for tryptophan and thus
lacks a functional catalytic domain
(7). Plasmids were amplified in
E. coli strain JM109 and purified with a Maxi Kit according to the
manufacturer's direction (Qiagen, Valencia, CA). Both T84 and Caco-2 cells
were plated at a density of 4 x 105 cells/cm2 in
six-well plates containing complete medium. After 72 h, cells were transfected
with 1 µg of plasmid by using Lipofectamine Plus (Invitrogen) following the
manufacturer's protocol. Cells were infected 48 h later with EPEC as described
in Bacterial strains and infection.
Immunofluorescent microscopy. Immunofluorescent staining was
performed on uninfected and infected monolayers of T84 and Caco-2 cells.
Monolayers were fixed with 3% paraformaldehyde pH 7.4 in PBS for 15 min,
rinsed with PBS, permeabilized with 0.2% Triton X-100 for 15 min, and blocked
in 1% BSA in PBS. Monolayers were incubated with antibodies against PKC
for 1 h followed by rhodamine-conjugated anti-rabbit IgG antibody for 1 h.
After washing, monolayers were mounted with Vectashield (Molecular Probes,
Eugene, OR) and assessed with a Nikon Opti-Phot microscope. Images were
captured with the Zeiss-RT Digital Imaging System.
Immunoblotting. After infection with EPEC, monolayers were washed
and proteins were extracted with RIPA buffer (in mM: 50 NaCl, 50 Tris, pH 7.4,
1 EGTA, 1 Na3VO4, 1 PMSF, and 1 NaF with 0.5% DOC, 0.1%
NP-40, 1 µg/ml aprotinin, and 1 µg/ml leupeptin). Proteins (100 µg)
were subjected to SDS-PAGE and then transferred onto 0.45-µm nitrocellulose
membranes (Bio-Rad, Hercules, CA). After blocking for 1 h at room temperature,
membranes were sequentially incubated for 1 h with primary antibody against
IB
(Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated
I
B
(New England Biolabs, Beverly, MA), PKC
, IKK, or p65
(Santa Cruz Biotechnology). The membranes were washed and incubated with
appropriate dilutions of secondary antibodies conjugated by alkaline
phosphatase or peroxidase for 1 h at room temperature. Color development was
achieved by alkaline phosphatase reaction with nitroblue
tetrazolium-5-bromo-4-chloro-indolyl phosphate solution (Zymed, San Francisco,
CA) or enhanced chemiluminescence (ECL; Pierce, Rockford, IL).
PKC translocation. PKC
translocation was
assessed as previously described
(5). Infected cells were washed
with buffer A (in mM: 25 Tris, pH 7.6, 1 EGTA, 10 NaCl), scraped into
buffer A+ (in mM: 25 Tris, pH 7.6, 1 EGTA, 10 NaCl, and 1 PMSF with
25 µg/ml leupeptin), and homogenized on ice with a Dounce homogenizer. The
homogenates were centrifuged at 15,000 g for 30 min at 4°C.
Supernatants represented the cytosolic fraction. Pellets were solubilized in
buffer A+ X-100 (in mM: 25 Tris, pH 7.6, 1 EGTA, 10 NaCl,
and 1 PMSF with 25 µg/ml leupeptin and 1% Triton X-100), homogenized with
10 strokes of the Dounce homogenizer, and centrifuged at 15,000 g for
30 min at 4°C. The resulting supernatants represented the membrane
fractions. Proteins extracted from both fractions were separated by 12%
SDS-PAGE, transferred to nitrocellulose membranes, and subjected to immunoblot
analysis with PKC
antibody. Immunoblots were quantitated by
densitometric analysis.
Immunoprecipitation. For immunoprecipitation, cells infected with
EPEC were harvested in lysis buffer (in mM: 40 Tris, pH 8.0, 300 NaCl, 6 EDTA,
6 EGTA, 10 -glycerophosphate, 10 NaF, 10
p-nitrophenylphosphate, 1 benzamidine, 2 PMSF, and 1 dithiothreitol
with 0.1% Nonidet P-40, 300 µM Na3VO4, 10 µg/ml
aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin). Extracts were
centrifuged at 15,000 g for 15 min, and 1 mg of whole cell lysate was
incubated with 10 µg of the indicated antibody for 1 h at 4°C. Protein
A beads (25 µg) were gently rotated for an additional hour at 4°C with
the protein-antibody mixture. Immunoprecipitates were washed five times with
lysis buffer, resolved by SDS-PAGE, transferred to nitrocellulose membranes,
and subjected to immunoblot analysis with the indicated antibody or used for
kinase activity assays.
Kinase activity assays. PKC and IKK activity assays were
performed as previously described
(41). Uninfected control and
infected cells were washed with PBS and lysed in buffer containing 50 mM Tris,
pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 1 mM CaCl2, 1% Triton
X-100, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin
by rocking for 30 min on ice. Extracts were centrifuged at 15,000 g
for 15 min at 4°C, and 1 mg of protein was incubated with 10 µl of
PKC
antibodies overnight at 4°C. The immune complexes were incubated
with 50 µl of protein A beads overnight at 4°C with gentle rotation.
Immunoprecipitates were washed seven times with lysis buffer modified to
contain 500 mM NaCl and incubated with 2 µg of myelin basic protein (MBP)
and 10 µCi of [
-32P]ATP for 30 min at 37°C in kinase
buffer (in mM: 35 Tris, pH 7.5, 10 MgCl2, 5 EGTA, 1
CaCl2, and 1 phenylphosphate) for PKC
activity assays.
Proteins were separated by 20% SDS-PAGE, gels were dried, and MBP was detected
as a 20-kDa band by autoradiography.
For IKK activity assays, immunoprecipitates were incubated with 2 µg of
glutathione S-transferase (GST)-tagged IB
produced in
E. coli (Santa Cruz Biotechnology) and 10 µCi of
[
-32P]ATP for 30 min at 37°C in kinase buffer (in mM: 35
Tris, pH 7.5, 10 MgCl2, 5 EGTA, 1 CaCl2, and 1
phenylphosphate). Kinase reaction mixtures were separated by 12% SDS-PAGE,
gels were dried, and phosphorylated rI
B
was detected as a 70-kDa
band by autoradiography.
Coimmunoprecipitation. Proteins were extracted for
coimmunoprecipitation with lysis buffer (in mM: 40 Tris, pH 8.0, 300 NaCl, 6
EDTA, 6 EGTA, 10 -glycerophosphate, 10 NaF, 10
p-nitrophenylphosphate, 1 benzamidine, 2 PMSF, and 1 dithiothreitol
with 0.1% Nonidet P-40, 300 µM Na3VO4, 10 µg/ml
aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin). One milligram of
whole cell lysate was incubated with ten micrograms of PKC
,
anti-phosphoserine, or p65 antibody (Zymed) and then twenty-five microliters
of protein A beads for 2 h at 4°C. Immunoprecipitates were washed five
times with lysis buffer. Samples were separated by 12% SDS-PAGE and
transferred to membranes, and immunoblot analysis was performed with the
appropriate antibody.
Statistical analysis. Data represent means ± SE. Data
comparisons were made with either ANOVA or Student's t-test.
Differences were considered significant at P 0.05.
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RESULTS |
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EPEC infection induces translocation of aPKC in
intestinal epithelial cells. The atypical isoform PKC
has been
reported to be involved in the activation of NF-
B
(12,
17,
28) and ERK
(6,
7). Both NF-
B and ERK
are activated by EPEC and involved in inflammation in intestinal epithelial
cells (11,
47). Therefore, we focused on
PKC
and its role in EPEC-induced inflammation in intestinal epithelial
cells. To initially address this issue, we examined the impact of EPEC
infection on the localization of PKC
. Cell lysates from control and
infected T84 and Caco-2 monolayers were collected at 30 and 60 min after
infection and then fractionated into cytosolic and membrane pools as
previously described (5). In
uninfected intestinal epithelial cells the majority of PKC
was found in
the cytosol, but after EPEC infection, progressive translocation to the
membrane fraction was seen (Fig. 2,
A and B). Immunostaining of control and
EPEC-infected T84 cells confirmed that the PKC
redistributed to the
periphery of cells, consistent with translocation to the membrane after
infection (Fig. 2C).
Similar PKC
translocation was obtained in Caco-2 cells after EPEC
infection (data not shown).
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EPEC infection activates PKC in intestinal epithelial
cells. Translocation of PKC
from the cytosolic to the membrane
fraction after EPEC infection suggests activation of this molecule. To
determine whether PKC
activity is increased after infection, PKC
kinase activity assays were performed.
Figure 3 shows that PKC
activity, determined as phosphorylation of MBP, increased 1.4 ±
0.2-fold by 15 min after infection, reached a plateau of 2.9 ± 1.2-fold
after 30 min, and remained activated for up to 60 min. It should be noted that
although PKC
kinase activity was significantly increased by 15 min after
infection, PKC
translocation did not reach statistical significance
until 60 min, although the trend was apparent at 30 min. It is possible that
the discrepancy between PKC
activity and translocation results from the
different sensitivities of the techniques used. That is, cell fractionation
and immunoblotting used to detect translocation are less sensitive than kinase
activity assays that use radiolabeled ATP to measure phosphorylation.
Nonetheless, these data show that EPEC infection induces not only the
translocation but also the activation of PKC
in intestinal epithelial
cells.
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EPEC-activated PKC is involved in regulation of the
NF-
B pathway. Many investigators using a variety of model
systems have reported a role for PKC
in the regulation of NF-
B
(12,
28,
39,
53). To define whether
PKC
is involved in the EPEC-induced activation of the NF-
B
pathway, we used three different approaches. First, we used the
pharmacological inhibitor rottlerin, which at high concentrations inhibits
PKC
. Figure 4A
shows that rottlerin inhibits PKC
translocation in response to EPEC
infection. Additionally, the same concentration of rottlerin significantly
suppressed EPEC-induced I
B
phosphorylation
(Fig. 4B), suggesting
the involvement of this isoform in the activation of the inflammatory cascade
by this enteric pathogen. However, the mechanisms by which rottlerin blocks
PKC are not defined. A recent publication
(52) suggests that rottlerin
decreases intracellular ATP, thus indirectly blocking PKC
while the
inhibitory mechanisms for the other isoforms remain unclear. Therefore, we
used a second, more specific approach to define the role of this isoform in
NF-
B activation. For these experiments, we used MYR-PKC
-PS, which
binds to the active center of PKC
, thus inhibiting its activity.
Figure 5A confirms the
inhibitory capacity of MYR-PKC
-PS by showing that treatment of cells
with this peptide decreases PKC
activity. The EPEC-induced increase in
PKC
activity was suppressed
40% by MYR-PKC
-PS. Furthermore,
inhibition of PKC
with MYR-PKC
-PS significantly attenuated
EPEC-induced I
B
phosphorylation
(Fig. 5B). After 30
min following EPEC infection, I
B
phosphorylation increased 2.5
± 0.1-fold above that seen in uninfected controls. Pretreatment with
MYR-PKC
-PS diminished this response to 1.5 ± 0.3-fold above that
measured in MYR-PKC
-PS-treated controls. The third approach used in this
study was transient transfection of cells with a plasmid containing the
PKC
gene harboring a mutation in kinase domain. Although the
transfection efficiency of T84 cells is low
(43), expression of this
nonfunctional PKC
attenuated by
30% the phosphorylation of
I
B
in response to EPEC (Fig.
5C). Similar results were obtained with Caco-2 cells
(data not shown).
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|
PKC is not involved in EPEC activation of the ERK
pathway in intestinal epithelial cells. We
(47) and others
(11) reported recently that
EPEC infection activates ERK, which is involved in the activation of the
NF-
B pathway in intestinal epithelial cells. The best-described pathway
leading to ERK activation is the Ras-Raf-MEK-ERK kinase cascade
(7,
12,
16,
17,
28,
53). Another possible
activator of MEK is PKC
(7,
12,
16,
17,
28,
53,
56b). Therefore, we questioned
whether EPEC activation of ERK was PKC
dependent or independent. To
address this, intestinal epithelial cells were preincubated with
MYR-PKC
-PS and EPEC-induced ERK phosphorylation was assessed. Although
this inhibitory peptide suppressed PKC
kinase activity and attenuated
I
B
phosphorylation, it had no effect on EPEC-induced ERK
phosphorylation (Fig.
6A). These data suggest that PKC
is not involved in
ERK activation by EPEC. To further substantiate that ERK activation was
independent of PKC
, the effect of EPEC infection on an upstream kinase
in the ERK pathway, Raf, was investigated.
Figure 6B shows that
EPEC indeed activates Raf, implying that ERK activation is a result of these
upstream regulators and not of PKC
. These data suggest, therefore, that
the upstream activation of PKC
and ERK is independent but the downstream
effects of these pathways converge to stimulate proinflammatory events.
|
EPEC-activated PKC regulates NF-
B by
interacting with and activating IKK
/
. There are two
possible mechanisms by which PKC
can regulate the NF-
B pathway:
1) through its interaction with and subsequent activation of IKK
(12,
28) and 2) by
phosphorylation of the p65 subunit of NF-
B, which increases its
transcriptional activity (1,
23,
31). To determine whether
PKC
interacts with IKK after infection with EPEC, coimmunoprecipitation
experiments were performed. Figure
7A shows that there is a 1.4-fold increase in PKC
and IKK association at 30 min and a 3-fold increase at 60 min after infection.
The only substrate of IKK is I
B
. To determine whether the
interaction of PKC
with IKK in response to EPEC infection led to its
activation, immunoprecipitated PKC
-IKK complexes from uninfected and
EPEC-infected intestinal epithelial cells were assayed for kinase activity
with recombinant GST-tagged I
B
as a substrate.
Figure 7B shows that
GST-I
B
phosphorylation increased 2.5-fold in T84 cells after 30
min of EPEC infection. These findings suggest that EPEC infection enhances the
interaction between IKK and PKC
and activates IKK, leading to increased
phosphorylation of the IKK-specific substrate I
B
.
|
The other mechanism by which PKC can influence NF-
B is through
the phosphorylation of several serine residues in the p65 subunit
(1,
23,
31). The effect of EPEC
infection on p65 serine phosphorylation was therefore examined.
Figure 8, A and
B, shows that there is a significant increase in the
serine phosphorylation of p65 in intestinal epithelial cells after EPEC
infection. Pretreatment of cells with MYR-PKC
-PS, however, did not
consistently reduce this response (Fig.
8C), suggesting that other signaling molecules are likely
involved at this level of NF-
B regulation.
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DISCUSSION |
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Other investigators have reported that EPEC infection activates
conventional, calcium-dependent PKC isoforms in host cells
(3,
10). The role of these
isoforms in EPEC pathogenesis is, however, unknown. Although the issue of
EPEC-induced elevation of intracellular calcium is controversial
(24),
we demonstrated (47) that EPEC
elevates intracellular calcium in T84 cells. We found
(47), however, that increased
intracellular calcium was not involved in the inflammatory response,
suggesting that conventional PKCs do not contribute to this process. We show
here instead that calcium-independent PKCs, specifically PKC,
participate in the EPEC-induced inflammatory response.
It was shown previously that aPKC isoforms play a crucial role in
NF-B activation (7,
12,
16). Thus the blockade of
aPKCs with pseudosubstrate peptide inhibitors
(16), antisense
oligonucleotides (16,
17), or transfection of
dominant negative mutants of PKC
(7,
12,
17) dramatically impairs
NF-
B activation. In this report, we show that blocking PKC
with a
pharmacological inhibitor, inhibitory pseudosubstrate peptide, or
kinase-defective enzyme significantly attenuated EPEC-induced I
B
phosphorylation in intestinal epithelial cells. A role for PKC
in a
pathogen-associated host response was recently demonstrated in the
Salmonella-induced stress signaling pathway in macrophages
(38). Here, however, we
demonstrate for the first time that an enteric pathogen activates PKC
in
intestinal epithelial cells and that it is involved in the inflammatory
cascade.
The mechanisms of NF-B regulation by PKC
are best defined in
response to TNF-
. In this case, PKC
interacts with IKK, thus
activating the latter (12,
17,
28,
53). Another kinase that can
activate the NF-
B pathway is NIK
(40,
57). NIK communicates with the
intracellular domain of TNF-
receptors, thus triggering additional
downstream signaling events that ultimately activate IKK
(27,
40). NIK can also interact
directly with IKK
and IKK
but phosphorylates and activates only
IKK
(27,
40,
57). The aPKCs can also bind
to both IKKs but, in contrast to NIK, activate only IKK
and have no
effect on IKK
(23).
Additionally, it was shown recently that IKK can be activated by MEK kinase.
However, MEK kinase 1 selectively activates IKK
and has no effect on
IKK
(36). Hence, there
appear to be specific kinase pathways upstream of the different IKKs that
control I
B phosphorylation and NF-
B activation. We have shown in
this article that PKC
regulates NF-
B through binding to and
activating IKK. Thus activated IKK phosphorylates I
B
, triggering
its degradation and release of the NF-
B molecule. Recently, another
mechanism by which PKC
can regulate NF-
B has been identified,
PKC
-dependent phosphorylation of serine residues in the p65 subunit of
NF-
B (1,
23,
31). Other signaling molecules
can directly phosphorylate p65, including IKK
(42a), Ras
(1), and the p38 pathway
(55a). We show here that EPEC
infection triggers the serine phosphorylation of p65 but could not
consistently demonstrate attenuation by inhibition of PKC
. These
findings suggest that other signaling molecules likely contribute to
EPEC-induced p65 phosphorylation. Nonetheless, our data clearly reveal the
involvement of PKC
in the upstream activation of NF-
B in
intestinal epithelial cells after EPEC infection.
Another level of PKC involvement in inflammation-related signaling is via
its effect on ERK. aPKCs have been shown to activate the ERK signaling pathway
through Raf-independent mechanisms by interacting with and activating MEK,
which then activates both IKK and IKK
(20,
26,
33,
34,
39). Although the upstream
signaling pathway by which EPEC activates ERK in intestinal epithelial cells
is not fully defined, our data suggest that activation of ERK is PKC
independent and that the Ras/Raf kinase cascade is responsible instead.
Therefore, EPEC activation of the proximal signaling pathways, PKC
and
ERK, is independent but converges downstream to ensure stimulation of the
proinflammatory response. A variety of pathways likely control the
EPEC-induced increase in p65 serine phosphorylation.
An additional role for PKC in epithelia is the regulation of tight
junctions (21,
37,
54). PKC
interacts with
a number of tight junction-associated proteins including PAR-6
(21,
54) and occludin
(37). We reported previously
(51) that the localization of
tight junction proteins is disrupted after EPEC infection, as is intestinal
epithelial barrier function. New data from our laboratory suggest that
EPEC-activated PKC
also participates in the perturbation of barrier
function (22), suggesting that
this particular signaling molecule, in contrast to MAP kinases
(11,
47), is involved in two major
physiological effects, inflammation and barrier function.
Over the past several years, a variety of signaling pathways activated in host cells by pathogenic bacteria have been described. Different pathogens can activate different signaling pathways, and a single pathogen can activate a variety of signaling cascades in host cells. The end points of these pathogen-activated signaling networks in host cells include perturbation of physiological processes such as inflammation, barrier function, ion secretion, and cell survival. Additionally, it is clear that a single signaling molecule may be involved in controlling different physiological events. Continued efforts focusing on the elucidation of these pathogen-activated signaling networks and their downstream impact on specific physiological responses will increase our understanding of microbial pathogenesis.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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