Department of Medicine, Section of Digestive and Liver Diseases, University of Illinois, West Side Veterans Affairs Medical Center, Chicago, Illinois 60612
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ABSTRACT |
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Enteropathogenic
Escherichia coli (EPEC) alters many functions of the
host intestinal epithelia. Inflammation is initiated by activation of
nuclear factor (NF)-B, and paracellular permeability is enhanced via
a Ca2+- and myosin light-chain kinase (MLCK)-dependent
pathway. The aims of this study were to identify signaling pathways
by which EPEC triggers inflammation and to determine whether these
pathways parallel or diverge from those that alter permeability.
EPEC-induced phosphorylation and degradation of the primary inhibitor
of NF-
B (I
B
) were tumor necrosis factor (TNF)-
and
interleukin (IL)-1
independent. In contrast to Salmonella
typhimurium, EPEC-stimulated I
B
degradation and IL-8
expression did not require Ca2+. Instead, extracellular
signal-regulated kinase (ERK)-1/2 was significantly and rapidly
activated. ERK1/2 inhibitors attenuated I
B
degradation and IL-8
expression. Although ERK1/2 can activate MLCK, its inhibition had no
impact on EPEC disruption of the tight junction barrier. In conclusion,
EPEC-induced inflammation 1) is TNF-
and IL-1
receptor
independent, 2) utilizes pathways differently from S. typhimurium, 3) requires ERK1/2, and 4)
employs signals that are distinct from those that alter permeability. This is the first time that EPEC-activated signaling cascades have been
linked to independent functional consequences.
permeability; nuclear factor-B; primary inhibitor of nuclear
factor-
B; interleukin-8; enteric pathogens; enteropathogenic
Escherichia coli; extracellular signal-regulated
kinase-1/2
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INTRODUCTION |
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WE HAVE PREVIOUSLY
SHOWN using an in vitro model that enteropathogenic
Escherichia coli (EPEC) infection alters host intestinal epithelial physiological function. For example, EPEC induces the transepithelial migration of acute inflammatory cells, neutrophils (37). The secretion of interleukin (IL)-8 from the basal
aspect of infected intestinal cells in part directs neutrophil
transmigration. Expression of IL-8 by intestinal epithelial cells
infected with EPEC or other enteric pathogens is regulated via
activation of nuclear factor (NF)-B (7, 36, 41, 45).
NF-
B is an inducible transcriptional factor located in the cytoplasm
and bound to inhibitory proteins that are part of the NF-
B inhibitor
(I
B) family, i.e., I
B
, I
B
, and I
B
(11,
49). There are many inducers of NF-
B activation, including
cytokines such as tumor necrosis factor (TNF)-
and IL-1
, that
trigger a cascade of events that results in I
B phosphorylation.
Subsequently, ubiquitin-dependent degradation of I
B
by
proteasomes (42, 46) allows the unbound NF-
B to translocate to the nucleus and function as a transcriptional activator. Various signaling pathways such as elevation of intracellular Ca2+ (10, 43), mitogen-activated protein (MAP)
kinases (23, 30), protein kinase C (PKC), protein kinase A
(PKA), and Raf-1 (38, 39, 43) have been reported to
participate in NF-
B activation. Recent publications show that
specific enzymes in the extracellular signal-regulated kinase (ERK)
pathway such as MAP or ERK kinase kinase (MEKK)-1, -2, and -3 can directly activate the NF-
B regulatory cascade (23, 29, 33,
50).
Infection of epithelial cells with microbial pathogens also triggers
various cellular signals that activate the NF-B pathway, resulting
in inflammation. For example, Pseudomonas aeruginosa (25) activates NF-
B via MAP kinases. Salmonella
typhimurium-induced MAP kinases and elevation of intracellular
Ca2+ participate in the activation of NF-
B and
subsequent upregulation of IL-8 expression (10, 16).
Recently, Warny et al. (47) have shown that
Clostridium difficile toxin A induces IL-8 expression and
inflammation by stimulating p38 MAP kinase.
Previous studies have also shown that EPEC infection of intestinal
epithelia disrupts barrier function. Paracellular permeability can be
regulated by phosphorylation of the 20-kDa myosin light chain
(MLC20) located in the perijunctional cytoskeletal ring (14, 44). We have shown that EPEC-induced perturbation of the intestinal epithelial tight junction barrier requires intracellular Ca2+ and myosin light-chain kinase (MLCK) leading to
MLC20 phosphorylation (48). Aside from
Ca2+-dependent activation of MLCK, recent studies have
shown that ERK can also activate this enzyme (21, 31). The
aims of this study, therefore, were to define the signaling pathways by
which EPEC activates NF-B, compare them with those utilized by
S. typhimurium, and determine whether these pathways are the
same or divergent from those that lead to the disruption of intestinal
epithelial permeability.
In this report, we show that EPEC infection of intestinal epithelial
cells induces phosphorylation and degradation of IB
in a
TNF-
/IL-1
receptor-independent manner. In contrast to S. typhimurium, EPEC-induced activation of NF-
B and upregulated IL-8 expression do not involve Ca2+. Instead, ERK1/2 are
rapidly phosphorylated and activated in intestinal epithelial cells
after EPEC infection, and enzymes from this cascade are directly
involved in NF-
B activation and IL-8 expression. In contrast, the
ERK pathway does not participate in the disruption of intestinal
epithelial tight junction barrier function associated with EPEC infection.
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MATERIAL AND METHODS |
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Cell culture. T84 cells were a generous gift from Dr. Kim Barrett (Univ. of California San Diego). Passages 35-55 were used for these studies and were grown in a 1:1 (vol/vol) mixture of Dulbecco-Vogt modified Eagle's medium (GIBCO-BRL) and Ham's F-12 (GIBCO-BRL) as described previously (28).
Bacterial strains and infection. EPEC, wild-type strains, E2348/69, RN587/1, and S. typhimurium [American Type Culture Collection (ATTC) no. 14028, Rockville, MD] were used. EPEC strain RN587/1 is a recent clinical isolate from Brazil obtained from Dr. Michael Donnenberg (Univ. of Maryland Baltimore). Commensal E. coli strains were isolated from the stool of healthy humans by the Clinical Microbiology Laboratory at the University of Illinois (Chicago, IL) (13). Overnight bacterial cultures, 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 midlog growth phase. Monolayers were infected as previously described (37) to yield a multiplicity of infection (MOI) of 100 after 1 h of infection. After infection, for the indicated time periods, medium was removed from monolayers and cells were washed three times with PBS.
Treatment with inhibitors.
To define the signaling pathways involved in activation of NF-B,
cells were pretreated for 1 h with select inhibitors before infection with pathogens. The MAP or ERK kinase (MEK) inhibitor PD-98059 (Calbiochem, La Jolla, CA), which at a concentration of 5 µM
inhibits MEK1 and at 50 µM inhibits MEK2, was used. Another specific
inhibitor of MEK1/2 employed for these studies was U0126 (Calbiochem)
at a concentration of 0.7 µM. MG-132 (Calbiochem), a proteasomal
inhibitor, was used at a concentration of 50 µM. Eukaryotic protein
synthesis was inhibited with 2 µM cycloheximide (Sigma, St. Louis,
MO). We have previously reported that this concentration blocks
75% of protein synthesis in T84 cells (15). Cell-permeant acetoxymethyl ester of
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM, 15 µM; Calbiochem) was used as a chelator of
intracellular Ca2+ as described previously by Gewirtz et
al. (10). In brief, studies were performed in Hanks'
balanced salt solution (HBSS) containing 0.25 mM CaCl2, in
contrast to the standard concentration of 1.25 mM CaCl2, to
maximize Ca2+ chelation but preserve the monolayer barrier.
Stimulation and blockage of TNF- and IL-1
receptors.
Cytokine-mediated pathways were stimulated by treatment of intestinal
epithelial cells with TNF-
(10 ng/ml) or IL-1
(5 ng/ml). To assess I
B
degradation, cells were treated for 15, 30, 45, and
60 min. Proteins were extracted, and the effect on I
B
degradation was analyzed by immunoblot as described below. TNF-
or IL-1
receptor activation by their respective ligands was blocked by preincubation of cells for 1 h with blocking antibody against either the TNF-
(6 µg/ml) or IL-1
receptor (1 µg/ml) (R&D
Systems, Minneapolis, MN). The cells were then stimulated with TNF-
or IL-1
for the indicated times. Proteins were extracted and
analyzed for I
B
degradation.
Immunoblot.
Whole cell lysates from control or treated T84 cells were extracted in
RIPA buffer [50 mM NaCl, 50 mM Tris, pH 7.4, 0.5% deoxycorticosterone (DOC), 0.1% NP-40, 1 mM EGTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM NaF, 1 µg/ml aprotinin,
and 1 µg/ml leupeptin]. Protein concentration was determined using a
Bradford protocol, and 100 µg of protein were separated by 12%
SDS-PAGE. The gels were transblotted and incubated with antibodies
recognizing total IB
(Santa Cruz Biotechnology, Santa Cruz, CA)
or phosphospecific I
B
(New England Biolabs, Beverly, MA). For ERK
detection, both general (Sigma) and phosphospecific (Promega, Madison,
WI) antibodies were used. Equal loading of protein was determined by
probing blots for total protein-tyrosine phosphatase-1D/SH2-containing phosphatase-2 (PTP1D/SHP2) concentration (Transduction
Laboratories, Lexington, KY), which does not change after EPEC
infection. Color development was achieved with a nitro blue
tetrazolium-5-bromo-4-chloro-indolylphosphate premixed solution (Zymed,
San Francisco, CA).
ERK1/2 activity assay.
For detection of ERK phosphorylation and activity, cells were serum
starved overnight. After infection, proteins were extracted with 50 mM
Tris, pH 7.5; 1 mM EDTA; 1 mM EGTA; 0.5 mM
Na3VO4; 0.1% 2-mercaptoethanol; 1% Triton
X-100; 50 mM sodium fluoride; 5 mM sodium pyrophosphate; 10 mM sodium
-glycerophosphate; 0.1 mM PMSF; 1 µM/ml of aprotinin, pepstatin,
and leupeptin; and 1 µM microcystein. The immunocomplex kinase
activity assay for ERK1/2 was performed according to the
manufacturer's protocol (Upstate Biotechnology, Lake Placid, NY).
Kinase activity was measured in a scintillation counter, and data were
expressed as mean ± SE increases over activity measured in
uninfected monolayers. ERK activity measured in control monolayers was
set at 1 to compare the results of independent assays.
Quantitation of IL-8.
T84 cells were grown on 1-cm2 Transwell filters (Costar,
Cambridge, MA) coated with collagen. The cells were then infected with
EPEC or treated with TNF- in the presence or absence of specific
inhibitors. Medium from the basolateral compartments was collected
after 6 h, and IL-8 was quantified by use of a dual-antibody ELISA
kit from R&D Systems, following the manufacture's protocol.
Measurement of intracellular Ca2+.
T84 cells were plated on coverslips constructed to fit into standard
fluorescent cuvettes. After 7 days, cells were incubated with 5 µM
acetoxymethyl ester of fura 2 (fura 2-AM; Calbiochem) for 1 h at
37°C and infected with EPEC for 30 min as described above. Cuvettes
containing coverslips were then placed into a spectrofluorometer
(Perkin Elmer, Buckinghamshire, UK) and exposed to fluorescence
emission read at 505 nm while excitation wavelength was switched
between 340 and 380 nm. Values of intracellular Ca2+
concentration ([Ca2+]i) were calculated via
the Grynciewitz equation: [Ca2+]i = Kd[(R Rmin)/(Rmax
R)], where Kd is dissociation
constant and R is the experimental ratio.
Rmax and Rmin were
measured by adding digitonin (10 µM) and then EGTA (20 µM), and
Kd was taken to be 2.54 × 10
7 M.
Electrophysiological studies. Cells were grown to confluence on 0.33-cm2 collagen-coated permeable supports (Transwells). With the use of a simplified apparatus described by Madara et al. (27), transepithelial electrical resistance was determined by passing 25 µA of current, measuring the resulting voltage deflection, and applying Ohm's law (V = IR; where V is voltage, I is current, and R is resistance).
Statistical analysis.
All data are represented as the means ± SE. Data comparisons were
made with Student's t-test. Differences were considered significant at P 0.05.
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RESULTS |
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Infection of intestinal epithelial cells by EPEC induces
phosphorylation/degradation of IB
.
We have previously shown that the upregulation of IL-8 expression by
intestinal epithelial cells in response to infection with EPEC is via
activation of NF-
B (36). NF-
B activation is preceded
by phosphorylation and degradation of the inhibitory molecule I
B
.
Increased I
B
phosphorylation was detected 15 min after infection
of T84 intestinal epithelial cells with EPEC and peaked at 60 min (Fig.
1A). By 2 h, however,
this signal was absent. I
B
degradation was detectable at 45 min postinfection and progressed until the protein was
nearly absent by 2-3 h, corresponding to the loss of the
phosphorylation signal. Furthermore, the proteasomal inhibitor
MG-132 prevented I
B
degradation, suggesting that EPEC stimulates
this classic pathway (Fig. 1B).
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Degradation of IB
by EPEC is independent of TNF-
and
IL-1
receptor activation and de novo protein synthesis.
The best-defined pathways leading to phosphorylation and degradation of
I
B
are those initiated by activation of cytokine-specific membrane receptors, in particular TNF-
and IL-1
receptors.
Infection of T84 cells by enteric bacterial pathogens has been shown to increase the expression of both TNF-
and IL-1
proinflammatory molecules (17). We therefore sought to exclude the
possibility that paracrine or autocrine responses from EPEC-stimulated
TNF-
or IL-1
production could account for activation of the
inflammatory cascade. Cells were preincubated with antagonistic
antibodies against TNF-
or IL-1
receptors and then infected with
EPEC. As shown in Fig. 2A,
neither antibody prevented I
B
degradation in response to
infection. The efficiency of this approach was confirmed by
demonstrating that TNF-
-induced I
B
degradation was prevented
by pretreatment with TNF-
receptor blocking antibodies (Fig.
2B). These data suggest that EPEC-activated NF-
B
signaling pathways in intestinal epithelial cells are TNF-
and
IL-1
receptor independent and that alternative mechanisms are
responsible. To define whether EPEC-induced expression of other
cytokines that may secondarily activate NF-
B signaling pathways was
involved, cells were preincubated with cycloheximide, an inhibitor of
eukaryotic protein synthesis, and infected with EPEC. Figure
2C shows that cycloheximide did not prevent EPEC-induced
I
B
degradation, suggesting that de novo protein synthesis is not
required to trigger the signaling cascades by which EPEC activates
inflammation-associated events.
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Chelation of intracellular Ca2+ has no effect on
EPEC-induced IB
degradation or IL-8 expression.
Gewirtz et al. (10) have recently demonstrated that
S. typhimurium-induced I
B
degradation in intestinal
epithelial cells is Ca2+ dependent. Although the published
results have been conflicting, EPEC has been shown to increase
intracellular Ca2+ in eukaryotic cells
(2-4). We therefore measured the concentration of
intracellular Ca2+ in uninfected control T84 cells and
cells infected with EPEC for 30 min. In uninfected cells, the
intracellular concentration of Ca2+ was determined to be
153 ± 34 nM (n = 9). After 30 min of infection, however, the intracellular Ca2+ concentration increased to
581 ± 75 nM (n = 5, P = 0.00004). To address whether this EPEC-induced elevation of intracellular Ca2+ was involved in activation of NF-
B and IL-8
expression, intestinal T84 cells were treated with the
cell-permeant Ca2+ chelator BAPTA-AM. Figure
3A shows that BAPTA-AM
did not prevent EPEC-induced I
B
degradation or IL-8 expression
(Fig. 3A). In contrast, BAPTA-AM did block I
B
degradation and IL-8 production in response to S. typhimurium (Fig. 3B) as was previously shown (10). These data suggest that activation of NF-
B and
increased IL-8 expression by EPEC are Ca2+ independent.
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EPEC activates ERK pathways in intestinal epithelial cells.
Several bacterial pathogens have been demonstrated to induce
inflammation via MAP kinase pathways (16, 18, 47).
Investigation of the impact of EPEC infection on the ERK pathway was of
particular interest, since recent reports suggest that some enzymes
from the ERK cascade are involved in the regulation of NF-B
(30). We therefore explored whether EPEC infection
triggered activation of ERK 1/2. Figure
4A shows that the
amount of total ERK1/2 in T84 cells remained unchanged after EPEC
infection. Equal loading of protein was determined by probing the
immunoblots for total PTP1D/SHP2 protein. Immunoblots were then
scanned, and densitometric analysis showed that the ratio between
ERK1/2 and PTP1D/SHP2 is unchanged after infection (~1:2).
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EPEC-activated ERK pathways participate in IB
degradation and
IL-8 production.
The next studies were aimed at determining whether the ERK1/2 pathway
is directly involved in EPEC-induced inflammatory events. Inhibitors of
specific kinases in the ERK pathway were used for these experiments:
PD-98059 at 5 µM was used to inhibit MEK1 and at 50 µM was used to
inhibit both MEK1 and -2. Both 5 and 50 µM PD-98059 attenuated
I
B
degradation (Fig. 5,
A and B) and IL-8 production (Fig. 5C)
associated with EPEC infection, suggesting that MEK1 and -2 are
involved in this pathway. To confirm the role of ERK1/2 in
EPEC-associated I
B
degradation, another MEK1/2 inhibitor, U0126,
was used. Figure 5D shows that U0126 significantly suppressed EPEC-induced ERK1/2 phosphorylation and blocked the subsequent I
B
degradation. These data support the contention that
the ERK arm of the MAP kinase pathway is stimulated by EPEC infection
and participates in I
B
degradation, which ultimately leads to
IL-8 expression.
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ERK1/2 are not involved in EPEC-induced disruption of the tight
junction barrier.
We have been shown previously that EPEC infection alters intestinal
epithelial tight junction barrier function in part by phosphorylating
MLC20 (48). The classically described pathway by which MLCK is activated requires the formation of
Ca2+-calmodulin complexes, which occurs as a result of
increased intracellular Ca2+ (1, 5). The
sequestration of intracellular Ca2+ by dantrolene and
inhibition of MLCK by
1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepene (ML-9) decreased both MLC20 phosphorylation and
the drop in transepithelial resistance (14). More
recently, however, ERK1/2 was also shown to activate MLCK, thus
enhancing MLC20 phosphorylation (21, 31). To
determine whether the ERK pathway is involved in the EPEC-induced
alteration of tight junction barrier function, the effect of PD-98059
on this functional consequence was tested. Figure
6 shows that neither 5 (MEK1) nor 50 µM
(MEK2) PD-98059 prevented the EPEC-associated disruption of barrier
function as assessed by transepithelial electrical resistance. These
data support the premise that EPEC-activated signaling pathways diverge and ultimately affect separate intestinal epithelial physiological functions (Fig. 6).
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DISCUSSION |
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EPEC infection of intestinal epithelial cells triggers two major
functional effects: inflammation and disruption of barrier function.
The inflammatory response is coordinated by the transcription factor
NF-B. The regulation of NF-
B activation is well studied, the
best-defined pathway being that stimulated by cytokines. TNF-
, for
example, binds to its receptor and recruits cytoplasmic adapter molecules such as NF-
B-inducing kinases (NIKs) (42,
46). NIK activates a large (~800 kDa) multiprotein complex
I
B
kinase (IKK) that then phosphorylates I
B
(42,
46). Phosphorylated I
B
is ubiquitinated and degraded by
26S proteasomes, freeing NF-
B to translocate to the nucleus and
initiate gene transcription. Enteropathogenic E. coli
induces I
B
phosphorylation/degradation in <1 h postinfection,
highlighting these as early events. EPEC-stimulated NF-
B activation
is TNF-
and IL-1
receptor independent, does not require de novo
protein synthesis, and employs pathways different from those
utilized by Salmonella, suggesting that specific
signals are induced by EPEC that lead to intestinal inflammation.
We show here that ERK1/2, but not Ca2+, is involved in this
process. Whether EPEC initiates the inflammatory cascade by direct
activation of MAP kinases or utilizes NIK is not known.
There are three parallel MAP kinase cascades: ERK, c-Jun
NH2-terminal kinase (JNK), and p38 (12, 22,
24). Many growth factors and hormones stimulate the ERK pathway
via receptor activation, whereas JNK and p38 cascades are triggered by
environmental stresses. Involvement of the ERK pathway in NF-B
activation has been demonstrated by showing that a dominant-negative
mutant of MEKK1 inhibited NF-
B activation, and overexpression of
active MEKK1 resulted in the phosphorylation of I
B
(23). In addition, MEKK2 and -3 have been shown to
activate the NF-
B pathway (50), demonstrating communication between ERK1/2 and the NF-
B pathway. We show here that
ERK is rapidly phosphorylated and activated after EPEC infection and
that inhibition of enzymes within this pathway prevents I
B
degradation and IL-8 production. These findings suggest that EPEC may
trigger an unidentified receptor to stimulate ERK and activate NF-
B.
Interestingly, one of the EPEC proteins secreted by the type III
secretory apparatus and injected to the host cells is the translocated
intimin receptor (Tir), which serves as a receptor for the outer
membrane EPEC protein intimin (6, 19). Whether Tir-intimin
interactions are required for activation of ERK has not been explored.
Certainly, other receptor-ligand interactions may contribute to EPEC
pathogenesis, including those that involve the bundle-forming pilus,
flagellin, and perhaps type I pili. In our model system, however, the
interaction of type I pili with its receptor is blocked by the addition
of mannose. The role of these other potential interactions has yet to
be explored. Several gastrointestinal pathogens have been shown to
utilize MAP kinases to initiate inflammation. Warny et al.
(47) have shown that C. difficile
toxin A induces IL-8 expression and inflammation by activating p38 MAP
kinases. Helicobacter pylori activates ERK, JNK, and p38,
and their involvement in regulating IL-8 expression has been
demonstrated (18). Surprisingly, H. pylori-activated MAP kinases are not involved in activating
NF-
B, suggesting that other signaling pathways initiate the
inflammatory response. In contrast, S. typhimurium activates
NF-
B and upregulates IL-8 production through MAP kinase cascades
(16). In this case, however, intracellular
Ca2+ is also involved (10).
Ca2+-dependent NF-
B activation has also been
demonstrated in response to other stimuli such as acetylcholine
receptor ligation (10), neuropeptide substance P
(26), and endoplasmic reticulum "overload" (32). In regards to EPEC, however, Ca2+ does
not appear to play a role in this response, although it is involved in
perturbing tight junction permeability (48). Together
these findings suggest that enteric pathogens employ different
signaling pathways to induce a common response: intestinal inflammation.
EPEC stimulates other signaling molecules in intestinal epithelial
cells including tyrosine kinases (34), PKC
(3), reactive oxygen intermediates (35), and
inositol 1,4,5-trisphosphate (8). The roles of these
molecules in the physiological changes that occur in EPEC-infected
cells have not been defined. Our preliminary findings suggest that in
addition to ERK1/2, these other signaling pathways may also participate
in triggering the inflammatory cascade (35). Whether these
have direct effects on NF-B activation or converge on MAP kinase
cascades has not been investigated. However, in other model systems,
each of these signaling molecules has been shown to activate MAP
kinases (40).
In addition to inducing the inflammatory response, infection with EPEC perturbs another major physiological function: intestinal epithelial permeability. Previous studies have shown that epithelial barrier function can be regulated by MLC20 phosphorylation, which causes contraction of the perijunctional cytoskeletal ring (14). In part, EPEC perturbs the intestinal epithelial tight junction barrier through this Ca2+/MLCK-dependent event (48). MLCK is a Ca2+/calmodulin-dependent enzyme that phosphorylates MLC20 (1, 5). Recent studies show, however, that MLCK can also be activated by different stimuli. For example, urokinase-type plasminogen activator activates MLCK function by the Ras/ERK pathway (31). Cell migration occurs via ERK-induced activation of MLCK (21). A more recent study suggests that MAP kinases can regulate barrier function in intestinal epithelial cells by modulating the expression of claudin-2 (20), a transmembrane tight junction protein involved in barrier formation (9). In contrast to the impact of MAP kinase inhibition on the EPEC-induced inflammatory responses, no role for these enzymes could be demonstrated with regards to EPEC-associated disruption of the tight junction barrier.
In summary, here we provide evidence that EPEC-induced signaling
pathways that initiate inflammation 1) are activated early and independently of TNF- or IL-1
receptors, 2)
involve ERK1/2, 3) are different from those employed by
S. typhimurium, and 4) are distinct from those
that disrupt paracellular permeability. This is the first demonstration
that the multiple signaling cascades stimulated by EPEC infection
diverge and ultimately alter distinct intestinal epithelial functions.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the Crohn's and Colitis Foundation of American (Research Fellowship Award to S. D. Savkovic), National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50694 (to G. Hecht), and Merit Review and Research Enhancement Awards from the Department of Veterans Affairs (to G. Hecht).
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FOOTNOTES |
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This work was presented in preliminary form at the annual meeting of the American Gastroenterological Association Digestive Disease Week in San Diego, CA, on May 21, 2000.
Address for reprint requests and other correspondence: G. Hecht, Univ. of Illinois, Dept. of Medicine, Section of Digestive and Liver Disease, 840 South Wood St., CSB Rm. 704 (m/c 787), Chicago, IL 60612 (E-mail: gahecht{at}uic.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.
Received 29 January 2001; accepted in final form 6 June 2001.
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