EPEC-activated ERK1/2 participate in inflammatory response but not tight junction barrier disruption

Suzana D. Savkovic, Akila Ramaswamy, Athanasia Koutsouris, and Gail Hecht

Department of Medicine, Section of Digestive and Liver Diseases, University of Illinois, West Side Veterans Affairs Medical Center, Chicago, Illinois 60612


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Enteropathogenic Escherichia coli (EPEC) alters many functions of the host intestinal epithelia. Inflammation is initiated by activation of nuclear factor (NF)-kappa 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-kappa B (Ikappa Balpha ) were tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta independent. In contrast to Salmonella typhimurium, EPEC-stimulated Ikappa Balpha 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 Ikappa Balpha 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-alpha and IL-1beta 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-kappa B; primary inhibitor of nuclear factor-kappa B; interleukin-8; enteric pathogens; enteropathogenic Escherichia coli; extracellular signal-regulated kinase-1/2


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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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)-kappa B (7, 36, 41, 45). NF-kappa B is an inducible transcriptional factor located in the cytoplasm and bound to inhibitory proteins that are part of the NF-kappa B inhibitor (Ikappa B) family, i.e., Ikappa Balpha , Ikappa Bbeta , and Ikappa Bepsilon (11, 49). There are many inducers of NF-kappa B activation, including cytokines such as tumor necrosis factor (TNF)-alpha and IL-1beta , that trigger a cascade of events that results in Ikappa B phosphorylation. Subsequently, ubiquitin-dependent degradation of Ikappa Balpha by proteasomes (42, 46) allows the unbound NF-kappa 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-kappa 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-kappa B regulatory cascade (23, 29, 33, 50).

Infection of epithelial cells with microbial pathogens also triggers various cellular signals that activate the NF-kappa B pathway, resulting in inflammation. For example, Pseudomonas aeruginosa (25) activates NF-kappa B via MAP kinases. Salmonella typhimurium-induced MAP kinases and elevation of intracellular Ca2+ participate in the activation of NF-kappa 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-kappa 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 Ikappa Balpha in a TNF-alpha /IL-1beta receptor-independent manner. In contrast to S. typhimurium, EPEC-induced activation of NF-kappa 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-kappa 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.


    MATERIAL AND METHODS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa 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-alpha and IL-1beta receptors. Cytokine-mediated pathways were stimulated by treatment of intestinal epithelial cells with TNF-alpha (10 ng/ml) or IL-1beta (5 ng/ml). To assess Ikappa Balpha degradation, cells were treated for 15, 30, 45, and 60 min. Proteins were extracted, and the effect on Ikappa Balpha degradation was analyzed by immunoblot as described below. TNF-alpha or IL-1beta receptor activation by their respective ligands was blocked by preincubation of cells for 1 h with blocking antibody against either the TNF-alpha (6 µg/ml) or IL-1beta receptor (1 µg/ml) (R&D Systems, Minneapolis, MN). The cells were then stimulated with TNF-alpha or IL-1beta for the indicated times. Proteins were extracted and analyzed for Ikappa Balpha 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 Ikappa Balpha (Santa Cruz Biotechnology, Santa Cruz, CA) or phosphospecific Ikappa Balpha (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 beta -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-alpha 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Infection of intestinal epithelial cells by EPEC induces phosphorylation/degradation of Ikappa Balpha . 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-kappa B (36). NF-kappa B activation is preceded by phosphorylation and degradation of the inhibitory molecule Ikappa Balpha . Increased Ikappa Balpha 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. Ikappa Balpha 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 Ikappa Balpha degradation, suggesting that EPEC stimulates this classic pathway (Fig. 1B).


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Fig. 1.   Immunoblot analysis of proteins extracted from intestinal epithelial cells infected with enteropathogenic Escherichia coli (EPEC) using antibodies to phosphospecific and general primary inhibitor of nuclear factor (NF)-kappa B (Ikappa Balpha ). A: Ikappa Balpha phosphorylation (Phosphor.) was detected by 15 min postinfection and peaked at 1 h. Progressive degradation of Ikappa Balpha after EPEC infection was correspondingly demonstrated. B: MG-132 (50 µM), a proteasomal inhibitor, prevented Ikappa Balpha degradation in EPEC-infected cells. C: EPEC strain RN587/1 induces phosphorylation of Ikappa Balpha in a time course similar to that seen with strain E2348/69. D: in contrast, 3 different commensal E. coli strains did not induce significant phosphorylation of Ikappa Balpha .

To determine whether Ikappa Balpha phosphorylation was unique to EPEC strain E2348/69, another wild-type EPEC strain, RN587/1, was tested and similar results were seen (Fig. 1C). In contrast, nonpathogenic human commensal E. coli strains failed to elicit this change (Fig. 1D). These findings confirm our previously published data that pathogenic but not nonpathogenic E. coli activate the NF-kappa B pathway (13).

Degradation of Ikappa Balpha by EPEC is independent of TNF-alpha and IL-1beta receptor activation and de novo protein synthesis. The best-defined pathways leading to phosphorylation and degradation of Ikappa Balpha are those initiated by activation of cytokine-specific membrane receptors, in particular TNF-alpha and IL-1beta receptors. Infection of T84 cells by enteric bacterial pathogens has been shown to increase the expression of both TNF-alpha and IL-1beta proinflammatory molecules (17). We therefore sought to exclude the possibility that paracrine or autocrine responses from EPEC-stimulated TNF-alpha or IL-1beta production could account for activation of the inflammatory cascade. Cells were preincubated with antagonistic antibodies against TNF-alpha or IL-1beta receptors and then infected with EPEC. As shown in Fig. 2A, neither antibody prevented Ikappa Balpha degradation in response to infection. The efficiency of this approach was confirmed by demonstrating that TNF-alpha -induced Ikappa Balpha degradation was prevented by pretreatment with TNF-alpha receptor blocking antibodies (Fig. 2B). These data suggest that EPEC-activated NF-kappa B signaling pathways in intestinal epithelial cells are TNF-alpha and IL-1beta receptor independent and that alternative mechanisms are responsible. To define whether EPEC-induced expression of other cytokines that may secondarily activate NF-kappa 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 Ikappa Balpha 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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Fig. 2.   Blocking antibodies against tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta receptors did not prevent Ikappa Balpha degradation in intestinal epithelial cells infected with EPEC. A: T84 cells were incubated with antibodies against TNF-alpha or IL-1beta receptors before and during infection, and then protein extracts were subjected to immunoblot analysis. These findings suggest that EPEC-induced Ikappa Balpha degradation is independent of TNF-alpha /IL-1beta receptor activation. B: TNF-alpha receptor blocking antibody (TNFalpha R Ab) prevented Ikappa Balpha degradation induced by TNF-alpha . These experiments were performed as a positive control to demonstrate the inhibitory potential of TNF-alpha receptor antibodies. C: inhibition of protein synthesis with cycloheximide did not affect EPEC-stimulated Ikappa Balpha degradation. T84 cells were preincubated with cycloheximide (2 µg/ml) and infected with EPEC. Immunoblot analysis showed that blocking protein synthesis did not prevent EPEC-induced Ikappa Balpha degradation.

Chelation of intracellular Ca2+ has no effect on EPEC-induced Ikappa Balpha degradation or IL-8 expression. Gewirtz et al. (10) have recently demonstrated that S. typhimurium-induced Ikappa Balpha 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-kappa 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 Ikappa Balpha degradation or IL-8 expression (Fig. 3A). In contrast, BAPTA-AM did block Ikappa Balpha degradation and IL-8 production in response to S. typhimurium (Fig. 3B) as was previously shown (10). These data suggest that activation of NF-kappa B and increased IL-8 expression by EPEC are Ca2+ independent.


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Fig. 3.   EPEC- but not Salmonella typhimurium-induced degradation of Ikappa Balpha and upregulation of IL-8 expression is Ca2+ independent. A: intestinal T84 cells were preincubated with the acetoxymethyl ester of 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) and infected with EPEC. Immunoblot analysis showed that Ca2+ chelation did not prevent EPEC-induced Ikappa Balpha degradation or significantly alter EPEC-stimulated IL-8 expression. Each experiment was repeated 3 times, and data represent means ± SE (P = 0.2 for EPEC vs. EPEC/BAPTA-AM). B: S. typhimurium-induced degradation of Ikappa Balpha and IL-8 expression are Ca2+ dependent, as Ca2+ chelation with BAPTA-AM significantly attenuated both events. Each experiment was repeated 3 times in triplicate, and data represent means ± SE (n = 3; *P < 0.001 for S. typhimurium vs. S. typhimurium/BAPTA-AM).

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-kappa 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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Fig. 4.   EPEC induces the phosphorylation and activation of extracellular signal-regulated kinase (ERK)-1/2 in host intestinal epithelial cells. T84 cells were infected with EPEC for 15, 30, 45, 60, and 180 min and then immunoblotted with antibodies against total ERK showing that the concentration of isoforms-1 and -2 remained unchanged in T84 cells after infection (A). Detection of protein-tyrosine phosphatase-1D/SH2-containing phosphatase-2 (PTP1D/SHP2) was performed on the same immunoblot. Densitometric analysis revealed the same ratio (~1:2) between ERK1/2 and PTP1D/SHP2 in all lanes. Phosphorylation of ERK1/2 was seen at 15 min postinfection and remained detectable for up to 1 h. Densitometric analysis suggested that after 30 min of infection, EPEC increased ERK1/2 phosphorylation by 4-fold compared with controls (P = 0.003). B: enzyme activity assays showed that EPEC infection increased ERK1/2 activity 4-fold, corresponding to the phosphorylation data. Data represent the means ± SE (n = 3; *P < 0.01 for EPEC at 45 min vs. control). C: EPEC strain RN587/1 induced phosphorylation of ERK1/2 in a time course similar to that seen with strain E2348/69. D: in contrast, 4 different commensal E. coli strains did not phosphorylate ERK1/2.

ERK1/2 activation by EPEC was demonstrated in two ways. First, phosphorylation of ERK1/2 increased as early as 15 min postinfection and at 30 min was fourfold higher than in uninfected control monolayers (Fig. 4A). Correspondingly, ERK1/2 enzyme activity assays revealed a nearly fourfold peak increase at 45 min postinfection (Fig. 4B). Infection with EPEC strain RN587/1 also induced ERK1/2 phosphorylation with a similar time course (Fig. 4C). Human commensal E. coli strains, however, did not stimulate ERK1/2 phosphorylation (Fig. 4D), suggesting that this is a pathogen-specific response.

EPEC-activated ERK pathways participate in Ikappa Balpha 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 Ikappa Balpha 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 Ikappa Balpha degradation, another MEK1/2 inhibitor, U0126, was used. Figure 5D shows that U0126 significantly suppressed EPEC-induced ERK1/2 phosphorylation and blocked the subsequent Ikappa Balpha degradation. These data support the contention that the ERK arm of the MAP kinase pathway is stimulated by EPEC infection and participates in Ikappa Balpha degradation, which ultimately leads to IL-8 expression.


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Fig. 5.   Inhibition of mitogen-activated protein (MAP) or ERK kinase (MEK)-1 and -2 (PD-98059) blocked Ikappa Balpha degradation in intestinal epithelial cells infected with EPEC. A: immunoblots probed with antibody against Ikappa Balpha show reduced degradation of this molecule in T84 cells infected with EPEC in the presence of these specific inhibitors. B: densitometric analysis of immunoblots, expressed as %Ikappa Balpha degradation, showed that both 5 and 50 µM PD-98059 inhibited this process. C: ERK1/2 inhibitors blocked the EPEC-mediated induction of IL-8 expression. Intestinal T84 cells were preincubated for 1 h with inhibitors of enzymes in the ERK pathway, MEK1 (5 µM PD-98059), or MEK2 (50 µM PD-98059). Each experiment was repeated 3 times in triplicate, and data represent means ± SE (*P < 0.005 for EPEC vs. EPEC + inhibitors). D: similarly, another inhibitor of the ERK pathway, U0126, significantly decreased EPEC-induced ERK1/2 phosphorylation and prevented Ikappa Balpha degradation, suggesting the coupling of these 2 events.

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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Fig. 6.   ERK1/2 inhibitors have no effect on the decrease in transepithelial electrical resistance associated with EPEC infection. T84 cells were preincubated for 1 h with specific inhibitors and then infected with EPEC. Transepithelial electrical resistance was measured before infection and 4 h postinfection. Data are presented as %change in resistance compared with baseline values and represent the means ±SE (n = 4; P = 0.4 for EPEC vs. EPEC + inhibitors).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa B. The regulation of NF-kappa B activation is well studied, the best-defined pathway being that stimulated by cytokines. TNF-alpha , for example, binds to its receptor and recruits cytoplasmic adapter molecules such as NF-kappa B-inducing kinases (NIKs) (42, 46). NIK activates a large (~800 kDa) multiprotein complex Ikappa Balpha kinase (IKK) that then phosphorylates Ikappa Balpha (42, 46). Phosphorylated Ikappa Balpha is ubiquitinated and degraded by 26S proteasomes, freeing NF-kappa B to translocate to the nucleus and initiate gene transcription. Enteropathogenic E. coli induces Ikappa Balpha phosphorylation/degradation in <1 h postinfection, highlighting these as early events. EPEC-stimulated NF-kappa B activation is TNF-alpha and IL-1beta 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-kappa B activation has been demonstrated by showing that a dominant-negative mutant of MEKK1 inhibited NF-kappa B activation, and overexpression of active MEKK1 resulted in the phosphorylation of Ikappa Balpha (23). In addition, MEKK2 and -3 have been shown to activate the NF-kappa B pathway (50), demonstrating communication between ERK1/2 and the NF-kappa B pathway. We show here that ERK is rapidly phosphorylated and activated after EPEC infection and that inhibition of enzymes within this pathway prevents Ikappa Balpha degradation and IL-8 production. These findings suggest that EPEC may trigger an unidentified receptor to stimulate ERK and activate NF-kappa 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-kappa B, suggesting that other signaling pathways initiate the inflammatory response. In contrast, S. typhimurium activates NF-kappa B and upregulates IL-8 production through MAP kinase cascades (16). In this case, however, intracellular Ca2+ is also involved (10). Ca2+-dependent NF-kappa 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-kappa 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-alpha or IL-1beta 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.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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