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Am J Physiol Gastrointest Liver Physiol 291: G959-G968, 2006. First published June 15, 2006; doi:10.1152/ajpgi.00274.2005
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HORMONES AND SIGNALING

Rapid activation of Na+/H+ exchange by EPEC is PKC mediated

Kim Hodges, Ravinder Gill, K. Ramaswamy, Pradeep K. Dudeja,* and Gail Hecht*

Section of Digestive Disease and Nutrition, Department of Medicine, University of Illinois at Chicago and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois

Submitted 15 June 2005 ; accepted in final form 7 June 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Enteropathogenic Escherichia coli (EPEC) increases sodium/hydrogen exchanger 2 (NHE2)-mediated sodium uptake by intestinal epithelial cells in a type III secretion-dependent manner. However, the mechanism(s) underlying these changes are not known. This study examines the role of a number of known secreted effector molecules and bacterial adhesins as well as the signaling pathways involved in this process. Deletion of the bacterial adhesins Tir and intimin had no effect on the increase in sodium/hydrogen exchanger (NHE) activity promoted by EPEC infection; however, there was a significant decrease upon deletion of the bundle-forming pili. Bacterial supernatant also failed to alter NHE activity, suggesting that direct interaction with bacteria is necessary. Analysis of the signal transduction cascades responsible for the increased NHE2 activity during EPEC infection showed that PLC increased Ca2+, as well as PKC{alpha} and PKC{epsilon} were involved in increasing NHE activity. The activation of PKC{epsilon} by EPEC has not been previously described nor has its role in regulating NHE2 activity. Because EPEC markedly increases NHE2 activity, this pathogen provides an exceptional opportunity to improve our understanding of this less-characterized NHE isoform.

sodium/hydrogen exchanger; enteropathogenic Escherichia coli; protein kinase C; epsilon; alpha

ENTEROPATHOGENIC Escherichia coli (EPEC) is a cause of severe diarrheal illness associated with high infant mortality. Despite extensive research, the pathophysiology of EPEC-induced diarrhea is not yet fully understood. Unlike the diarrhea associated with Vibrio cholerae or other types of E. coli infection, that associated with acute EPEC infection is neither toxin mediated nor a result of electrogenic Cl– secretion (20). Several factors that influence fluid accumulation and ion imbalance during EPEC infection have been previously described; these include the disruption of tight junctions with alteration in paracellular permeability and increased polymorphonuclear leukocyte transmigration (38, 55, 59). Additionally, prolonged EPEC infection leads to an increase in expression of the receptor for galanin-1, a neuropeptide that regulates Cl– secretion and fluid accumulation within the intestinal lumen (21, 42). Although all of these factors contribute to the development of EPEC-induced diarrhea, the onset of diarrhea occurs more rapidly than would be predicted for any of these factors.

In some pathogens, early-onset diarrhea is due to alterations in ion transport including ion channel activation. The most commonly cited example is the effect of cholera toxin on electrogenic Cl– secretion through the activation of cystic fibrosis transmembrane conductance regulator (CFTR). Although acute EPEC infection does not stimulate electrogenic Cl– secretion, a decrease in electroneutral NaCl absorption in response to EPEC infection could also result in diarrhea. Our studies suggest that both electroneutral Na+/H+ and Cl–/OH– exchange activities are severely altered at early time points after EPEC infection (18, 19). An increase in sodium/hydrogen exchange (NHE) activity occurs as early as 20 min postinfection (19). The net increase in apical NHE activity is due to effects on NHE2, which opposes a concomitant decrease in NHE3 activity (19). In parallel assays, we noted that DIDS-sensitive Cl–/OH– exchange activity was significantly decreased after EPEC infection (18). The uncoupling of NHE activity from that of apical anion exchange has the potential to create alterations in Na+ and Cl– concentration in addition to alterations in pH without altering short circuit current.

To better understand the processes underlying our reported alterations in ion exchange processes, we sought to determine the signal transduction cascades that contribute to the increase in total NHE activity and to identify the specific bacterial protein(s) responsible for the increased Na+ uptake. Our previous studies showed that the increase in NHE activity was dependent on a functional type III secretion system (TTSS) (19). In this study, we assessed the roles of six secreted effector proteins on NHE activity. These proteins include EspF, known to disrupt tight junctions (47); EspH, which alters pedestal morphology and filopodia formation (63); EspG and its homolog from the gene orf3, which disrupt microtubules and produce subtle alterations in barrier function (14, 45, 61); Map, which alters mitochondrial membrane potential (32); and the translocated intimin receptor (Tir), the primary factor mediating intimate adherence and pedestal formation (30). After injection via TTSS, membrane-associated Tir forms a hairpinlike receptor for the bacterial outer membrane protein intimin leading to actin rearrangements necessary for intimate adherence. In addition to Tir-intimin interactions, EPEC also adhere to host cells via bundle-forming pili (BFP) (16). This pilus-mediated attachment is thought to play an important role in initial attachment of the bacteria to host cells (57, 60).

After EPEC infection, a number of signal transduction pathways are activated including phospholipase C-{gamma} (PLC) (28, 31), protein kinase C{alpha} (PKC{alpha}) (9, 39), myosin light chain kinase (MLCK) (66), and the small G-proteins Cdc42 (63) and Rho (45). EPEC also inhibits the activity of phosphoinositide-3 kinase (PI3K) (6). A number of these proteins alter NHE activity. For example, PKC activation increases NHE1 and NHE2 activity but decreases NHE3, a pattern similar to that induced by EPEC infection (29). In the case of NHE3, PLC acts in concert with PKC to downregulate activity (50). Cdc42 and Rho activate NHE1 via distinct pathways (22). Herein, we describe our findings that further dissect the effects of EPEC infection on sodium uptake and the signal transduction mechanisms involved in this process.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
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Materials. Radionuclide 22Na was obtained from Perkin-Elmer (Boston, MA). Caco-2 cell lines were obtained from American Type Culture Collection (Manassas, VA) or from Dr. Jerrold Tuner (University of Chicago, Chicago, IL). HOE-694 was received from Dr. Hans-J. Lang (Aventis, Pharma Deutschland; Chemical Research; Frankfurt/main, Germany). All of the chemical and peptide inhibitors used in this study were from Calbiochem (San Diego, CA). All other chemicals were of at least reagent grade and were obtained from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).

Cell culture. Caco-2 cells were grown at 37°C in a 5% CO2 environment. The culture medium consisted of high-glucose DMEM, 10% fetal bovine serum, 20 mM HEPES, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were used for experiments at day 7–10 postplating. The night before infection, cells were placed into serum-free and antibiotic free T84 media consisting of a 1:1 mixture of DMEM and F-12 media supplemented with 14 mM NaHCO3, 15 mM HEPES, and 5% mannose.

Bacterial culture and cell infection. Insertion mutants are denoted with appropriate antibiotic resistance and those with {Delta} are deletions whereas those mutants with both {Delta} and an antibiotic resistance are gene replacements. The following EPEC strains and mutants were used (Table 1): wild type (wt) EPEC strain E2348/69, {Delta}espF- orf32::Km (UMD874) (46), espG (Km) (SE1114) (14), SE896 (Km) (tir Km insertion, expresses the NH4 terminus of Tir, created by suicide vector) (13), JAC719 (Cm) ({Delta}tir, full deletion/Cm replacement, created by suicide vector) (10), KC14 ({Delta}tir, full deletion/Cm insertion, created by {lambda} Red method) (4), eae (intimin) (CVD206) (11), JPN15 (EAF–) (25), espH (Km) (SE874), escN (Km) (CVD452) (24), and map (Km) (SE882) provided by Dr. James Kaper (University of Maryland). KC14+Tir was recreated in our laboratory by using KC14 and the plasmid pKC17 (4). Strains were grown overnight in the presence of appropriate antibiotics. On the day of experimentation, 30 µl of bacterial culture were transferred per 1 ml of serum- and antibiotic-free T84 cell culture medium supplemented with 0.5% mannose; 300 µl of overnight culture was used to inoculate 10 ml of T84 media. Bacteria were grown ~3 h to an OD600 of 0.4. Cultures were centrifuged at 2,500 rpm for 5 min in a Beckman GS-6 centrifuge and resuspended in a 10-ml volume of fresh serum-free T84 media. Caco-2 cells on 24 well plates were inoculated with 200 µl of this solution per well for 30 min followed by a PBS wash to remove nonadherent bacteria.


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  		Table 1. Bacterial strains

 
Inhibitor use. Unless otherwise indicated, cells were treated with either inhibitor or vehicle control for 1 h before infection and new inhibitor was added at the time of infection and during acid load. Peptide inhibitors were added only before and not during infection. PKC{alpha} peptide was myristoylated and therefore cell permeable and was applied to cells for 1 h before infection. PKC{epsilon} inhibitor peptide is not cell permeable, and therefore cells required permeabilization on ice and subsequent healing of the membrane following the method of Johnson et al. (27). When PKC{alpha} and {epsilon} inhibitors were used together, the cells were permeabilized and treated with PKC{epsilon} inhibitor as usual, and then cells were treated with PKC{alpha} inhibitor peptide during the 30-min recovery period in conditioned media at 37°C.

Assay of Na+/H+ exchange. NHE activity was determined as the EIPA-sensitive 22Na+ uptake as previously described (15). NHE2 specific activity was determined as the HOE-694 (50 µM)-sensitive portion of 22Na+ uptake. NHE1 activity was not detectable in cells cultured on 24-well plastic plates rather than permeable supports on the basis of previous experiments. After infection, confluent cell monolayers were incubated for 30 min at room temperature in acid load solution containing 50 mM NH4Cl, 70 mM choline chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, and 15 mM MOPS (pH 7.0). The cells were then washed with 120 mM choline chloride and 15 mM Tris-HEPES, pH 7.5 before sodium uptake. The 5 min of 22Na+ uptake were carried out in a solution containing 3 mM NaCl, 110 mM choline chloride, 1 mM MgCl2, 2 mM CaCl2, 20 mM Tris-HEPES (pH 7.4), and 1 µCi/ml of 22Na+, with or without 50 µM EIPA or 50 µM HOE-694. Cells were then washed twice with ice-cold PBS. The cells were then lysed overnight by incubation with 0.5 M NaOH. Radioactive sodium was then quantified using a Packard TR1900 scintillation counter. The protein content of cell lysates was determined by the method of Bradford (3). 22Na+ uptake was expressed as nanomoles per milligram protein per 5 min.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Intimate adherence is not necessary for increased NHE activity by EPEC. Intimin is the binding partner for the injected effector protein Tir, and mutation of either tir or eae, the intimin gene, results in a loss of intimate adherence. Tir is the primary effector involved in actin pedestal formation, and a number of actin-binding/regulatory proteins are recruited to facilitate this process. Although Tir-intimin interactions are important for intimate attachment, the overall adherence rate in vitro is not decreased in an intimin mutant (53). Intimin mutation had no effect on the NHE phenotype compared with wt EPEC (Fig. 1A). The BFP are the primary adhesin in vitro and during early infection, and BFP mutants are severely impaired for attachment (16). Strain JPN15, which lacks the EAF plasmid that encodes for BFP, caused significantly less stimulation of NHE activity compared with that of wt EPEC (Fig. 1A). Attachment to host cells via BFP has been shown to be required for effective delivery of TTSS effector molecules across the host cell membrane (16, 65). Impaired TTSS effector delivery explains the reduced impact of the BFP mutant JPN15 on NHE activity. Typically nonadherent EPEC cannot deliver TTSS secreted effector molecules, unlike the closely related diffusely adhering E. coli, which secrete effectors into the media and can form translocation pores independently of attachment. Considering the poor adherence of JPN15 and the known decrease in TTSS-mediated delivery of effectors, we were surprised that alterations in NHE activity were not more impaired; therefore, we considered the possibility that the secreted TTSS effectors in the bacterial supernatant might have an effect without having to be translocated across the host membrane. Comparisons of filtered supernatant from a log phase culture grown as for infections for wt or escN, a TTSS defective mutant that does not secrete effectors into the media, showed no increase in activity compared with uninfected controls (Fig. 1B). These data suggested that the effector protein responsible for the NHE phenotype requires translocation into host cells. Therefore, disruption of either intimate adherence or BFP-mediated adherence alone does not fully prevent the delivery of TTSS effector molecules into host cells.


Figure 1
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  		Fig. 1. Human colonic epithelial cells, Caco-2, were infected with enteropathic Escherichia coli (EPEC) for 30 min followed by a 30-min period of acid load allowing for a total of 1 h of infection. Total sodium-hydrogen exchanger (NHE) activity is described as the EIPA-inhibitable portion of 22Na+ uptake expressed as nanomoles per milligram protein per 5 min. In A and C, the NHE activity has been described in terms of the percentage of NHE activity relative to wild-type (wt) EPEC. A: disruption of intimate adherence through mutation of eae, the gene for intimin, did not prevent the increase in NHE activity; however, preventing early attachment events mediated by bundle-forming pili in the JPN15 mutant caused a small but significant drop in NHE activity compared with wt EPEC controls. B: filtered bacterial supernatants (sup) had no effect on NHE activity, suggesting that direct bacterial contact is necessary. Supernatants from wt EPEC were comparable to those from an escN mutant, which is incapable of secreting type III effector molecules into the media because escN encodes for a putative ATPase that drives type III secretion. C: a number of EPEC mutant strains were tested to determine whether individual type III secretion system (TTSS) effectors play a role in the increase in NHE activity seen with EPEC infection. Infection of cells with espF, espH, espG, or map mutants increases NHE activity to levels comparable to wt EPEC infection. Data represent an n of 4 performed in triplicate, with the exception of B, which is an n of 2 in triplicate. *Significantly different from wt EPEC by Student’s t-test, P < 0.05.

 
Secreted effector molecules EspG, Orf3, EspH, EspF, and Map are not responsible for the EPEC-induced increase in NHE activity in human intestinal epithelial cells. Our earlier studies showed that total NHE activity was not altered by nonpathogenic E. coli or EPEC mutants that prevent type III secretion including EscN, a putative ATPase, or EspB and EspD, which combine to form the pore through which effector molecules are injected into the host cell (19). From these data it was unclear whether structural components of the TTSS or the injected effector molecules stimulated NHE activity. EPEC produces a number of secreted effector molecules that are translocated into eukaryotic cells; these include EspF, EspG, EspH, EspI, Tir, Cif, Map, Orf3, and EspB. Although the complete set of secreted effector molecules encoded within the locus of enterocyte effacement (LEE) pathogenicity island is believed to have been described (63), additional effector molecules encoded outside of the LEE are still being discovered, making the task of testing every potential effector molecule difficult. In previous work, EspB was found to be necessary to increase NHE activity; however, EspB is both a structural component of the TTSS that combines with EspD to form the pore in the host cell membrane and a secreted effector molecule that is injected into the host cell cytoplasm. Cif was not included in this study because only a partial fragment of the cif gene is present in our wild-type strain E2348/69 (41). Mutation of espF, involved in barrier disruption, espG or orf3, involved in microtubule loss, espH, involved in pedestal elongation and filopodia formation, or map, which alters mitochondrial membrane potential, had no effect on EPEC-induced increase in NHE activity (Fig. 1C). EspG and orf3 were tested as a double knockout since they are highly homologous and can compensate for one another in microtubule disruption; however, mutation of both genes did not prevent the increase in NHE activity in response to infection.

An unidentified effector promotes increased NHE activity. During the course of our experiments we found one mutant that failed to increase NHE activity, the {Delta}tir mutant KC14 (Fig. 2A). However, although complementation with tir, cesT, a chaperone for Tir, and eae, the members of the tir operon, restored pedestal formation (data not shown), the NHE phenotype was not complemented (Fig. 2B, KC14+Tir). In addition, two separate tir mutants created in a different manner, SE896 and JAC719, increased NHE activity like wt EPEC (Fig. 2A). The mutant KC14 was created using the {lambda} Red method, which is known to promote secondary mutations outside of the region being targeted (49). The {lambda} Red method consists of phage gene expression that stabilizes linear DNA, allowing PCR products to be used directly for creating deletion mutants. Although this is a random mutation that cannot be easily identified, these data support our hypothesis that a currently unidentified effector molecule that was unintentionally mutated in the KC14 background is responsible for the increase in NHE activity.


Figure 2
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  		Fig. 2. Three tir mutants were tested in terms of NHE activity, shown as a percent of wt EPEC infected cell activity. As in other experiments, Caco-2 cells were infected for 30 min followed by a 30-min acid load procedure and 5 min of 22Na+ uptake. A: Tir mutation does not prevent the increase in NHE activity as shown by SE896 and JAC719, which are both tir mutants; however, the tir mutant KC14 does not increase NHE activity, B: complementation of KC14 with the tir operon including tir, cesT, and eae did not restore its ability to increase NHE activity, suggesting a second site mutation. n = 4, performed in triplicate. *Significantly different from wt EPEC by Student’s t-test, P < 0.05.

 
PKC activation is necessary to increase NHE activity. EPEC activates a number of signal transduction molecules including PKC. PKC activation is consistent with the pattern of NHE activity caused by EPEC infection. Specifically, PKC activation causes an increase in NHE1 and NHE2 activity with a concomitant decrease in NHE3 activity, which mirrors that occurring in EPEC-infected cells. Although little is known about the PKC-mediated regulation of NHE2, NHE3 is known to be regulated by PKC{alpha} (34). A number of chemical inhibitors with known specificity for the distinct PKC isoforms were therefore used to identify the responsible PKC isoforms. The class of chemical inhibitors used here functions by competitively inhibiting the ATP-binding domain of PKC isoforms (62).

PKC isoforms are classified as conventional, novel, or atypical. Conventional PKCs such as PKC{alpha}, beta, or {gamma} require both Ca2+ and diacylglycerol (DAG) for activity whereas novel PKCs such as PKC{delta} or {epsilon} require only DAG. The conventional and novel PKC inhibitor bisindolylmaleimide I (BIM, 5 µM) (PKC{alpha}, betaI, {delta}, and {epsilon}) significantly blocked the EPEC-mediated increase in NHE activity, suggesting that PKC is in part responsible (Fig. 3A). However, at the levels of inhibitor necessary to block activity in epithelial cells, there is the inherent possibility that additional kinases can also be blocked. For example, PKA is known to be inhibited by 2 µM BIM. Therefore, we utilized a highly specific PKA inhibitor, KT5720, to determine whether PKA is involved. As shown in Fig. 3B, PKA inhibition had no effect on the EPEC-mediated increase in NHE activity. Another broad-specificity PKC inhibitor, Gö6983 (PKC{alpha}, beta, {gamma}, {delta}, and {zeta}), was used to confirm the data obtained with BIM. As can be seen in Fig. 3C, Gö6983 prevented the EPEC-mediated increase in total NHE activity.


Figure 3
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  		Fig. 3. Inhibition of PKC prevents the increase in NHE activity whereas inhibition of PKA had no effect. PKC inhibitors were used at the concentrations indicated during a 1-h preincubation in addition to during infection and acid load. A: the classical and novel PKC inhibitor bisindolylmalemide I (BIM) (PKC{alpha}, betaI, {delta}, and {epsilon}) was used at a 5 µM concentration and blocked the increase in NHE activity seen with EPEC infection. B: the PKA inhibitor KT5720 (100 µM) had no effect on the increase in NHE activity seen with EPEC infection. C: the broad-spectrum PKC inhibitor Gö6983 (PKC{alpha}, beta, {gamma}, {delta}, and {zeta}) (1 µM) was also capable of inhibiting the increase in NHE activity whereas basal NHE activity levels remained unchanged. *Data where infected cells treated with inhibitor were significantly lower than infected cells treated with DMSO vehicle (P < 0.05). Uninfected cells treated with inhibitor were not statistically different from vehicle-treated controls.

 
Classical PKC inhibitor prevents increased NHE activity. Because BIM and Gö6983 inhibit both novel and conventional PKCs, we used an additional PKC inhibitor, Gö6976, to narrow the class of PKC involved. Gö6976 is a specific inhibitor of conventional PKCs. The effect of EPEC on total NHE activity was significantly decreased by Gö6976, which specifically inhibits PKC{alpha}, betaI, and µ (Fig. 4A). The response pattern to these isoform-selective inhibitors was consistent with the involvement of conventional PKCs in this process, and these data strongly implicate PKC{alpha}, known to be activated by EPEC infection (9, 39). Since conventional PKCs are dependent on both Ca2+ and DAG, with Ca2+ being a unique requirement, we tested whether Ca2+ was required by using BAPTA-AM, an intracellular Ca2+ chelator (Fig. 4B). BAPTA-AM caused 53% reduction in the increase in NHE activity, showing that Ca2+ is required and supporting the conclusion that a conventional PKC isoform is involved in the increased NHE activity associated with EPEC infection.


Figure 4
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  		Fig. 4. PKC{alpha} is implicated in increasing NHE2 activity as seen by the effectiveness of Gö6976, a classical PKC inhibitor, as well as a peptide inhibitor specific to PKC{alpha} and beta and the calcium chelator BAPTA-AM. A: the conventional PKC inhibitor Gö6976 (PKC{alpha}, beta, and µ) (10 µM) was also capable of effectively inhibiting the EPEC mediated increase in NHE activity. B: Ca2+ is required for the increase in NHE activity as shown by inhibition in the presence of the Ca2+ chelator BAPTA-AM. Cells were pretreated with the myristoylated PKC{alpha}/beta pseudosubstrate peptide inhibitor 20–28 at 8 µM for 1 h before infection as well as during infection and acid load. Sodium uptake was quantitated in the presence of either EIPA (50 µM; C) a general NHE inhibitor, or HOE-694 (50 µM; D) preferentially inhibits NHE2, suggesting that PKC{alpha} specifically increases NHE2 activity leading to the increase in total NHE activity. This effect was specific for PKC{alpha} because the PKCbeta inhibitor (C2-4) had no effect on NHE activity (data not shown). *Values where inhibitors significantly decreased NHE activity compared with wt EPEC infected cells (P < 0.05 by Student’s t-test).

 
PKC{alpha} is required to increase NHE2 activity. Our data indicated a role for PKC{alpha} in EPEC-induced activation of NHE2; therefore a myristoylated peptide inhibitor derived from the pseudosubstrate sequence of PKC{alpha} and beta was used to further verify the involvement of this isoform. This peptide inhibitor functions by binding to the substrate-binding domain in the catalytic site of PKC{alpha} rather than the ATP-binding domain as for BIM and its derivates Gö6976 and Gö6983. The pseudosubstrate inhibitor was preincubated with cells for 1 h before infection as well as during infection and acid load. This isoform-specific inhibitor blocked the increase in EPEC-stimulated NHE activity by 51% (Fig. 4C). A PKCbeta inhibitor, C2–4 (52), which prevents RACK1 (receptor for activated C kinase 1) interaction with PKCbeta, had no effect on the increase in NHE activity after EPEC infection, indicating that PKCbeta is not involved. Although we previously attributed the increase in NHE activity to NHE2, we wanted to verify that the PKC-dependent portion of NHE activity was in fact due to NHE2 activity. A similar {alpha}/beta-peptide inhibitor experiment was performed in the presence of HOE-694 (50 µM), which inhibits NHE2 but not NHE3 (Fig. 4D). Similar to the data obtained with EIPA, peptide inhibition of PKC{alpha} blocked the increase in NHE2-specific activity by 43%.

Inhibition of PKC{epsilon} decreases NHE activation. EPEC-induced NHE-2 activation was not completely prevented by inhibition of conventional PKCs; therefore two additional PKC isoforms were tested, PKC{epsilon} because of its known role in altering Cl–/OH– exchange (54) and PKC{zeta} because of its known activation by EPEC. The peptide inhibitor V1-2 inhibits PKC{epsilon} translocation rather than inhibiting activity directly. In contrast to the previously used peptide and chemical inhibitors, V1-2 interacts completely outside of the catalytic domain of PKC, binding to the translocation domain, which is required for correct membrane localization of activated PKC. PKC can have kinase activity, but without correct translocation to the membrane, it is nonfunctional (26). V1-2 blocked NHE activity by 31% at 10 µM and 42% at 100 µM (Fig. 5A and data not shown). Since the effect of EPEC infection on PKC{epsilon} has not been examined, we investigated the impact of EPEC on PKC{epsilon} translocation from the cytosol to the membrane as an indicator of PKC activation. As can be seen in Fig. 5, B and C, PMA and EPEC infection markedly decreased cytosolic PKC{epsilon}. Reciprocally, the membrane-associated PKC{epsilon} increased with either PMA or EPEC treatment. We attempted to inhibit PKC{epsilon} using siRNA; however, the transfection reagent activated PKC{epsilon} in controls negating this as an alternative approach. Knowing that PKC{alpha} and {epsilon} are activated by EPEC infection, we considered the possibility that these two isoforms acted in concert to promote the increased level of NHE activity. Therefore, experiments were performed in the presence of the PKC{alpha} pseudosubstrate peptide or the PKC{epsilon} translocation inhibitor in combination. There was no additive effect from the combination of inhibitors (Fig. 5D), suggesting that PKC{alpha} and {epsilon} do not act in concert to activate NHEs, but instead one protein acts upstream of the other.


Figure 5
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  		Fig. 5. Increase in NHE activity seen during EPEC infection was blocked through use of a specific peptide inhibitor of PKC{epsilon} (V1-2), leading us to determine that PKC{epsilon} is activated during EPEC infection as seen through its translocation to the membrane fraction of infected cells. A: in addition to PKC{alpha}, PKC{epsilon} also plays a role in the increased NHE activity seen during EPEC infection of epithelial cells. The PKC translocation inhibitor V1-2 (100 µM) caused a significant decrease in the NHE activity of infected cells. B: PKC{epsilon} translocates to the membrane fraction of Caco-2 cells after 30 min of infection, as shown by a representative Western blot. PMA is included as a positive control for PKC{epsilon} translocation. C: densitometry for 3 Western blots, a representative of which is shown above in B. D: cells were treated with a scrambled peptide or PKC{epsilon}-specific inhibitor peptide (V1-2) alone or in combination with a peptide specific for PKC{alpha} and then infected with EPEC. As shown, all combinations of specific {alpha}/{epsilon} inhibitors were significantly reduced compared with controls, which were treated with scrambled peptide before infection; however, the combination of {alpha} and {epsilon} inhibitors did not significantly decrease the NHE activity compared with cells treated with either inhibitor alone. This experiment was performed 7 times, in triplicate. Note: there was substantial variation in the effectiveness of different lots of peptides. *P < 0.05 by Student’s t-test of inhibitor treated cells compared with appropriate control cells; in the case of densitometry, * represents those values where the average percentage of 3 bands were significantly (P < 0.05) different than uninfected control values.

 
Activation of PKC is TTSS dependent. Although activation of PKC{alpha} and {epsilon} was clearly required for the increase in NHE activity after infection with EPEC, it was not known whether PKC activation was TTSS dependent. PKC activity and an effector-mediated process could be independent requirements for activation of NHE2; therefore, we directly tested PKC activation by the TTSS defective strain escN (Fig. 6). Although both PMA and EPEC activated PKC{alpha} and {epsilon}, the escN mutant failed to activate either PKC isoform compared with uninfected controls. Therefore, the TTSS or an effector secreted by the TTSS directly activates PKC{alpha} and {epsilon}.


Figure 6
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  		Fig. 6. Activation of PKC{alpha} and {epsilon} is dependent on an intact TTSS. Western blots for PKC{alpha} and {epsilon} in Caco-2 cytoplasmic and membrane fractions at 1 h after infection or treatment with PMA. Active PKC isoforms are mobilized from the cytoplasmic fraction to the membrane fraction as can been seen through stimulation with PMA or infection with wt EPEC. An escN mutant that has a nonfunctional TTSS does not promote translocation or activation of PKC{alpha} and {epsilon}. Data are representative of 3 independent experiments.

 
PLC is involved but not ML-7, Rho, or PI3K. Although the pattern of NHE activity is most consistent with PKC activation, and we have implicated both PKC{alpha} and {epsilon} in this process, a number of additional signal transduction cascades are known to be both activated in EPEC-infected cells and to regulate NHE activity; therefore, we investigated whether any of these alternate pathways played roles in the EPEC-mediated increase in NHE activity. Phospholipase C has been shown to decrease NHE3 activity in concert with PKC downstream from G-protein activation (50). We found that phospholipase C also contributes to the EPEC-mediated increase in NHE activity, although this is most likely through its known downstream activation of PKC as in the case of NHE3 regulation (50). The phospholipase inhibitor ET-18-OCH3 (80 µM) was used on the basis of previous studies carried out with EPEC, and it caused a 50% decrease in the increase in NHE activity after EPEC infection (P < 0.001) (31). In addition to PLC, a number of EPEC-activated kinases and G-proteins are also known to alter NHE activity. PI3K has been shown to increase NHE3-mediated Na+ absorption and EPEC infection is known to block PI3K activation consistent with the decrease in NHE3 activity seen in our previous studies (6, 33). However, inhibition of PI3K with LY29004 had no effect on the increase in total NHE activity seen in Caco-2 cells (data not shown). Both the small G protein Rho and MLCK have been implicated in regulating NHE activity, but inhibition of small G-proteins with the myristolylation inhibitor lovastatin or MLCK with ML-7 had no effect on the increase in NHE activity seen in EPEC infected cells (data not shown).


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 ABSTRACT
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EPEC infection has been shown to alter host physiology in a number of ways. Perhaps the most notable change is the formation of attaching and effacing lesions resulting in the loss of microvilli and disruption of the epithelial barrier promoting paracellular permeability. Despite the contribution of these factors to illness, the early onset of diarrhea (3–4 h after ingestion) has not been explained suggesting that there are additional contributing factors. EPEC does not possess toxins similar to cholera toxin or the heat-stable enterotoxins that promote electrogenic Cl– secretion. Because electrogenic secretion is not implicated in early diarrhea, we opted to study alterations in electrolyte absorption. In previous work, we reported that total NHE activity is increased and that Cl–/OH– exchange activity is decreased, suggesting that the typical balance seen between Na+ and Cl– is perturbed within EPEC infected cells (18, 19). Although Cl– secretion is the most typical form of imbalance leading to fluid accumulation, a reduction in either Na+ or Cl– uptake by a host cell has the same end point in terms of ion imbalance. In the case of Na+, the net increase in NHE activity is expected to be antidiarrheal and may represent an adaptive response by the host cell to counteract decreases in Cl–/OH– exchange and in NHE3 activity or other early events. Alternatively, effectors may modulate NHE2 and NHE3 differentially with the net result being attenuation of diarrhea. In studying the signal transduction cascades and bacterial effectors involved, we have gained further insight into the underlying mechanisms of this cellular response.

Our previous work showed that a functional TTSS is required for the effect of EPEC on NHE activity in Caco-2 intestinal epithelial cells (19). In this study, we ruled out EspG, Orf3, Tir, EspH, Map, and EspF in addition to the adhesin intimin as effectors that can regulate NHE2 activity. Although we were unable to determine the effector responsible for increased NHE activity, the tir mutant strain KC14 was identified as being defective for the phenotype. Multiple attempts at complementing the tir deletion, including the appropriate chaperone and intimin gene, restored Tir functionality as assessed by actin staining, but failed to increase NHE activity. Two additional tir mutant strains had no impact on NHE activity, suggesting that there is a second site mutation in KC14. The {lambda} Red method is known to promote additional mutations if the phage genes are expressed in an unregulated manner as was the case for construction of this strain (49). Because these mutations are random and unmarked, a more global search must be performed to find altered genes. We generated a library using suppression subtractive hybridization, which differentiates between two strains at the whole genome level; however, we were unable to identify a specific mutation in KC14 (data not shown, Ref. 2). This is not entirely unexpected because suppression subtractive hybridization only recognizes deletions and not point mutations or frameshifts. Although the specific secondary mutation in KC14 has not been identified, this strain supports our contention that an unidentified secreted effector protein rather than a structural component of the TTSS is involved in the EPEC-mediated increase in NHE2 activity as this strain has a functional TTSS, secretes the pore-forming components EspB and EspD, and forms actin pedestals upon complementation with tir (data not shown).

When NHE2 is transfected into Chinese hamster ovary cells, PS120 fibroblasts, or LLC-PK1 cells (derived from the proximal tubule), there is an increase in NHE2 activity in response to stimulation with PMA (29, 35, 56). In contrast, NHE2 transfected into the Caco-2 cell derivative C2/bbe was unresponsive to PMA stimulus (48). There are several differences between this study and our own. First, C2/bbe cells are a subclone and represent a small subpopulation of the parental Caco-2 cell line that is heterogeneous and undergoes constant differentiation. Individual Caco-2 subclones are highly diverse and are not always expected to respond in the same manner as the parental Caco-2 line. Second, the C2/bbe cells described were transfected with NHE2 whereas our study looked at only endogenous NHE2. Third, EPEC are a more complex stimulus than PMA and activate several signal transduction cascades. There may be a required contribution of both PKC and an additional stimulatory signal for PKC-mediated increase in NHE2 activity observed in our present studies.

A number of ion exchangers and ion channels are regulated by PKC isoforms. NHE1 and NHE3 are both regulated by PKC{alpha} activity (34, 40). NHE3 is regulated primarily by changing the amount of protein expressed at the cell surface. The level of cell surface protein is determined through fusion of recycling endosomes containing NHE3 or through the stimulation of apical recycling removing NHE3 from the surface (1, 12, 23). This process is mediated in part via PKC{alpha} interaction with E3KARP (NHERF2), a cytoskeletal anchor (34). This type of regulation is unlikely to occur in the case of NHE2 for several reasons. First, NHE2 does not have the E3KARP/actin-mediated linkage described for NHE3 but instead is linked directly to {alpha}-spectrin (8). Second, NHE2 that is removed from the cell surface is targeted to the lysosome rather than a recycling endosome on the basis of lysosomal inhibitor data and cell surface expression (5). Apical Cl–/OH– exchange decreased in response to PKC{epsilon} activation (54). CFTR activity is regulated in response to PKC{epsilon} interactions with NHERF1 (EBP50) and RACK1 and the Na+/K+/2Cl– cotransporter NKCC1 is regulated through an interaction of PKC{delta} with actin (36, 37). In the case of CFTR, PKC regulation is twofold. Phosphorylation of NHERF1 by PKC disrupts the interaction of NHERF1 with CFTR decreasing the open probability of the channel (51). However, direct phosphorylation of CFTR by PKC can be either inhibitory or activating depending on the site phosphorylated (7). Although there appears to be a common theme in regulating ion exchangers and channels through PKC interaction, typically through regulation of cytoskeletal linkage or phosphorylation, the unusual anchoring of NHE2 to {alpha}-spectrin precludes direct comparison with these pathways. However, PKC is known to interact with spectrin as well as the spectrin/actin-associated protein adducin, which contains a myristoylated alanine-rich C kinase substrate-related domain (43, 44, 64). Although its possible that NHE2 activity is altered in response to other PKC-mediated changes within the cell, it is not likely to be due to the decrease in NHE3 activity given that we have seen similar results in both HT-29 and T-84 cells that are derived from colonic crypts and do not express NHE3 (19).

Our data suggest that EPEC inject an unidentified secreted effector molecule into the host cytoplasm that leads to activation of PLC and an increase in Ca2+ levels. Isoform-specific inhibitor data suggest that both PKC{alpha} and {epsilon} are required for the increase in NHE activity and that NHE2 is the specific target. The activation of PKC{alpha} and {epsilon} appears to be sequential rather than additive, and this could reflect activation of another channel or transporter before activation of NHE2 (Fig. 7). Although PKC isoforms are not known to directly activate each other, Hassouna et al. (17) found in the case of potassium-sensitive mitochondrial ATP channels (KATP), that PKC{epsilon} acts upstream of KATP channel opening, which results in PKC{alpha} activation during ischemic preconditioning. Recent data also suggest that the correct targeting of active PKC{delta} requires actin remodeling initiated by PKC{epsilon} (58). PKC isoforms including PKC{epsilon} translocate to the membrane of cells during activation. Translocation data showed that EPEC induces activation of PKC{epsilon} as early as 30 min and continued to increase for at least 1 h, a time equivalent to the assay of NHE activity. This is consistent with the initial onset of increased NHE activity by 20–30 min (18). In addition, the isoform-specific involvement of both PKC{alpha} and {epsilon} with NHE2 activity has not been previously reported as only limited work has been carried out on the regulation of NHE2, in contrast to NHE3. These studies of sodium transport shed light not only on the complex processes underlying EPEC-mediated disease but also on the signal transduction cascades being activated within infected epithelial cells and the ensuing changes in downstream target molecules such as NHE2.


Figure 7
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  		Fig. 7. Proposed model of the effect of EPEC on NHE2 stimulation. EPEC attach to host epithelial cells and utilize the TTSS to inject effector molecules. An unidentified effector molecule activates PLC and Ca2+ which assist in the activation of PKC{epsilon} and PKC{alpha}. Here we hypothesize that PKC{epsilon} acts upstream from PKC{alpha} through an unknown intermediate, potentially another ion exchanger, and that PKC{alpha} directly activates NHE2.

 

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These studies were supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-050694 (G. Hecht), DK-68324 (P. K. Dudeja), DK-54016 (P. K. Dudeja), DK-33349 (K. Ramaswamy), and DK-67990 (K. Ramaswamy) in addition to Veterans Affairs Merit Review Awards (G. Hecht, P. K. Dudeja, and K. Ramaswamy).


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Hecht, Digestive Diseases and Nutrition, Univ. of Illinois, Clinical Science Bldg. (MC 716), #704, 840 S. Wood St., 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.

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