1Department of Pathology, University of Chicago, Chicago, Illinois 60637; and 2Epithelial Pathology Unit, Department of Pathology and Laboratory Medicine, Emory University, School of Medicine, Atlanta, Georgia 30322
Submitted 25 November 2003 ; accepted in final form 30 January 2004
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ABSTRACT |
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host defense; inflammation; cellular proliferaton
It has recently been observed that interactions between the intestinal epithelium and nonpathogenic Salmonella strains can attenuate the host innate immune response by preventing the ubiquitination of IB
and thus the activation of the NF-
B proinflammatory pathway (17). Although the NF-
B and
-catenin/TCF signaling pathways are independent, both I
B and
-catenin are regulated by phosphorylation at similar consensus NH2-terminal serines (4, 25). Phosphorylation at this site targets both proteins for ubiquitination by the same ubiquitin ligase complex, E3-SCF
-TrCP, ultimately resulting in proteasomal degradation of the ubiquitinated proteins (4, 25). Interestingly, the consequences of E3-SCF
-TrCP regulation of these two pathways can be very different. E3-SCF
-TrCP-mediated degradation of I
B leads to the induction of NF-
B activity and proinflammatory responses, whereas E3-SCF
-TrCP-mediated degradation of cytoplasmic
-catenin inhibits TCF-dependent transcriptional activation (4, 25). It is likely that the nonpathogenic bacterial-induced effect is mediated by inhibition of E3-SCF
-TrCP. In a recent publication (3), it was shown that the blockade of I
B ubiquitination can be attributed to the bacterial effector protein AvrA, which is injected into the host cell by the type III secretory system of nonvirulent Salmonella. AvrA is a novel target of the centisome 63 type III protein secretion system of S. enterica. It shares sequence similarity with YopJ of the animal pathogen Yersinia pseudotuberculosis and AvrRxv of the plant pathogen Xanthomonas campestris pv. Vesicatoria (5). AvrA protein from S. typhimurium inhibits activation of the key proinflammatory NF-
B transcription factor in human epithelial cells. Besides I
B, the other known substrate of the E3 ligase is
-catenin, which has been implicated in epithelial growth control. Thus we hypothesize that bacterial-epithelial interactions that block NF-
B degradation could also influence
-catenin-mediated signaling of epithelial cell growth.
In the present study, we have used the human epithelia HeLa cell model system to examine the effects of nonpathogenic bacteria on the -catenin signaling pathway. HeLa cells provide an excellent model system because they are known to have an intact
-catenin signaling pathway, grow rapidly, and are easy to transiently transfect. In addition, a great deal of literature has been published concerning the
-catenin pathway in HeLa cells, providing a good foundation for comparison. We also used human intestinal cell lines T84, HT29-C19A, and rat small intestinal epithelial cell IEC-18 to further determine our hypothesis. We report here that the nonpathogenic bacteria S. typhimurium PhoPc activates the
-catenin signaling pathway by attenuating
-catenin ubiquitination and enhances cell proliferation by allowing
-catenin-TCF interactions that result in increased c-myc expression. For the first time, we show that Salmonella effector, AvrA, is involved in modulating this
-catenin activation. Our observations suggest that bacterial-host interactions might induce long-term cellular responses in addition to the more transient inhibition of acute inflammatory responses, utilizing bacteria-derived factors to activate host machinery.
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MATERIALS AND METHODS |
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Bacteria strains include wild-type (WT) S. typhimurium ATCC 14028s, S. typhimurium PhoPc, Salmonella PhoPc mutant strain lacking the AvrA gene (PhoPc AvrA), and PhoPc AvrA transcomplemented with a plasmid encoding WT AvrA (PhoPc AvrA/AvrA+). S. typhimurium InvA (an invasion deficient), and Escherichia coli F18 (a flagellated nonpathogenic strain) were used as controls in some experiments. Bacterial growth conditions were as follows: nonagitated microaerophilic bacterial cultures were prepared by inoculating 10 ml of Luria Bertanibroth with 0.01 ml of a stationary phase culture followed by overnight incubation (18 h) at 37°C as previously detailed (10).
Cell culture.
HeLa cells and HT29-C19A were grown in DMEM (high glucose, 4.5 g/l) containing 5% (vol/vol) fetal bovine serum, 50 µg/ml streptomycin, and 50 U/ml penicillin. T84 epithelial cells (American Type Culture Collection, Manassas, VA) were grown in 1:1 DMEM and Ham's F-12 medium supplemented with 15 mM HEPES (pH 7.5), 14 mM NaHCO3, antibiotics, and 5% neonatal calf serum. The rat small intestinal IEC-18 cell line was grown in DMEM (high glucose, 4.5 g/l) containing 5% (vol/vol) fetal bovine serum, 0.1 U/ml insulin, 50 µg/ml streptomycin, and 50 U/ml penicillin. IEC-18 cells were used at 6070% confluence between passages 20 and 30 in 96-well plates for cell proliferation assay.
Immunoblotting of ubiquitinated proteins.
Equal numbers of cells were plated on six-well plates. Cells were colonized as previously detailed (8) with equal numbers of the indicated bacteria in HBSS for 30 min before the addition of 0.5 µM MG262 for the times indicated. Cells were rinsed twice in ice-cold HBSS, and lysed in protein loading buffer (50 mM Tris, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol). Equal volumes of total cell lysis were separated by SDS-PAGE, transferred to nitrocellulose, and processed for immunoblotting with ubiquitin-protein conjugates antibody (Affiniti Research Products, Exeter, UK), and -catenin antibody (Transduction Laboratories). Immune complexes were visualized with the appropriate peroxidase-conjugated antibodies and developed by using an ECL kit (Amersham Biosciences).
Immunoblotting.
Cells were colonized with equal numbers of the indicated bacteria for 30 min, washed with HBSS, and incubated in DMEM containing gentamicin (500 µg/ml) for the times indicated. Cells were rinsed twice in ice-cold HBSS, lysed in a lysis buffer [in mM: 50 Tris, pH 8.0, 150 NaCl, 5 EDTA with a protease inhibitor cocktail and 1% Triton X-100, and complete Mini (1 tablet/10 ml, Roche)], and sonicated. Protein concentrations were measured with Bio-Rad protein assay solution. Equal amounts of total cell proteins were loaded and separated by SDS-PAGE and processed for immunoblotting with -catenin, c-myc,
-actin (H-196) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), or
-actin (AC-15) antibody from Sigma.
Immunostaining.
Cells were incubated with WT S. typhimurium or PhoPc for 30 min, washed, and incubated in medium containing gentamicin for 24 h. As a positive control for -catenin nuclear translocation, some cells were incubated with the proteasome inhibitor 25 µM N-acetyl-leu-leu-norleucinal (Affiniti, UK) for 12 h before staining. Cells were rinsed three times in PBS, fixed for 10 min in 3.7% paraformaldehyde, and then rinsed three times in PBS. The cells were then permeabilized for 10 min with 0.2% Triton X-100 and rinsed three times with PBS containing 10% bovine serum albumin. Permeabilized cells were then incubated with mouse anti-
-catenin antibodies (Transduction Laboratories) for 1 h at 37°C. After cells were stained with primary antibodies, they were rinsed three times with PBS and incubated with the appropriate FITC-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA) for 1 h at 37°C. The sample was covered with 10 µl of SlowFade reagent (Molecular Probes, Eugene, OR) followed by a coverslip, and the edges were sealed to prevent drying. Specimens were examined with a Zeiss LSM410 scanning laser confocal microscope.
EMSA.
Cells were scraped in a lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1 mM DTT) containing a protease-inhibitor cocktail, and were incubated for 15 min on ice. Nuclei were collected by centrifugation at 2,000 g for 5 min at 4°C and resuspended in 50 µl of the same buffer without KCl but with 420 mM NaCl. After a 30-min incubation on ice, nuclear debris were removed by centrifugation at 13,000 g for 10 min at 4°C, and the supernatants were collected and stored at 80°C before use. The DNA sequences of the double-stranded oligonucleotide specific of TCF were 5'-TCGACCCTTTGATCTTACC-3' and 5'-TCGAGGTAAGATCAAAGGG-3' (based on Ref. 9a). Complementary strands were annealed and double-stranded oligonucleotides were labeled with [32P]dCTP by using the Klenow fragment of DNA polymerase (GIBCO-BRL). Five micrograms of nuclear proteins were incubated for 10 min at 4°C in a binding buffer (in mM: 20 Tris·HCl, pH 7.9, 5 MgCl2, 0.5 DTT, and 0.5 EDTA with 20% glycerol) in the presence of 2 µg of poly(dI-dC). The extracts were then incubated for 30 min at 4°C with 10,000 cycles/min of 32P-labeled TCF probes. The samples were loaded on a 5% native polyacrylamide gel and run in 0.5x TBE buffer. The -catenin/TCF specific band was confirmed by competition with a 500-fold excess of the respective unlabeled probe and mouse anti-
-catenin antibody.
TCF transcriptional activity assay.
Cells were transiently cotransfected with 1-µg pGL3-OT (TCF-responsive reporter with WT TCF binding site), or pGL3-OF (mutant TCF binding site) using lipofectin reagent according to the manufacturer's instructions (Life Technology). PRL-TK vector (Promega) was used as an internal control reporter. Cells were colonized with equal numbers of bacteria for 30 min, washed, and incubated in DMEM for 6 h. Luciferase activity was monitored by using the dual luciferase assay system (Promega).
Cell proliferation assays.
Equal numbers of cells were plated on 96-well plates. Cells were colonized as previously detailed (8) with equal numbers of the indicated bacteria in HBSS for 30 min. Cells were rinsed twice in ice-cold HBSS and incubated in DMEM containing gentamicin (500 µg/ml) for 24 h, and then incubated for an additional 4 h in medium containing MTT or 5-bromo-2'-deoxyuridine (BrdU). Proliferation assays were performed according to the manufacturer's specifications for the MTT cell proliferation kit (Roche) and cell proliferation BrdU labeling kit (Roche).
RT-PCR.
Total bacterial RNA was purified with Qiagen RNeasy Mini Kit. Qiagen Onestep RT-PCR kit was used for AvrA and Malate dehydrogenase amplification. Primers for AvrA PCR were designed from the published sequence (GenBank accession no. AE008830), as follows: 5' primers (5'-ACCGCTATCGCTGTTCC-3') and 3' primer (5'-GTATTATTTGCCATCGTGTTTATT-3'). Use of the primers yielded a 1200-bp segment. For amplification of Malate dehydrogenase (2), an 849-bp segment, the PCR primers are as follows: the 5' primers (5'-ATGAAAGTCGCAGTCCTC-3') and the 3' primer (5'-ATATCTYTTCAGCGTATCCAGC-3'). DNA sequencing was used to confirm the identity of the PCR products.
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RESULTS |
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We first investigated whether colonization of epithelial cells with Salmonella would promote an increase in the higher molecular weight ubiquitinated forms of -catenin as has been previously seen for the I
B
protein (17). To be able to visualize ubiquitinated proteins, cells had to be treated with a proteasome inhibitor (MG262 or MG132), because all ubiquitin-modified proteins are targeted for rapid proteasomal degradation, making them inherently short-lived and difficult to capture. Treating cells with the proteasome inhibitor prevents degradation of ubiquitinated proteins and results in the appearance of multiple higher molecular weight forms when probed by immunoblot. HeLa cells were incubated with or without Salmonella for 30 min, washed, and then treated with the proteasome inhibitor MG262 for the times indicated. To prevent protein degradation after cell lysis, cells were lysed directly in SDS-reducing loading buffer (12). A
-catenin immunoblot of equal amounts of total cellular protein revealed that in control cells treated with MG262 alone, ubiquitinated
-catenin persisted during the 3-h incubation as expected (Fig. 1A, MG262, arrowheads). During the same interval, cells colonized with WT Salmonella showed a slight increase in ubiquitinated forms of
-catenin (Fig. 1A). In contrast, cells colonized by the nonpathogenic Salmonella strain PhoPc completely lacked the ubiquitinated
-catenin bands (Fig. 1A). This indicates that PhoPc colonization had blocked
-catenin ubiquitination before any stabilization by the proteasome inhibitor. Using an antibody to ubiquitin-protein conjugates, we also determined that general cellular protein ubiquitination was unaffected by bacterial colonization (data not shown), suggesting that the effect of PhoPc was specific for
-catenin. Our finding in the HeLa cell line is consistent with the previous report from our laboratory (17) in the T84 cell line. When T84 model epithelia were colonized by WT S. typhimurium, followed by the addition of proteasome inhibitor, ubiquitination of
-catenin could also be seen. However, colonization with the anti-inflammatory Salmonellae, again followed by addition of proteasome inhibitor, totally abrogated the appearance of ubiquitinated
-catenin over 3 h (17). These results support our hypothesis that S. typhimurium PhoPc mediate an anti-inflammatory state by inhibition of ubiquitination and could suggest that the effects of this class of bacteria may extend beyond reduction of inflammation and influence
-catenin pathway.
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We then sought to determine whether the observed blockade of -catenin ubiquitination by nonpathogenic Salmonella could also prevent
-catenin degradation. Cells were colonized with bacteria for 30 min, washed, and incubated in medium containing gentamicin to prevent further bacterial growth. We examined
-catenin levels by immunoblot over a 24-h period, and as expected,
-catenin protein levels remained constant in untreated cells and decreased in a time-dependent fashion in cells colonized with WT Salmonella (Fig. 1, B and C). This decrease in
-catenin stability in cells incubated with WT Salmonella correlates nicely with the slight increase in ubiquitinated
-catenin seen in Fig. 1, A and B. In contrast, we saw
-catenin initially decrease from control levels and then rebound in cells colonized with PhoPc (Fig. 1, B and C). Because bacterial colonization has many effects on the cells, we could not directly compare the total
-catenin levels between treated and untreated cells, but rather were interested in whether the levels remained constant (no treatment), decreased (WT), or increased (PhoPc) over time. It is known that the cellular stores of
-catenin are continually being ubiquitinated and degraded at a rate equivalent to that of synthesis, thus maintaining steady-state levels. However, because ubiquitination is inhibited by PhoPc colonization,
-catenin synthesis is able to recover quickly from the initial loss of the previously ubiquitinated
-catenin. We examined
-catenin levels over a 24-h period in the human intestinal T84 cell line. We found the similar trend of
-catenin expression as in HeLa cell line after bacterial colonization. (Fig. 2).
-catenin protein levels remained constant in untreated cells and decreased in a time-dependent fashion in cells colonized with WT Salmonella (Fig. 2, A and B). In contrast, we saw
-catenin initially decrease from control levels and then rebound in cells colonized with PhoPc (Fig. 2, A and B). These data demonstrate that colonization with nonpathogenic Salmonella PhoPc inhibits the
-catenin degradation by attenuating its ubiquitination.
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Our next objective was to determine whether the protective effects of PhoPc colonization on -catenin would alter its cellular distribution. In cells receiving no treatment,
-catenin immunofluorescent staining was mainly localized to the cell periphery at the plasma membrane (Fig. 3). Cells that were colonized by WT Salmonella for 24 h exhibited changes in cell morphology, and
-catenin was not as clearly membrane-localized, but was still excluded from nuclei. However, colonization with nonpathogenic PhoPc induced nuclear translocation of
-catenin (Fig. 3) that was very similar to the positive control cells treated with the proteasome inhibitor ALLN (Fig. 3, bottom). Taken together, these results clearly demonstrate that PhoPc colonization not only stabilizes
-catenin by preventing its degradation but also allows its accumulation and translocation into the nucleus.
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Under normal circumstances, when -catenin binds to the transcription factor TCF and activates target genes, cell proliferation is enhanced. We then sought to determine how cell proliferation would be influenced by bacterial colonization. The MTT cell proliferation assay measures cellular metabolic activity on the basis of the reduction of the tetrazolium salt MTT to a water-insoluble formazan dye by viable cells. Cells were incubated for 30 min with equal numbers of the indicated bacteria and then assayed 24 h later. We found that cell proliferation was enhanced only with nonvirulent Salmonella PhoPc but not by colonization with WT or nonpathogenic Salmonella InvA bacteria (Fig. 5A). Additionally, we also measured BrdU incorporation into newly synthesized DNA to confirm the cell proliferation effects seen with the MTT assay. HeLa cells colonized with PhoPc incorporated as much BrdU as the cells that received no treatment, a level which was approximately eightfold greater than cells colonized with the WT Salmonella in HeLa cell line (Fig. 5B). Human intestinal epithelial HT29-C19A cells colonized with PhoPc incorporated as much BrdU as the control HT29-C19A cells, a level that was approximately fourfold greater than cells colonized with the WT. Because IEC-18 has lower baseline of c-myc expression than HeLa and HT29-C19A cells, it was shown that cells colonized with PhoPc incorporated more BrdU than the HT29-C19A cells without treatment and the cells colonized by the WT Salmonella or the noninvasive InvA. These data demonstrate that PhoPc is able to override the negative effects of bacterial colonization seen in cells incubated with WT or the noninvasive InvA.
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During colonization of epithelial cells, Salmonella injects a number of bacterial effector proteins that usurp host-cell machinery, allowing bacteria to invade the normally nonphagocytic epithelial cell. Whereas many of these effector proteins have been assigned functions, the role of others such as AvrA have yet to be determined. We (3) recently reported that Salmonella effector protein AvrA inhibits the activation of the key proinflammatory transcription factor NF-B by preventing the ubiquitination of I
B
. To determine whether expression of AvrA also mediates the inhibition of
-catenin ubiquitination (Fig. 1), cells were colonized with either Salmonella PhoPc, PhoPc lacking AvrA (PhoPc AvrA), or PhoPc AvrA transcomplemented with a plasmid encoding WT AvrA (PhoPc AvrA/AvrA+). When cells were colonized with PhoPc AvrA, ubiquitinated
-catenin levels similar to cells infected with WT Salmonella were observed (Fig. 6, AvrA). Exposure to PhoPc AvrA/AvrA+ decreased the ubiquitinated
-catenin to levels similar to those seen with PhoPc (Fig. 6). Because the transcomplemented AvrA is not expressed to the same level as endogenous AvrA in PhoPc, we did not expect a complete reversal of ubiquitination in the presence of PhoPc AvrA/AvrA+. These data suggest that the expression of Salmonella AvrA effector in PhoPc is able to prevent the ubiquitination of
-catenin, because its removal restores normal ubiquitination.
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DISCUSSION |
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Our observations presented here suggest that bacterial-host interactions might induce long-term cellular responses in addition to the more transient inhibition of acute inflammatory responses, utilizing bacteria-derived factors to activate host machinery. Signaling through -catenin is controlled by its regulated degradation. In normal cells,
-catenin is constitutively phosphorylated by Gsk-3
kinase, ubiquitinated by E3 ligase, and degraded in the proteasome (Fig. 8A). However, after nonpathogenic bacterial colonization, ubiquitination is inhibited, presumably via AvrA inhibition of the ubiquitin ligase E3 by a currently undetermined mechanism. As a result,
-catenin is not degraded but rather accumulates and enters the nucleus. Once in the nucleus,
-catenin binds to the transcription factor TCF and activates multiple target genes, including c-myc (Fig. 8B). The effects of these target genes have been shown by others (15) to regulate cell proliferation, apoptosis, and cell fate.
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For the first time, we show that Salmonella effector protein AvrA regulates the -catenin/TCF pathway in human epithelia. It is clear that AvrA inhibits ubiquitination, which in turn influence the ubiquitin-mediated proteolysis of
-catenin, perhaps Bcl-2 and p53, which are also regulated by the ubiquitin-proteolysis system (21, 23). Thus our current focus is to understand the mechanism by which AvrA regulates the activity of the E3 ligase and how by doing so, also modulates the stability several important substrates.
PhoP-PhoQ is a two-component regulatory system that controls the expression of over 40 genes essential for S. typhimurium virulence and survival within macrophages. PhoPc is a PhoP-PhoQ constitutive mutation that increases the expression of PhoP-activated genes and represses the synthesis of 20 proteins encoded by PhoP-repressed genes (11). The apparent differences in
-catenin ubiquitination between WT Salmonella and nonpathogenic PhoPc suggest that bacterial genes regulated by the PhoP-PhoQ regulon are involved in the inhibition of
-catenin ubiquitination. Currently, few of the genes controlled by the PhoP-PhoQ regulon have been assigned functions (reviewed in Ref. 7), and it is not known whether any of them may regulate AvrA; however, this is an active area of investigation.
In summary, we have established that nonpathogenic Salmonella do influence -catenin signaling. Using epithelial model systems, we have demonstrated that S. typhimurium PhoPc requires AvrA expression to inhibit the constitutive ubiquitination and degradation of
-catenin. This bacterial-induced inhibition allows
-catenin/TCF stimulated expression of c-myc and enhances cell proliferation. Although the exact mechanism of AvrA action is not known, further characterization of AvrA may provide insight into how aberrant signals from nonpathogenic gut bacteria may lead to long-term changes in cell proliferation and possibly oncogenesis. These questions are currently being examined in polarized epithelial cell model systems.
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GRANTS |
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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