Crosstalk between NF-
B and
-catenin pathways in bacterial-colonized intestinal epithelial cells
Jun Sun,1
Michael E. Hobert,1
Yingli Duan,1
Anjali S. Rao,1
Tong-Chuan He,2
Eugene B. Chang,3 and
James L. Madara1
1Department of Pathology, 3The Inflammatory Bowel Disease Research Center, Department of Medicine, The University of Chicago, and 2Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, Illinois
Submitted 16 November 2004
; accepted in final form 8 March 2005
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ABSTRACT
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Salmonella-epithelial cell interactions are known to activate the proinflammatory NF-
B signaling pathway and have recently been found to also influence the
-catenin signaling pathway, an important regulator of epithelial cell proliferation and differentiation. Here, using polarized epithelial cell models, we demonstrate that these same bacteria-mediated effects also direct the molecular crosstalk between the NF-
B and
-catenin signaling pathways. Convergence of these two pathways is a result of the direct interaction between the NF-
B p50 subunit and
-catenin. We show that PhoPc, the avirulent derivative of a wild-type Salmonella strain, attenuates NF-
B activity by stabilizing the association of
-catenin with NF-
B. In cell lines expressing constitutively active
-catenin, I
B
protein was indirectly stabilized and NF-
B activity was repressed after wild-type Salmonella colonization. Accordingly, constitutively active
-catenin was found to inhibit the secretion of IL-8. Thus our findings strongly suggest that the crosstalk between the
-catenin and NF-
B signaling pathways is an important regulator of intestinal inflammation.
bacteria; inflammation; nuclear factor-
B; interleukin-8
IT HAS BECOME INCREASINGLY apparent that a dynamic molecular crosstalk exists between the microflora of the gut and mucosal epithelial cells. Patients with inflammatory bowel disease (IBD) have higher amounts of bacteria attached to their epithelial surfaces than do healthy people. These bacteria are from diverse genera, and some of them, especially bacteroides, were identified within the epithelial layer, in some instances, intracellularly (12, 37). Evidence indicates that IBD results from aberrant immune activation of the intestinal mucosa by normal, usually commensal, host bacterial flora, and/or their products (29, 32). Compelling evidence for this hypothesis can be found in a number of murine strains engineered to lack immune regulators (IL-2, IL-10, etc.). Such mice are symptom free when raised in germ-free conditions, but they will develop acute inflammatory colitis when bacterial flora is introduced (27, 28, 31, 34), thus indicating that bacteria or their products act as inflammatory-inciting events in the background of immune dysregulation.
Enteric pathogens like Salmonella typhimurium cause acute intestinal inflammation by activating the NF-
B pathway, which requires the ubiquitination and degradation of the inhibitory molecule I
B
. A previous report (21) has demonstrated that PhoPc, the avirulent derivative of wild-type (WT) Salmonella strain 14028s, is able to attenuate the host innate immune response by preventing ubiquitination of I
B
. It appears that this effect is mediated by a Salmonella effector protein that prevents I
B
ubiquitination by the ubiquitin ligase E3. Whereas NF-
B has been intensely studied for its involvement in immunity and inflammation (25), this transcription factor is also known to play an important role in cell proliferation, apoptosis, and cell migration (3, 10, 11, 41). Genes known to be regulated by NF-
B include proinflammatory cytokines (IL-1
, IL-2, IL-6, TNF-
, and IFN-
), chemokines (IL-8, MCP-1), adhesion molecules (E-selectin, VCAM-1, and ICAM-1), antimicrobial peptides, FAS, TRAF1, COX2, and matrix metalloproteinase (16, 42). Therefore, it is not surprising that NF-
B has been shown to be involved in several types of cancer (2, 10, 11, 41).
In addition to I
B
, another well-characterized protein that undergoes ubiquitination by the E3 ligase is
-catenin. It plays an essential role as transducer of the Wnt signaling pathway and is also an important component of the cadherin cell-adhesion complex. In the absence of a Wnt signal,
-catenin is rapidly degraded in the proteasome. Wnt stimulation leads to the stabilization of
-catenin, which is subsequently translocated into the nucleus and activates genes involved in cell-cycle regulation. The importance of
-catenin signaling in colorectal carcinogenesis was first suggested by the protein's association with the adenomatous polyposis coli (APC) tumor suppressor. This was further highlighted by the discovery of oncogenic mutations of the
-catenin genes in the majority of colon cancers that contain WT APC (35). In the vast majority of human colorectal cancers, loss-of-function mutations in APC or oncogenic mutations in
-catenin lead to stabilization of the
-catenin protein. Once in the nucleus,
-catenin binds to the transcription factor LEF/TCF and activates multiple target genes, such as c-myc (14), cyclin D1 (33, 38), and matrix metalloproteinase 7 (MMP-7) (3), all of which help to modulate the proliferation and differentiation of epithelial cells (44). Therefore,
-catenin is also involved in several types of cancer (24, 26, 30).
We have recently demonstrated that bacterial epithelial cell interactions influence
-catenin signaling in a previously unsuspected manner (36). PhoPc is a PhoP-PhoQ constitutive mutation of a WT Salmonella strain that increases the expression of PhoP-activated genes and represses the synthesis of
20 proteins encoded by the PhoP-repressed genes (20). PhoPc activates the
-catenin signaling pathway of human epithelia via a blockade of
-catenin degradation. The normal
-catenin ubiquitination necessary for constitutive
-catenin degradation is abolished, allowing the accumulation and translocation of
-catenin into the nucleus. Transcriptional activation mediated by the
-catenin/TCF complex increases c-myc expression and enhances cell proliferation. 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 and are targeted for ubiquitination by Skp1-Cdc53-F-box receptor E3 ubiquitin ligase (SCF
-TrCP E3) complex, followed by proteasomal degradation. However, the consequences of this SCF
-TrCP E3 regulation are very different. Although the E3 ligase-mediated degradation of I
B
leads to the induction of NF-
B activity, it also mediates the degradation of
-catenin and inhibits the activity of the Wnt pathway (9, 43). In addition, glycogen synthase kinase-3
(GSK-3
) contributes to the degradation of
-catenin and represses
-catenin/TCF signaling, but the activity of NF-
B is enhanced by GSK-3
(15, 22). The I
B kinase (IKK) complex that phosphorylates I
B
contains three family members: IKK
, IKK
, and IKK
. Recent studies show that IKK
can function independently and also phosphorylate
-catenin, thus contributing to the regulation of
-catenin-mediated TCF-dependent gene transcription (1). In addition, both NF-
B and
-catenin pathways drive the proliferation of epithelial cells depending on c-myc and cyclin D1 expression (4, 16, 23). Deng and colleagues (7) first suggested that
-catenin interacted with and inhibited NF-
B in human colon and breast cancer cells. They found that
-catenin could physically complex with NF-
B, resulting in a reduction of NF-
B DNA binding, transactivation activity, and target gene expression (7). In a more recent report (8), the activity of the APC/GSK-3
complex, working through
-catenin, may cross-regulate the NF-
B signaling pathway.
Because S. typhimurium PhoPc both inhibits activation of the proinflammatory transcription factor NF-
B and regulates the
-catenin pathway in human epithelial cells, we hypothesized that a potential crosstalk exists between the NF-
B and
-catenin/TCF pathways during bacterial-epithelial cell interaction. In the present study, we used the human intestinal epithelia cell lines T84, HT29-C19A, HCT116 CTNNB1WT/
45, and its
-catenin knockout derivative lines HCT116 CTNNB1WT/ and HCT116 CTNNB1/
45 to test our hypothesis. We also used the nontransformed rat intestinal epithelial cell line IEC-18 to further test our hypothesis. We report here that there is a direct molecular crosstalk between the NF-
B and
-catenin signaling pathways, convergence of which is a result of the direct interaction between the NF-
B p50 subunit and
-catenin. Colonization with the nonvirulent Salmonella strain PhoPc showed attenuated NF-
B activity and stabilization of the association between
-catenin and NF-
B. To demonstrate the direct importance of
-catenin, cell lines expressing constitutively active
-catenin were used. These cell lines showed indirect I
B
stabilization and repressed NF-
B activity even after WT Salmonella colonization. Additionally, we have found that stabilization of
-catenin resulted in a reduction of NF-
B DNA binding and almost completely abolished Salmonella-induced IL-8 secretion. Taken together, our data strongly suggest that the Salmonella-induced proinflammatory NF-
B pathway is cross-regulated by the activity of
-catenin.
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MATERIALS AND METHODS
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Bacterial strains and growth conditions.
Bacteria strains included WT S. typhimurium ATCC 14028s and S. typhimurium mutant PhoPc. Bacterial growth conditions were as follows: nonagitated microaerophilic bacterial cultures were prepared by inoculating 10 ml of Luria-Bertani broth with 0.01 ml of a stationary phase culture followed by overnight incubation (
18 h) at 37°C, as previously described (19).
Cell culture.
HCT116 CTNNB1WT/
45 and its
-catenin knockout derivative lines HCT116 CTNNB1WT/ and HCT116 CTNNB1/
45 (kindly provided by Kenneth Kinzler and Bert Vogelstein) (5) were cultured in McCoy's 5A medium supplemented with 10% (vol/vol) fetal bovine serum. 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. HT29-C19A cells were seeded on collagen-coated, permeable polycarbonate filters (0.4-µm pore size) with surface areas of 5 or 45 cm2 (Costar, Cambridge, MA). 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% fetal bovine serum. T84 cells were seeded on collagen-coated, permeable polycarbonate filters (0.4-µm pore size). 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.
Immunoprecipitation.
Cells were rinsed twice in ice-cold HBSS and lysed in cold immunoprecipitation buffer [1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, pH 8.0, 0.2 mM sodium ortho-vanadate, protease inhibitor cocktail (Boehringer-Mannheim)]. Samples were precleared with protein A-agarose. Precleared lysates were then incubated with 2 µg of anti-p50 (Santa Cruz Biotechnology) for 1 h at 4°C. A 50% slurry of protein A-agarose (Life Technologies) was added to the lysate and incubated for 30 min with agitation at 4°C, then washed with cold immunoprecipitation buffer. The pellet was resuspended in 0.1 M glycine (pH 2.5) and incubated with agitation for 10 min at 4°C, then centrifuged at 9,000 g for 2 min at 4°C. The supernatant was removed and neutralized with 1 M Tris (pH 8.0). Concentrated electrophoresis (5x) sample buffer (125 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2%
-mercaptoethanol) was added to each sample, boiled for 5 min, separated by SDS-PAGE, and transferred to nitrocellulose. Blots were probed with anti-
-catenin Ab (Transduction Laboratories), stripped, and reprobed with anti-p50, anti-I
B
(Santa Cruz Biotechnology), and anti-
-catenin (Zymed) antibodies and visualized by enhanced chemiluminescence (ECL).
Immunoblotting.
Cells were rinsed twice in ice-cold HBSS, lysed in protein loading buffer (50 mM Tris, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol), and sonicated. Equal volumes of total cell lysis were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-
-catenin (Transduction Laboratories), anti-I
B
(Santa Cruz Biotechnology), or
-actin (Sigma) antibodies, and visualized by ECL.
Immunostaining.
HCT116 Cells were incubated with or without S. typhimurium for 30 min, washed, and incubated in medium containing gentamicin for 24 h. 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 Ab (BD Transduction Laboratories) antibodies and goat anti-NF-
B p50 Ab (C-19; Santa Cruz Biotechnology) for 1 h at 37°C. Cells were rinsed three times with PBS and incubated with the appropriate Alexa Fluor594 anti-goat secondary antibodies, A488 anti-mouse FITC-conjugated secondary antibodies (Molecular Probes, Eugene, OR) and DAPI (Jackson Immunoresearch Laboratories, West Grove, PA) for 1 h at 37°C. The sample was covered with 10 µl of SlowFade reagent (Molecular Probes) followed by a coverslip, and the edges were sealed to prevent drying. Specimens were examined with a Zeiss LSM410 scanning laser confocal microscope.
NF-
B activation DNA binding ELISA.
Confluent HCT116 cells were incubated with Salmonella-containing HBSS (1.6 x 1010 bacteria/ml) for 30 min, washed three times in HBSS, and nuclear protein was extracted with Active Motif Nuclear Extract kit (Active Motif North America, Carlsbad, CA). NF-
B activation was measured using the TransAM NF-
B p50 Transcription Factor Assay kit (Active Motif North America), which specifically measures NF-
B binding to its consensus site (5'-GGGACTTTCC-3') according to the manufacturer's instructions.
Salmonella-induced human IL-8 Secretion.
Confluent HCT116 cells were incubated with Salmonella-containing HBSS (1.6 x 1010 bacteria/ml) for 30 min, washed three times in HBSS and incubated at 37°C for 4 h. Cell supernatants were removed and assayed for IL-8 by ELISA in 96-well plates (Linbro/Titertek; ICN Biochemicals, Costa Mesa, CA) as previously described (19).
Salmonella-induced GRO/CINC-1 (rat IL-8) secretion.
Confluent IEC-18 cells were seeded on 24-well plates and treated with 100 µl of Salmonella-containing HBSS (1.6 x 1010 bacteria/ml) for 30 min. The cells were washed with HBSS three times and incubated in 200 µl HBSS at 37°C for 4 h after adding the bacteria. Cell supernatants were collected, diluted (1:100), and assayed for IL-8 using a rat GRO/CINC-1 (rat IL-8) TiterZyme Enzyme Immunometric Assay (EIA) kit (Assay Designs, Ann Arbor, MI) according to the manufacturer's instructions.
Statistical analysis.
Data are expressed as means (SD). Differences were analyzed by Student's t-test. P values of <0.05 were considered significant.
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RESULTS
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-Catenin physically interacts with NF-
B.
The human intestinal epithelial cell lines T84 and HT29 provide excellent model systems for studying the effects of bacterial-epithelial crosstalk on epithelial cell function. T84 cells are known to display phenotypic similarities to crypt epithelia of the large intestine (18), and HT29 cells have been used extensively to study the
-catenin signaling pathway. Previous studies done in T84 cells have shown that Salmonella strain PhoPC prevents both I
B
(21) and
-catenin degradation (36) through a common mechanism. Taken together with the evidence that
-catenin and NF-
B can physically complex in colon and breast cancer cells (7), we therefore asked whether cross-regulation between the
-catenin and NF-
B signaling pathways might occur in bacteria-infected intestinal epithelial cells.
To examine the possibility that cross-regulation of the
-catenin and NF-
B signaling pathways may occur through physical interaction, we first determined whether
-catenin can form a complex with NF-
B and then asked whether colonization by Salmonella might modulate complex formation. With the use of a coimmunoprecipitation assay, we found that the NF-
B p50 subunit was able to form a complex with
-catenin in untreated monolayers of T84 cells and HT29 cells (Fig. 1, A and B). Interestingly, cells colonized with WT Salmonella over 24 h showed a significant decrease in the coimmunoprecipitation of
-catenin by p50 (Fig. 1, A-D; WT). In contrast, cells colonized for 24 h by the nonvirulent Salmonella strain PhoPc showed a significant increase in the amount of
-catenin pulled down by p50 (Fig. 1, A-D, PhoPc). There is no significant difference in expression between p50 and I
B
in cells colonized by either of the Salmonella strains after 24 h. Under normal conditions (i.e., Salmonella colonization of epithelial cells), I
B
degradation becomes apparent after 30 min of bacterial colonization and then returns to control levels by 3 h postcolonization. Because the data we present here (Fig. 1, A and B) is for bacterial colonization over 24 h, it is well beyond the point at which I
B
levels have returned to baseline, and I
B
degradation is not observed. Although there is clearly a physical interaction between
-catenin and NF-
B in both T84 and HT29 cells without any treatment, the amount of
-catenin binding to NF-
B varied depending on the strain of Salmonella with which the cells had been colonized. These results are entirely consistent with our previous report demonstrating that nonvirulent PhoPc colonization stabilizes total
-catenin expression, whereas WT Salmonella decreases total
-catenin expression (36). Additionally, we found no apparent interaction of p50 with
-catenin (Fig. 1, A and B), nor with a nonspecific IgG (data not shown), indicating that the physical interaction between
-catenin and NF-
B is specific. Thus our data demonstrate a clear physical interaction between
-catenin and NF-
B that can be altered by bacterial colonization.
Expression of constitutively active
-catenin inhibits I
B
degradation.
To address the possible biochemical and physiological effects of
-catenin on the NF-
B pathway during bacterial-epithelial cell interaction, we chose to use the human colonic epithelial cell line HCT116, expressing either the mutant (
45) or (WT) CTNNB1 allele (
-catenin gene). Parental HCT116 (CTNNB1WT/
45) cells possess the monoallelic
-catenin mutation
45 and WT CTNNB1, and its somatic CTNNB1 knockout line HCT116 CTNNB1/
45 possesses only mutant CTNNB1, whereas HCT116 CTNNB1WT/ has only WT CTNNB1 (5). For simplicity, the HCT116 cell lines will be referred to as CTNNB1WT/
45, CTNNB1/
45, and CTNNB1WT/. Cells expressing only WT
-catenin (CTNNB1WT/) showed decreased colony-forming ability when plated at low density, although their growth was similar to that of parental cells (CTNNB1WT/
45) when grown under routine conditions. Previous immunohistochemistry and cell-fractionation studies suggested that the CTNNB1/
45
-catenin activity was distinguished primarily by its cellular localization and not by protein degradation (5). Less CTNNB1/
45
-catenin bound to E-cadherin than did CTNNB1WT/
-catenin, and the membranous localization of CTNNB1WT/ and CTNNB1/
45
-catenin was accordingly distinct (5). The
-catenin signaling pathway has also been shown to be constitutively active in the CTNNB1/
45
-catenin cell line (5). These genetically modified cell lines provided us with the necessary tools to examine the effects of
-catenin signaling on Salmonella-induced activity of the NF-
B pathway.
The process of NF-
B activation involves phosphorylation of the inhibitory molecule I
B
by IKK, followed by ubiquitination and subsequent degradation in a proteasome. Degradation of I
B
serves as an excellent indicator of activation of the NF-
B signaling pathway. As shown in Fig. 2A, WT Salmonella colonization led to a significant degradation of I
B
in both CTNNB1WT/
45 and CTNNB1WT/ cells. However, when CTNNB1/
45 cells possessing only mutant
-catenin were incubated with either WT or PhoPC Salmonella, levels of I
B
were more directly comparable with levels seen in untreated control cells. Although constitutively active
-catenin (/
45) does not completely prevent I
B
degradation after WT Salmonella colonization, it was able to reduce I
B
degradation by twofold. These results indicate that expression of constitutively active
-catenin inhibits I
B
degradation during bacteria-host cell interaction. We also found similar protection of I
B
after TNF-
stimulation in CTNNB1/
45 cells possessing only mutant
-catenin (data not shown). This indicates that the protective effect of constitutively active
-catenin (/
45) is not strictly limited to Salmonella-induced I
B
degradation. (Fig. 2, A and B).
To determine the possible cause of I
B
stabilization in CTNNB1/
45, we examined whether there is a direct physical interaction between I
B
and
-catenin. Coimmunoprecipitation showed that
-catenin was unable to form a complex with I
B
(data not shown), whereas p50 antibody was able to pull down both
-catenin and I
B
in CTNNB1/
45 cells (Fig. 1, A and B). Taken together, these results indicate that whereas
-catenin is able to bind directly to NF-
B p50, I
B
may be stabilized by a mechanism that is independent of direct
-catenin binding.
Expression of mutation
-catenin
45 does not change NF-
B/
-catenin binding.
Our next objective was to determine whether the NF-
B/
-catenin interaction could be influenced by the
-catenin mutation
45. With the use of the coimmunoprecipitation assay, we found that the NF-
B p50 subunit was able to form a complex with
-catenin in untreated CTNNB1WT/
45, CTNNB1/
45, and CTNNB1WT/ HCT116 cells (data not shown). To our surprise, there is no significant increase in the amount of
-catenin pulled down by p50 in CTNNB1/
45. We also found equivalent levels of total
-catenin protein in CTNNB1WT/
45, CTNNB1/
45, and CTNNB1WT/ HCT116 cells, which is consistent with the report from Chan et al. (5). These data indicated that the mutant
-catenin
45 did not alter the physical binding between NF-
B and
-catenin.
Expression of constitutively active
-catenin inhibits NF-
B DNA binding.
In quiescent cells, NF-
B is bound to I
B
and held in the cytoplasm in a form that is unable to bind DNA. Colonization of cells with WT Salmonella colonization results in the dissociation of NF-
B from I
B
allowing NF-
B to translocate to the nucleus where it binds to its recognition sites in the promoter region of a wide variety of genes, including IL-8. Because we have shown that expression of constitutively active
-catenin can alter I
B
stability and increase
-catenin/NF-
B complex formation after bacterial colonization, we then asked whether activation of the NF-
B pathway might also be inhibited. One way to determine activation of the NF-
B pathway is to measure NF-
B DNA binding. This method has been shown to be 10-fold more sensitive than the gel shift assay for measuring NF-
B DNA binding. In CTNNB1WT/
45 and CTNNB1WT/ cells, WT Salmonella colonization increased NF-
B DNA binding, whereas PhoPc colonization had reduced DNA binding compared with WT. In contrast, the NF-
B DNA binding was almost nonexistent in CTNNB1/
45 colonized by either WT or PhoPc Salmonella (Fig. 3A). These results clearly demonstrate that Salmonella-induced NF-
B DNA binding activity can be inhibited by
-catenin pathway activation.
Localization of
-catenin and NF-
B during inflammation.
The signaling events triggered in response to bacterial colonization include activation of the IKK complex, nuclear translocation of the transcription factor NF-
B, and nuclear signaling.
-catenin is regulated by both degradation and its subcellular localization. With the activation of the
-catenin pathway, stabilized
-catenin protein is also translocated into the nucleus. To investigate the subcellular distribution of
-catenin and NF-
Bp50, we stained the cells with anti-
-catenin and anti-NF-
Bp50 antibodies in HCT116 CTNNB1WT/
45, CTNNB1/
45, and CTNNB1WT/ cells with or without bacterial colonization. In cells receiving no treatment,
-catenin was distributed diffusely throughout the membranes, with faint nuclear staining visible in both parental CTNNB1WT/
45 and CTNNB1WT/ cells, and there is no p50 nuclear staining (Fig. 4
-catenin WT/
45 and WT/). In contrast, untreated CTNNB1/
45 had a striking nucleus staining of both
-catenin and p50 (Fig. 4;
-catenin /
45). As expected, colonization with PhoPc induced nuclear translocation of
-catenin in CTNNB1WT/
45 (Fig. 4; PhoPc
-catenin WT/
45). This is consistent with our previous finding that PhoPc colonization not only stabilizes
-catenin by preventing its degradation, but also allows its accumulation and translocation into the nucleus (36). In the same cells, nuclear staining of p50 colocalized with
-catenin. Interestingly, CTNNB1/
45 cells that were colonized by WT Salmonella for 24 h exhibited changes in cell morphology due to the toxicity of WT Salmonella. In these cells,
-catenin was not as clearly membrane-localized, but it did colocalize with p50 in both the cytoplasm and nucleus (Fig. 4; WT Salmonella
-catenin /
45). These results clearly demonstrate the colocalization of p50 and
-catenin during Salmonella-induced inflammation. We have shown that in CTNNB1WT/
45, PhoPc colonization had reduced DNA binding, and the NF-
B DNA binding was almost nonexistent in CTNNB1/
45 colonized by WT Salmonella. Taken together, these data suggest that nuclear localized NF-
B may not necessarily be transcriptionally active when the
-catenin level is high in the nucleus.
Activation of
-catenin pathway inhibits Salmonella-induced IL-8 secretion.
S. typhimurium colonization of the intestinal epithelium initiates biochemical crosstalk between pathogen and host that activates the NF-
B pathway and results in the secretion of IL-8. We next sought to determine whether the
-catenin-mediated blockade of NF-
B activity could also influence the cell's ability to secrete IL-8 in response to Salmonella colonization. In CTNNB1WT/
45 and CTNNB1WT/ cells, IL-8 secretion was observed after WT Salmonella colonization, and as expected, less IL-8 secretion after avirulent PhoPc colonization. This pattern of Salmonella-induced IL-8 secretion is identical to the response seen in various epithelial cell lines including T84, HT29, and MDCK cells. In contrast, there was nearly 20-fold less IL-8 secretion after WT Salmonella colonization in CTNNB1/
45 cells and no IL-8 secretion after PhoPc colonization (Fig. 5). These results show that activation of the
-catenin pathway alters the host cell response to Salmonella colonization by reducing I
B
degradation, inhibiting NF-
B DNA binding, and preventing NF-
B-mediated IL-8 secretion.

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Fig. 5. -catenin expression negatively regulates Salmonella-induced IL-8 secretion. HCT116 and HT29C19A cells were incubated without () or with WT or PhoPc Salmonella for 30 min, rinsed, and incubated in HBSS for an additional 4 h. IL-8 secretion in cell culture supernatants was measured by ELISA as described in MATERIALS AND METHODS. Data presented are the means (SD) from a single experiment assayed in triplicate and are representative of results obtained in 3 separate experiments. *P < 0.05 vs. cells without treatment; **P < 0.001 vs. cells without treatment.
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To further examine the effects of
-catenin stabilization on Salmonella-induced activation of the NF-
B pathway, we used the nontransformed rat small intestine cell line IEC-18. This cell line provides an excellent model system because the IEC-18 cells are known to possess only WT
-catenin. Because these cells have not been genetically altered, an alternative method to stabilize
-catenin is through incubation with LiCl for 24 h, which is known to inhibit GSK-3
and prevent
-catenin degradation (17). In IEC-18 cells, pretreatment with LiCl allowed stabilized
-catenin to accumulate increasing total
-catenin (Fig. 6A). When untreated IEC-18 cells were challenged with WT Salmonella or PhoPc, they secreted IL-8 in a manner consistent with other epithelial cells (Fig. 6B). LiCl stabilization of
-catenin significantly decreased the amount of IL-8 secretion by sixfold in cells colonized with WT Salmonella, thus confirming our results seen in CTNNB1/
45 cells (Fig. 6B). In cells colonized with PhoPc, IL-8 secretion also decreased significantly. These data further confirm that activation/stabilization of
-catenin can negatively regulate the proinflammatory NF-
B pathway.
Activation of
-catenin does not influence NF-
B target genes Fas and TNF receptor-associated factor 1.
To further investigate the physiological relevance of the crosstalk between the
-catenin and NF-
B pathways, we examined whether inhibition of NF-
B in CTNNB1/
45 cells would affect the expression of Fas and TNF receptor-associated factor 1 (TRAF1), other important target genes of NF-
B. Fas is a member of cytokine TNF receptor family and is expressed at high levels in normal colonic epithelial cells (39). TRAF1 is a downstream target gene of NF-
B that is associated with apoptosis (42). To our surprise, there was no detectable difference in the basal expression levels of Fas or TRAF1 in CTNNB1WT/
45, CTNNB1/
45, or CTNNB1WT/ cells. Bacterial colonization by either WT or PhoPc Salmonella did not alter the expression levels of Fas or TRAF1 (data not shown). This would seem to indicate that activation of
-catenin is not involved in regulation of Fas and TRAF1 during the bacterial epithelial interaction.
 |
DISCUSSION
|
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The goal of this study was to determine how Salmonella-induced proinflammatory signaling might be influenced by activation of the
-catenin pathway. This was achieved by examining Salmonella-epithelial cell interactions using physiologically relevant epithelial cell model systems. In summary, we have shown that activation of
-catenin inhibits NF-
B signaling during bacterial-host cell interaction. Our results demonstrate that
-catenin forms a complex with NF-
B p50 in polarized human epithelial intestinal cells. Expression of constitutively active
-catenin prevents Salmonella-induced NF-
B activity, indirectly stabilizes I
B
, and inhibits the secretion of IL-8. Using biochemical and genetic techniques, we have demonstrated that crosstalk between the
-catenin and NF-
B signaling pathways may play an important regulatory role in intestinal inflammation.
WT S. typhimurium activates the proinflammatory NF-
B pathway (21) and enhances
-catenin degradation (36), whereas the nonvirulent Salmonella strain PhoPc inhibits activation of NF-
B and attenuates IL-8 secretion by inhibiting I
B
degradation (21). Incubation of cells with PhoPc also activates the
-catenin signaling pathway via a blockade of
-catenin degradation (36). It is the direct cross-regulation between these two pathways that is likely to contribute to the observed attenuation of NF-
B signaling and IL-8 secretion in cells colonized by PhoPc. We propose that the Salmonella-induced proinflammatory cascade involves both
-catenin and NF-
B pathways (Fig. 7). It is well accepted that WT Salmonella triggers the proinflammatory program including I
B
degradation, NF-
B activation, and IL-8 secretion in epithelial cells. However, we have shown that the same stimulation by WT Salmonella also causes
-catenin degradation (Fig. 7A) (35), which may enhance the proinflammatory response by preventing
-catenin interference with NF-
B signaling. This idea is in complete agreement with our results showing that stabilization of
-catenin by mutation (CTNNB1/
45) or LiCl-pretreatment increases both its binding of NF-
B and the colocalization of
-catenin and NF-
B p50 in the nucleus. Thus stabilization of
-catenin represses NF-
B pathway activation and inhibits IL-8 secretion during bacterial colonization (Fig. 7B). Our data also suggest that nuclear NF-
B may not necessarily be transcriptionally active when
-catenin is located in the nucleus. Interestingly, others (8) have reported similar observations in human tumor tissue samples. Thus activated
-catenin may play a role in the inhibition of NF-
B target gene expression in vivo.
In this study, we used polarized HT29-C19A and T84 cells to address the potential bacteria-mediated cross-regulation of
-catenin and NF-
B pathways. Our data clearly demonstrate that
-catenin can bind with NF-
B p50 but does not bind to I
B
. Using HCT116 (CTNNB1WT/
45) and its somatic CTNNB1 knockout lines HCT116/
45 and HCT116WT/, we have shown that
-catenin can inhibit NF-
B signaling via a mechanism that also prevents I
B
-degradation (Fig. 2). In contrast, recent reports using human embryonic kidney cells that overexpress
-catenin have shown that
-catenin can inhibit NF-
B in an I
B
-independent manner (7). Likewise, in the nontransformed rat small intestinal cell IEC-18, we found that activation of
-catenin is involved in attenuation of IL-8 secretion but that I
B
degradation induced by WT Salmonella colonization was not inhibited by LiCl pretreatment (data not shown). It is reported that LiCl treatment has many nonspecific effects (17) in addition to inhibition of GSK3
, whereas the
-catenin
45 mutation directly affects
-catenin stability. The apparent differences in these observations may not be a direct indicator of the importance of I
B
degradation. Further investigation is required to determine whether the regulatory effect of
-catenin on the NF-
B pathway during bacterial epithelial interaction is truly I
B
independent.
It has been suggested that the crosstalk between the Wnt cascade and NF-
B signaling is mediated through the E3-ligase
-TrCP1 in vascular smooth muscle cells (40). Stimulation of the Wnt cascade through either the upregulation of Wnt or degradation-resistant
-catenin significantly enhances both baseline and TNF-
-induced NF-
B activity. In contrast, others (8) and we have observed that the activation of the
-catenin pathway negatively regulates the NF-
B pathway in tumor cells and in response to bacterial stimulation in intestinal epithelial cells. Taken together, the combined evidence suggests that crosstalk occurs between the Wnt cascade and NF-
B signaling. Whether
-catenin cross-regulation positively or negatively regulates NF-
B signaling may be dependent on the model system and/or the agonist studied. Others (7) have shown that the interaction between
-catenin and NF-
B can be indirect and that an additional adaptor protein is required, implying that the interaction between
-catenin and NF-
B may be subject to another level of regulation. Thus identification and characterization of the interacting domain in
-catenin and the adaptor protein(s) will provide important information for understanding the detailed mechanism by which
-catenin cross-regulates NF-
B signaling.
Others (7) have demonstrated that both Fas and TRAF1, target genes of NF-
B pathway, are regulated by TNF-
stimulation and that
-catenin can inhibit the expression of these genes. However, during Salmonella epithelial cell interaction, we found that activation of the
-catenin pathway did not influence Fas or TRAF1 expression, suggesting TNF-
and Salmonella activate the proinflammatory cascade differentially. Although both Salmonella and TNF-
are potent activators of intestinal epithelial cell proinflammatory gene expression, there seem to be distinct differences in the specific patterns of gene expression induced by these stimulators.
This study represents the first report demonstrating cross-regulation between
-catenin and NF-
B pathways during bacterial-epithelial interactions and, as such, provides a potential new avenue of investigation into the role of
-catenin in intestinal inflammation. Both our published and unpublished data suggested that
-catenin and NF-
B pathways are involved during interactions with normal commensal bacteria and during specific pathogenic infections. Our previous studies (13) have demonstrated that AvrA, a previously identified Salmonella effector protein, plays a novel role in the activation of the
-catenin pathway and regulation of the NF-
B pathway (6, 35). Whether AvrA actually modulates the crosstalk between
-catenin and NF-
B remains to be determined. It will be interesting to ascertain the exact mechanism of action of AvrA, because it is expressed in both Salmonella and commensal E. coli strains (unpublished observations). Thus activation of host cell machinery by bacterial-derived factors may be able to induce long-term cellular responses in addition to the more transient inhibition of acute inflammation.
 |
GRANTS
|
---|
This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-47662 and DK-35932 (to J. L. Madara), and the Pilot & Feasibility Award DK-42086 (to J. Sun).
 |
ACKNOWLEDGMENTS
|
---|
We thank Kenneth Kinzler and Bert Vogelstein of Johns Hopkins Medical Institution, Baltimore, MD, for providing human colorectal cancer line HCT116 (CTNNB1WT/
45) and its somatic CTNNB1 knockout lines HCT116/
45 and HCT116WT/, We also want to thank Pam Fegan and Yingmin Wang for their technical assistance.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: J. Sun, Dept. of Pathology, Univ. of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637 (E-mail: jsun{at}bsd.uchicago.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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REFERENCES
|
---|
- Albanese C, Wu K, D'Amico M, Jarrett C, Joyce D, Hughes J, Hulit J, Sakamaki T, Fu M, Ben-Ze'ev A, Bromberg JF, Lamberti C, Verma U, Gaynor RB, Byers SW, and Pestell RG. IKK
regulates mitogenic signaling through transcriptional induction of cyclin D1 via Tcf. Mol Biol Cell 14: 585599, 2003.[Abstract/Free Full Text]
- Bours V, Bentires-Alj M, Hellin AC, Viatour P, Robe P, Delhalle S, Benoit V, and Merville MP. Nuclear factor-
B, cancer, and apoptosis. Biochem Pharmacol 60: 10851089, 2000.[CrossRef][ISI][Medline]
- Brabletz T, Jung A, Dag S, Hlubek F, and Kirchner T. Beta-catenin regulates the expression of the matrix metalloproteinase-7 in human colorectal cancer. Am J Pathol 155: 10331038, 1999.[Abstract/Free Full Text]
- Cao Y, Bonizzi G, Seagroves TN, Greten FR, Johnson R, Schmidt EV, and Karin M. IKK
provides an essential link between RANK signaling and cyclin D1 expression during mammary gland development. Cell 107: 763775, 2001.[CrossRef][ISI][Medline]
- Chan TA, Wang Z, Dang LH, Vogelstein B, and Kinzler KW. Targeted inactivation of CTNNB1 reveals unexpected effects of beta-catenin mutation. Proc Natl Acad Sci USA 99: 82658270, 2002.[Abstract/Free Full Text]
- Collier-Hyams LS, Zeng H, Sun J, Tomlinson AD, Bao ZQ, Chen H, Madara JL, Orth K, and Neish AS. Cutting edge: Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-
B pathway. J Immunol 169: 28462850, 2002.[Abstract/Free Full Text]
- Deng J, Miller SA, Wang HY, Xia W, Wen Y, Zhou BP, Li Y, Lin SY, and Hung MC.
-Catenin interacts with and inhibits NF-
B in human colon and breast cancer. Cancer Cell 2: 323334, 2002.[CrossRef][ISI][Medline]
- Deng J, Xia W, Miller SA, Wen Y, Wang HY, and Hung MC. Crossregulation of NF-
B by the APC/GSK-3beta/beta-catenin pathway. Mol Carcinog 39: 139146, 2004.[CrossRef][ISI][Medline]
- Fuchs SY, Chen A, Xiong Y, Pan ZQ, and Ronai Z. HOS, a human homolog of Slimb, forms an SCF complex with Skp1 and Cullin1 and targets the phosphorylation-dependent degradation of I
B and beta-catenin. Oncogene 18: 20392046, 1999.[CrossRef][ISI][Medline]
- Ghosh S, May MJ, and Kopp EB. NF-
B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 16: 225260, 1998.[CrossRef][ISI][Medline]
- Gilmore TD, Koedood M, Piffat KA, and White DW. Rel/NF-
B/I
B proteins and cancer. Oncogene 13: 13671378, 1996.[ISI][Medline]
- Guarner F and Malagelada JR. Gut flora in health and disease. Lancet 361: 512519, 2003.[CrossRef][ISI][Medline]
- Hardt WD and Galan JE. A secreted Salmonella protein with homology to an avirulence determinant of plant pathogenic bacteria. Proc Natl Acad Sci USA 18: 98879892, 1997.[CrossRef]
- He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B, and Kinzler KW. Identification of c-MYC as a target of the APC pathway. Science 281: 15091512, 1998.[Abstract/Free Full Text]
- Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O, and Woodgett JR. Requirement for glycogen synthase kinase-3beta in cell survival and NF-
B activation. Nature 406: 8690, 2000.[CrossRef][ISI][Medline]
- Karin M, Cao Y, Greten FR, and Li ZW. NF-
B in cancer: from innocent bystander to major culprit. Nature Cancer 2: 303310, 2002.
- Klein PS and Melton DA. A molecular mechanism for the effect of lithium on development. Proc Natl Acad Sci USA 93: 84558459, 1996.[Abstract/Free Full Text]
- Madara JL and Dharmsathaphorn K. Occluding junction structure-function relationships in a cultured epithelial monolayer. J Cell Biol 101: 21242133, 1985.[Abstract]
- McCormick BA, Colgan SP, Delp-Archer C, Miller SI, and Madara JL. Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils. J Cell Biol 123: 895907, 1993.[Abstract]
- Miller JJ and Mekalanos SI,. Constitutive expression of the phoP regulon attenuates Salmonella virulence and survival within macrophages. J Bacteriol 172: 24852490, 1990.[ISI][Medline]
- Neish AS, Gewirtz AT, Zeng H, Young AN, Hobert ME, Karmali V, Rao AS, and Madara JL. Prokaryotic regulation of epithelial responses by inhibition of I
B-alpha ubiquitination. Science 289: 15601563, 2000.[Abstract/Free Full Text]
- Polakis P. Wnt signaling and cancer. Genes Dev 14: 18371851, 2000.[Free Full Text]
- Polakis P. More than one way to skin a catenin. Cell 105: 563565, 2000.[CrossRef][ISI]
- Rimm DL, Caca K, Hu G, Harrison FB and Fearon ER. Frequent nuclear/cytoplasmic localization of beta-catenin without exon 3 mutations in malignant melanoma. Am J Pathol 154: 325-329, 1999.[Abstract/Free Full Text]
- Rogler G, Brand K, Vogl D, Page S, Hofmeister R, Andus T, Knuechel R, Baeuerle PA, Scholmerich J, and Gross V. Nuclear factor
B is activated in macrophages and epithelial cells of inflamed intestinal mucosa. Gastroenterologist 115: 357369, 1998.
- Samowitz WS, Powers MD, Spirio LN, Nollet F, van Roy F, Slattery ML. Beta-catenin mutations are more frequent in small colorectal adenomas than in larger adenomas and invasive carcinomas. Cancer Res 59: 14421444, 1999.[Abstract/Free Full Text]
- Sartor RB. Insights into the pathogenesis of inflammatory bowel diseases provided by new rodent models of spontaneous colitis. Inflamm Bowel Dis 1: 6475, 1995.
- Sartor RB. Pathogenesis and immune mechanisms for chronic inflammatory bowel diseases. Am J Gastroenterol 92, Suppl 12: 5S11S, 1997.
- Sartor RB and Rath H. Microbial factors in chronic intestinal inflammation. Current Opin Gastroenterol 12: 327333, 1996.[ISI]
- Sawyer EJ, Hanby AM, Rowan AJ, Gillett CE, Thomas RE, Poulsom R, Lakhani SR, Ellis IO, Ellis P, and Tomlinson IP. The Wnt pathway, epithelial-stromal interactions, and malignant progression in phyllodes tumours. J Pathol 196: 437444, 2002.[CrossRef][ISI][Medline]
- Sellon RK, Tonkonogy S, Schultz M, Dieleman LA, Grenther W, Balish E, Rennick DM, and Sartor RB. Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice. Infect Immun 66: 52245231, 1998.[Abstract/Free Full Text]
- Shanahan F. Inflammatory bowel disease: immunodiagnostics, immunotherapeutics, and ecotherapeutics. Gastroenterology 120: 622635, 2001.[ISI][Medline]
- Shtutman M, Zhurinsky J, Simcha I, Albanese C, D'Amico M, Pestell R, and Ben-Ze'ev A. The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc Natl Acad Sci USA 96: 55225527, 1999.[Abstract/Free Full Text]
- Strober W and Ehrhardt R. Chronic intestinal inflammation: an unexpected outcome in cytokine or T-cell mutant mice. Cell 75: 203205, 1993.[CrossRef][ISI][Medline]
- Su LK, Vogelstein B, and Kinzler KW. Association of the APC tumor suppressor protein with catenins. Science 262: 17341737, 1993.[ISI][Medline]
- Sun J, Hobert ME, Rao AS, Neish AS, and Madara JL. Bacterial activation of beta-catenin signaling in human epithelia. Am J Physiol Gastrointest Liver Physiol 287: G220G227, 2004.[Abstract/Free Full Text]
- Swidsinski A, Ladhoff A, Pernthaler A, Swidsinski S, Loening-Baucke V, Ortner M, Weber J, Hoffmann U, Schreiber S, Dietel M, and Lochs H. Mucosal flora in inflammatory bowel disease. Gastroenterology 122: 4454, 2002.[ISI][Medline]
- Tetsu O and McCormick F. Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398: 422426, 1999.[CrossRef][ISI][Medline]
- Von Reyher U, Strater J, Kittstein W, Gschwendt M, Krammer PH, and Moller P. Colon carcinoma cells use different mechanisms to escape C.D95-mediated apoptosis. Cancer Res 58: 526534, 1998.[Abstract]
- Wang CY, Mayo MW, Korneluk RG, Goeddel DV, and Baldwin AS Jr. NF-
B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 281: 16801683, 1998.[Abstract/Free Full Text]
- Wang W, Abbruzzese JL, Evans DB, Larry L, Cleary KR, and Chiao PJ. The nuclear factor-
B RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells. Clin Cancer Res 5: 119127, 1999.[Abstract/Free Full Text]
- Wang X, Adhikari N, Li Q, Guan Z, and Hall JL. The role of
-transducin repeat-containing protein ([beta]-TrCP) in the regulation of NF-
B in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 24: 8590, 2004.[Abstract/Free Full Text]
- Winston JT, Strack P, Beer-Romero P, Chu CY, Elledge SJ, and Harper JW. The SCF-
-TRCP ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in I
B and
-catenin and stimulates I
B ubiquitination in vitro. Genes Dev 3: 270283, 1999.
- Wong NA and Pignatelli M.
-Catenina linchpin in colorectal carcinogenesis? Am J Pathol 160: 389401, 2002.[Abstract/Free Full Text]
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