Intestinal Epithelial Cells Exposed to Bacteroides fragilis Enterotoxin Regulate NF-κB Activation and Inflammatory Responses through β-Catenin Expression

Abstract

The Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), interacts with intestinal epithelial cells and can provoke signals that induce mucosal inflammation. Although β-catenin signaling is reported to be associated with inflammatory responses and BFT is known to cleave E-cadherin linked with β-catenin, little is known about the β-catenin-mediated regulation of inflammation in ETBF infection. This study was conducted to investigate the role of β-catenin as a cellular signaling intermediate in the induction of proinflammatory responses to stimulation of intestinal epithelial cells with BFT. Expression of β-catenin in intestinal epithelial cells was reduced relatively early after stimulation with BFT and then recovered to normal levels relatively late after stimulation. In contrast, phosphorylation of β-catenin in BFT-exposed cells occurred at high levels early in stimulation and decreased as time passed. Concurrently, late after stimulation the nuclear levels of β-catenin were relatively higher than those early after stimulation. Suppression of β-catenin resulted in increased NF-κB activity and interleukin-8 (IL-8) expression in BFT-stimulated cells. However, suppression or enhancement of β-catenin expression neither altered the phosphorylated IκB kinase α/β complex nor activated activator protein 1 signals. Furthermore, inhibition of glycogen synthase kinase 3β was associated with increased β-catenin expression and attenuated NF-κB activity and IL-8 expression in BFT-exposed cells. These findings suggest the negative regulation of NF-κB-mediated inflammatory responses by β-catenin in intestinal epithelial cells stimulated with BFT, resulting in attenuation of acute inflammation in ETBF infection.

Keywords: Bacteroides fragilis; NK-κB; enterotoxin; intestinal epithelial cells; β-catenin.

FIG 1

β-Catenin expression in intestinal epithelial cells stimulated with BFT. (A) HCT 116 cells were treated with BFT (300 ng/ml) for the indicated periods of time. The total cellular proteins β-catenin, phospho-β-catenin, and actin were analyzed by immunoblotting. (B) The β-catenin and lamin B in the nuclear fraction were analyzed by immunoblotting. (C and D) Immunoblots for β-catenin levels in CCD 841 CoN cells under the same treatment conditions used for the assays for which the results are shown in panels A and B are shown beneath each time point. (E) HCT 116 cells were treated with each concentration of BFT for 18 h, after which nuclear fractions were obtained. Nuclear β-catenin and lamin B were analyzed by immunoblotting. All results are representative of those from more than three independent experiments.

FIG 2

Effects of β-catenin on NF-κB activation in intestinal epithelial cells stimulated with BFT. (A) HCT 116 cells were treated with BFT (300 ng/ml) for the indicated periods of time. (Top) The expression of IκBα, phospho-IκBα, and actin in whole-cell lysates was analyzed by immunoblotting. (Bottom) Immunoblotting results for phospho-p65 and lamin B in nuclear extracts tested under the same conditions used for the assays for which the results are provided in the top panels. (B) HCT 116 cells were transfected with β-catenin-specific siRNA or nonsilencing control siRNA (NS-RNA) as a control. After 48 h, the expression of β-catenin and actin was analyzed by immunoblotting. The results are representative of those from three independent experiments. (C) The transfected cells were stimulated with BFT (300 ng/ml) for 18 h, and the expression of β-catenin and actin was analyzed by immunoblotting. (D) The transfected cells were stimulated with BFT (300 ng/ml) for 18 h. The expression of phospho-p65, β-catenin, and lamin B in the nuclear fractions was analyzed by immunoblotting. The results shown are representative of those from more than three independent experiments. (E) Transfected and untransfected cells were treated with BFT (300 ng/ml) for 18 h. The activities of NF-κB were measured by an ELISA kit. The data are expressed as the mean fold induction ± SEM relative to that for the untreated controls (n = 5). *, P < 0.05. (F) Transfected and untransfected cells were treated with BFT (300 ng/ml) for the indicated periods of time. The levels of IL-8 were analyzed by quantitative RT-PCR. Values are expressed as the mean ± SD (n = 5). *, P < 0.05 compared to the results for BFT-treated untransfected cells. In this experimental system, the β-actin mRNA levels in each group remained relatively constant throughout the same periods (∼10⁶ transcripts/μg total RNA). (G) Transfected and untransfected cells were treated with BFT (300 ng/ml) for 24 h. The amount of IL-8 in the culture supernatants was measured by an ELISA kit. The data are expressed as the mean ± SEM (n = 5). *, P < 0.05. (H) Transfected and untransfected cells were treated with BFT (300 ng/ml) for 1 h. The activities of NF-κB were measured by an ELISA kit. The data are expressed as the mean fold induction ± SEM relative to that for the untreated controls (n = 5). NS, statistically nonsignificant; *, P < 0.05.

FIG 2

Effects of β-catenin on NF-κB activation in intestinal epithelial cells stimulated with BFT. (A) HCT 116 cells were treated with BFT (300 ng/ml) for the indicated periods of time. (Top) The expression of IκBα, phospho-IκBα, and actin in whole-cell lysates was analyzed by immunoblotting. (Bottom) Immunoblotting results for phospho-p65 and lamin B in nuclear extracts tested under the same conditions used for the assays for which the results are provided in the top panels. (B) HCT 116 cells were transfected with β-catenin-specific siRNA or nonsilencing control siRNA (NS-RNA) as a control. After 48 h, the expression of β-catenin and actin was analyzed by immunoblotting. The results are representative of those from three independent experiments. (C) The transfected cells were stimulated with BFT (300 ng/ml) for 18 h, and the expression of β-catenin and actin was analyzed by immunoblotting. (D) The transfected cells were stimulated with BFT (300 ng/ml) for 18 h. The expression of phospho-p65, β-catenin, and lamin B in the nuclear fractions was analyzed by immunoblotting. The results shown are representative of those from more than three independent experiments. (E) Transfected and untransfected cells were treated with BFT (300 ng/ml) for 18 h. The activities of NF-κB were measured by an ELISA kit. The data are expressed as the mean fold induction ± SEM relative to that for the untreated controls (n = 5). *, P < 0.05. (F) Transfected and untransfected cells were treated with BFT (300 ng/ml) for the indicated periods of time. The levels of IL-8 were analyzed by quantitative RT-PCR. Values are expressed as the mean ± SD (n = 5). *, P < 0.05 compared to the results for BFT-treated untransfected cells. In this experimental system, the β-actin mRNA levels in each group remained relatively constant throughout the same periods (∼10⁶ transcripts/μg total RNA). (G) Transfected and untransfected cells were treated with BFT (300 ng/ml) for 24 h. The amount of IL-8 in the culture supernatants was measured by an ELISA kit. The data are expressed as the mean ± SEM (n = 5). *, P < 0.05. (H) Transfected and untransfected cells were treated with BFT (300 ng/ml) for 1 h. The activities of NF-κB were measured by an ELISA kit. The data are expressed as the mean fold induction ± SEM relative to that for the untreated controls (n = 5). NS, statistically nonsignificant; *, P < 0.05.

FIG 3

Effects of β-catenin overexpression on NF-κB activation and IL-8 expression in intestinal epithelial cells stimulated with BFT. (A) HCT 116 cells were transfected with lentivirus containing wild-type (WT) β-catenin-overexpressing or control plasmids. The expression of β-catenin proteins was analyzed by immunoblotting using an anti-DDK Ab. The results are representative of those from three independent experiments. (B) WT β-catenin-overexpressing cells were treated with BFT (300 ng/ml) for 1 h. The expression of phospho-p65 signals in the nuclear factions was assessed by immunoblotting. The results are representative of those from three independent experiments. (C) β-Catenin-overexpressing cells were stimulated with BFT (300 ng/ml) for 1 h. The activities of NF-κB were measured by an ELISA kit. The data are expressed as the mean fold induction ± SEM relative to that for the untreated controls (n = 5). *, P < 0.05. (D) Transfected and untransfected cells were treated with BFT (300 ng/ml) for the indicated periods of time. The levels of IL-8 were analyzed by quantitative RT-PCR. Values are expressed as the mean ± SD (n = 5). *, P < 0.05 compared to the results for BFT-treated untransfected cells. The β-actin mRNA levels in each group remained relatively constant throughout the same periods (∼10⁶ transcripts/μg total RNA). (E) β-Catenin-overexpressing cells were stimulated with BFT (300 ng/ml) for 24 h. The amount of IL-8 in the culture supernatants was measured by an ELISA kit. The data are expressed as the mean ± SEM (n = 5). *, P < 0.05.

FIG 4

Suppression of β-catenin enhances NF-κB activity and the secretion of IL-8 in intestinal epithelial cells stimulated with BFT. (A) CCD 841 CoN cells were pretreated with the β-catenin inhibitor IWP-4 (100 nM) for 30 min, after which the cells were stimulated with BFT (300 ng/ml) for the indicated periods of time. The activities of NF-κB were measured by an ELISA kit. The data are expressed as the mean fold induction ± SEM relative to that for the untreated controls (n = 5). *, P < 0.05 in the group treated with BFT and IWP-4 combined compared with the group treated with BFT alone. (B) Cells were pretreated with the β-catenin inhibitor IWP-4 (100 nM) for 30 min, after which cells were stimulated with BFT (300 ng/ml) for 24 h. The amounts of IL-8 in the culture supernatant were measured by an ELISA kit. The data are expressed as the mean ± SEM (n = 5). *, P < 0.05.

FIG 5

Relationship between β-catenin and IKK activation in intestinal epithelial cells stimulated with BFT. (A) HCT 116 cells were transfected with β-catenin-specific siRNA or nonsilencing siRNA (NS-RNA) as a control for 48 h. The transfected cells were combined with BFT (300 ng/ml) for 1 h, and the expression of phospho-IKKα/β was analyzed by immunoblotting. The results shown are representative of those from more than three independent experiments. (B) HCT 116 cells were transfected with lentivirus containing wild-type (WT) β-catenin-overexpressing plasmids. Transfected cells were stimulated with BFT (300 ng/ml) for 1 h, and the phospho-IKKα/β protein was then analyzed by immunoblotting. The results are representative of those from three independent experiments. (C) HCT 116 cells transfected with β-catenin siRNA or NS-RNA were stimulated with BFT (300 ng/ml) for the indicated periods of time. IKK activity was measured by a phospho-IκBα kinase assay kit. The data are expressed as the mean fold induction ± SEM of phosphorylated IκBα relative to that for the untreated controls (n = 5). (D) CCD 841 CoN cells were pretreated with the β-catenin inhibitor IWP-4 (100 nM) for 30 min, after which the cells were stimulated with BFT (300 ng/ml) for the indicated periods of time. IKK activities were measured by a phospho-IκBα kinase assay kit. The data are expressed as the mean fold induction ± SEM of phosphorylated IκBα relative to that for the untreated controls (n = 5).

FIG 6

Effects of β-catenin suppression on AP-1 activation in intestinal epithelial cells stimulated with BFT. (A) HCT 116 cells were treated with BFT (300 ng/ml) for the indicated periods of time. The expression of phospho-c-Jun and lamin B in nuclear extracts was analyzed by immunoblotting. The results shown are representative of those from more than three independent experiments. (B) HCT 116 cells were transfected with β-catenin-specific siRNA or nonsilencing siRNA (NS-RNA) as a control for 48 h. Transfected and untransfected cells were treated with BFT (300 ng/ml) for the indicated periods of time. The activities of AP-1 were measured by an ELISA kit. The data are expressed as the mean fold induction ± SEM relative to that for the untreated controls (n = 5). (C and D) CCD 841 CoN cells were pretreated with the β-catenin inhibitor IWP-4 (100 nM) for 30 min, after which the cells were stimulated with BFT (300 ng/ml) for 1 h (C) and 18 h (D). The activities of AP-1 were measured by an ELISA kit. The data are expressed as the mean fold induction ± SEM relative to that for the untreated controls (n = 5). NS, statistically nonsignificant.

FIG 7

Effects of GSK-3β inhibition on β-catenin expression, NF-κB activation, and IL-8 expression in intestinal epithelial cells stimulated with BFT. (A) HCT 116 cells were transfected with GSK-3β-specific siRNA or nonsilencing siRNA (NS-RNA) as a control for 48 h. The expression of GSK-3β and actin was analyzed by immunoblotting. The results are representative of those from three independent experiments. (B) The transfected cells were combined with BFT (300 ng/ml) for 1 h, after which GSK-3β, IκBα, β-catenin, and β-actin were analyzed by immunoblotting. The results shown are representative of those from more than three independent experiments. (C) Transfected and untransfected cells were treated with BFT (300 ng/ml) for 1 h. The phospho-p65 and lamin B signals in the nuclear fractions were examined by immunoblotting. The results shown are representative of those from more than three independent experiments. (D and E) Transfected and untransfected cells were treated with BFT (300 ng/ml) for 1 h. The levels of β-catenin (D) and NF-κB (E) were analyzed by the use of the respective ELISA kits. The data are expressed as the mean percent induction ± SEM relative to that for the untreated controls (n = 5, β-catenin) and the mean fold induction ± SEM relative to that for the untreated controls (n = 5, NF-κB). *, P < 0.05. (F) Transfected HCT 116 cells were treated with BFT (300 ng/ml) for the indicated periods of time. The levels of IL-8 mRNA were analyzed by quantitative RT-PCR. Values are expressed as the mean ± SD (n = 5). The β-actin mRNA levels in each group remained relatively constant throughout the same periods (∼10⁶ transcripts/μg total RNA). *, P < 0.05 compared with untransfected cells treated with BFT. (G) Transfected and untransfected cells were treated with BFT (300 ng/ml) for 18 h. The amount of IL-8 in the culture supernatants was measured by an ELISA kit. The data are expressed as the mean ± SEM (n = 5). *, P < 0.05.

FIG 8

Effects of GSK-3β inhibition on NF-κB activation and IL-8 expression in CCD 841 CoN cells stimulated with BFT. CCD 841 CoN cells were pretreated with LiCl (30 nM) for 24 h, after which the cells were stimulated with BFT (300 ng/ml) for 1 h (NF-κB) or 24 h (IL-8). (A) The activity of NF-κB was measured by an ELISA kit. The data are expressed as the mean fold induction ± SEM relative to that for the untreated controls (n = 5). (B) The amount of IL-8 in culture supernatants was measured by an ELISA kit. The data are expressed as the mean ± SEM (n = 5). *, P < 0.05.