Muc1 cell surface mucin attenuates epithelial inflammation in response to a common mucosal pathogen

Abstract

Helicobacter pylori infection of the gastric mucosa causes an active-chronic inflammation that is strongly linked to the development of duodenal and gastric ulcers and stomach cancer. However, greater than 80% of individuals infected with H. pylori are asymptomatic beyond histologic inflammation, and it is unknown what factors influence the incidence and character of bacterial-associated gastritis and related disorders. Because previous studies demonstrated that the Muc1 epithelial glycoprotein inhibited inflammation during acute lung infection by Pseudomonas aeruginosa, we asked whether Muc1 might also counter-regulate gastric inflammation in response to H. pylori infection. Muc1(-/-) mice displayed increased bacterial colonization of the stomach and greater TNF-alpha and keratinocyte chemoattractant transcript levels compared with Muc1(+/+) mice after experimental H. pylori infection. Knockdown of Muc1 expression in AGS human gastric epithelial cells by RNA interference was associated with increased phosphorylation of IkappaBalpha, augmented activation and nuclear translocation of NF-kappaB, and enhanced production of interleulin-8 compared with Muc1-expressing cells. Conversely, Muc1 overexpression was correlated with decreased NF-kappaB activation, reduced interleulin-8 production, and diminished IkappaB kinase beta (IKKbeta)/IKKgamma coimmunoprecipitation compared with cells expressing Muc1 endogenously. Cotransfection of AGS cells with Muc1 plus IKKbeta, but not a catalytically inactive IKKbeta mutant, reversed the Muc1 inhibitory effect. Finally, Muc1 formed a coimmunoprecipitation complex with IKKgamma but not with IKKbeta. These results are consistent with the hypothesis that Muc1 binds to IKKgamma, thereby inhibiting formation of the catalytically active IKK complex and blocking the ability of H. pylori to stimulate IkappaBalpha phosphorylation, NF-kappaB activation, and downstream inflammatory responses.

FIGURE 1.

Muc1^−/− mice exhibit increased colonization of the gastric mucosa by H. pylori. A, mice were infected with H. pylori strain SS1 by gastric intubation, and bacterial CFUs in the stomach were quantified at 4 weeks post-infection. B, H. pylori urease C gene copy numbers in the stomach were quantified by PCR at 4 weeks post-infection. H. pylori-reactive serum IgG (C) and IgA (D) levels were measured by ELISA in uninfected mice or mice infected with H. pylori SS1 at 4 weeks post-infection. Values are the means ± S.D. of triplicate samples. NS, not significant. The results are representative of two independent experiments.

FIGURE 2.

Muc1^−/− mice display increased TNF-α and KC transcript levels after H. pylori gastric infection. A, Muc1^+/+ and Muc1^−/− mice were uninfected or infected with 10⁷ CFUs/mouse of H. pylori SS1, and transcripts encoding TNF-α and KC were measured by quantitative reverse transcription-PCR in gastric tissue homogenates at 4 weeks post-infection. B and C, Muc1^+/+ and Muc1^−/− mice were unimmunized and unchallenged (U/U), unimmunized and challenged (U/C), or immunized and challenged (I/C). Intranasal immunization at 1, 2, 3, and 4 weeks was with a bacterial lysate of H. pylori SS1 (100 μg/mouse). Unimmunized mice received cholera toxin adjuvant alone in the absence of H. pylori lysate. Challenge infection was by gastric intubation on 2 consecutive days with 10⁷ CFUs/mouse of SS1 at 2 weeks after the last immunization. At 4 weeks post-infection, transcripts encoding TNF-α (B) and KC (C) were measured by quantitative reverse transcription-PCR in gastric tissue homogenates. Values are the means ± S.D. of 4–6 replicates per sample. The results are representative of two independent experiments.

FIGURE 3.

Knockdown of Muc1 expression increases IL-8 production. A, AGS cells were treated for the indicated times with H. pylori SS1 or H. pylori 26695, and IL-8 levels in culture media were measured by ELISA. B, AGS cells were transfected with a Muc1 targeting siRNA or a nontargeting control siRNA, the cells were incubated for 12 (lane 1), 24 (lane 2), 48 (lane 3), or 72 (lane 4) h, and equal protein aliquots of cell lysates were analyzed by Muc1 Western blotting. As a loading control, the blot was stripped and probed for β-actin. IB, immunoblot. C, AGS cells were transfected with Muc1 or control siRNAs for 72 h and treated for the indicated times with H. pylori 26695, and IL-8 levels in culture media were measured by ELISA. The results are representative of three independent experiments. *, p < 0.05; **, p < 0.02; ***, p < 0.01 comparing H. pylori-treated, Muc1 siRNA-transfected cells with H. pylori-treated, control siRNA-transfected cells.

FIGURE 4.

Overexpression of Muc1 decreases IL-8 production. A, AGS cells were transfected with the pcDNA empty vector or a Muc1 expression plasmid (pMuc1), the cells were incubated for 72 h, and equal protein aliquots of cell lysates were analyzed by Muc1 Western blotting (IB). As a loading control, the blot was stripped and probed for β-actin. B, shown is densitometric quantification of the Muc1 immunoreactive bands in A. C, AGS cells were transfected with pcDNA empty vector or pMuc1 for 72 h and treated for the indicated times with H. pylori 26695, and IL-8 levels in culture media were measured by ELISA. The results are representative of three independent experiments. *, p < 0.05 comparing H. pylori-treated, pcDNA-transfected cells with H. pylori-treated, pMuc1-transfected cells.

FIGURE 5.

Muc1 expression correlates inversely with NF-κB activation. AGS cells were transfected with Muc1 or control siRNAs (A) or with pcDNA empty vector or pMuc1 (B). Cells were incubated for 48 h and cotransfected with the pGL4.32 plasmid containing NF-κB response elements linked to a P. pyralis (firefly) luciferase reporter gene plus the pRL-SV40 plasmid encoding Renilla luciferase. The cells were incubated for an additional 24 h either untreated or treated with H. pylori 26695 for the indicated times, and luciferase activities were measured in cell lysates. Each point represents the mean ± S.D. value of triplicate samples. *, p < 0.05; **, p < 0.01 comparing H. pylori-treated, Muc1 siRNA-transfected cells with H. pylori-treated, control siRNA-transfected cells (A) or comparing H. pylori-treated, pcDNA- transfected cells with H. pylori-treated, pMuc1-transfected cells (B). The results are representative of two independent experiments.

FIGURE 6.

Muc1 siRNA increases H. pylori-stimulated IκBα phosphorylation that is inhibited by Bay 11-7082. A, AGS cells were transfected with Muc1 or control siRNAs, incubated for 48 h, and treated with H. pylori strain 26695 for the indicated times, and phospho-IκBα (pIκBα; upper panel) and total IκBα (lower panel) levels were determined by Western blotting (IB). B and C, AGS cells were pretreated with 10 μm Bay 11-7082 (Calbiochem) or medium control for 1 h, washed, and treated with H. pylori 26695 for the indicated times. Cytoplasmic levels of pIκBα and total IκBα (B) and nuclear levels of NF-κB (C) were determined by Western blotting. As loading controls, the blots were stripped and probed for β-actin or lamin B1. The results are representative of two independent experiments.

FIGURE 7.

A, Muc1 overexpression increases nuclear translocation of NF-κB in response to H. pylori treatment. AGS cells were transfected with pcDNA empty vector or pMuc1 for 72 h and treated for 6 h with H. pylori 26695, and NF- κB in nuclear and cytoplasmic fractions was detected by Western blotting (IB). B, shown is densitometric quantification of the NF-κB bands in A. C, Bay 11-7082 inhibits H. pylori-stimulated IL-8 production. AGS cells were pretreated with medium alone or with 10 or 20 μm Bay 11-7082 for 1 h. Cells were washed and either untreated or treated with H. pylori 26695 for the indicated times, and IL-8 levels in culture media were determined by ELISA. Each point represents the mean ± S.D. value of triplicate samples. The results are representative of two independent experiments.

FIGURE 8.

A and B, IKKβ phosphorylates IκBα after H. pylori treatment. A, AGS cells were transfected with plasmids encoding wild type IKKβ (WT) or catalytically inactive IKKβ (K44M), the cells were untreated or treated with H. pylori 26695 for 6 h, and pIκBα and IKKβ levels were determined by Western blotting (IB). As a loading control, the blot was stripped and probed for β-actin. The results are representative of two independent experiments. B, shown is densitometric quantification of the IKKβ bands in A normalized to the loading controls. *, p < 0.05. C and D, overexpression of IKKβ reverses Muc1-mediated reduction in H. pylori-stimulated NF-κB activation. C, AGS cells were transfected with the pcDNA empty vector, pMuc1, or plasmids encoding wild type IKKβ (pIKKβ) or catalytically inactive pIKKβ (K44M), and the cells were incubated for 24 h and cotransfected with the NF-κB-luciferase reporter plasmid plus pRL-SV40. The cells were incubated for an additional 24 h and treated with H. pylori 26695 for the indicated times, and luciferase activities were measured in cell lysates. D, AGS cells were transfected with empty vector, pMuc1, wild type pIKKβ, or pIKKβ (K44M), the cells were incubated for 24 h and treated with H. pylori 26695, and IL-8 levels in culture media were determined by ELISA. Each point represents the mean ± S.D. value of duplicate samples. *, p < 0.05 comparing pcDNA- or pMuc1/pIKKβ-transfected cells with pMuc1- or pMUC1/pIKKβ (K44M)-transfected cells. The results are representative of two independent experiments.

FIGURE 9.

Muc1 inhibits IKKβ·IKKγ coIP and binds to IKKγ. AGS cells were transfected with the pcDNA empty vector or pMuc1 and incubated for 24 h, and equal protein aliquots of cell lysates were subjected to coIP analysis as indicated (left panels). As controls, the blots were stripped and probed with the respective immunoprecipitating antibodies (right panels). IP, immunoprecipitation; IB, immunoblot. The results are representative of two independent experiments.

FIGURE 10.

Muc1 inhibits H. pylori-stimulated NF-κB activation through its intracellular COOH terminus. A, HEK293 cells were transfected with plasmids encoding the CD8/Muc1 fusion protein (pCD8/Muc1), CD8 ectodomain, and transmembrane domains alone (pCD8) or pCD8/Muc1 containing seven tyrosine-to-phenylalanine substitutions in the Muc1 COOH terminus (pCD8/7YF). The cells were incubated for 24 h, cotransfected with the NF-κB-luciferase reporter plasmid plus pRL-SV40, incubated for an additional 24 h, untreated or treated with H. pylori 26695 for the indicated times, and luciferase activities were measured in cell lysates. Each point represents the mean ± S.D. value of duplicate samples. *, p < 0.05; **, p < 0.01 comparing H. pylori-treated, pCD8- or pCD8/7YF-transfected cells with H. pylori-treated, pCD8/Muc1-transfected cells. B, HEK293 cells were transfected with the pCD8/Muc1 or pCD8 constructs and incubated for 24 h, and equal protein aliquots of cell lysates were immunoprecipitated (IP) with IKKγ antibody and immunoblotted (IB) with antibody against the CD8 ectodomain (left panel). As a control, the blot was stripped and probed with the immunoprecipitating antibodies (right panel). C, HEK293 cell lysates were examined for IKKβ/CD8/Muc1 coIP as described in (B).