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Helicobacter pylori Activates HMGB1 Expression and Recruits RAGE into Lipid Rafts to Promote Inflammation in Gastric Epithelial Cells

View Article: PubMed Central - PubMed

ABSTRACT

Helicobacter pylori infection is associated with several gastrointestinal disorders in the human population worldwide. High-mobility group box 1 (HMGB1), a ubiquitous nuclear protein, mediates various inflammation functions. The interaction between HMGB1 and receptor for advanced glycation end-products (RAGE) triggers nuclear factor (NF)-κB expression, which in turn stimulates the release of proinflammatory cytokines, such as interleukin (IL)-8, and enhances the inflammatory response. However, how H. pylori activates HMGB1 expression and mobilizes RAGE into cholesterol-rich microdomains in gastric epithelial cells to promote inflammation has not been explored. In this study, we found that HMGB1 and RAGE expression increased significantly in H. pylori-infected cells compared with -uninfected cells. Blocking HMGB1 by neutralizing antibody abrogated H. pylori-elicited RAGE, suggesting that RAGE expression follows HMGB1 production, and silenced RAGE-attenuated H. pylori-mediated NF-κB activation and IL-8 production. Furthermore, significantly more RAGE was present in detergent-resistant membranes extracted from H. pylori-infected cells than in those from -uninfected cells, indicating that H. pylori exploited cholesterol to induce the HMGB1 signaling pathway. These results indicate that HMGB1 plays a crucial role in H. pylori-induced inflammation in gastric epithelial cells, which may be valuable in developing treatments for H. pylori-associated diseases.

No MeSH data available.


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Role of cholesterol-rich microdomains in H. pylori-induced HMGB1 and RAGE expression. AGS cells were untreated or pretreated with 5 mM MβCD at 37°C for 1 h. Cells were then washed and infected with H. pylori at an MOI of 100 for 6 h. (A) Detergent-resistant membrane (DRM) and detergent-soluble (S) fractions were prepared and subjected to cold detergent extraction using 1% Triton X-100 at 4°C followed by centrifugation. Each fraction was analyzed by dot blot or Western blot using cholera toxin subunit B (CTX-B) conjugated to horseradish peroxidase or antibodies against HMGB1 and RAGE, respectively. Protein expression levels of (B) HMGB1 and (C) RAGE were quantified by densitometric analysis (*P < 0.05).
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Figure 7: Role of cholesterol-rich microdomains in H. pylori-induced HMGB1 and RAGE expression. AGS cells were untreated or pretreated with 5 mM MβCD at 37°C for 1 h. Cells were then washed and infected with H. pylori at an MOI of 100 for 6 h. (A) Detergent-resistant membrane (DRM) and detergent-soluble (S) fractions were prepared and subjected to cold detergent extraction using 1% Triton X-100 at 4°C followed by centrifugation. Each fraction was analyzed by dot blot or Western blot using cholera toxin subunit B (CTX-B) conjugated to horseradish peroxidase or antibodies against HMGB1 and RAGE, respectively. Protein expression levels of (B) HMGB1 and (C) RAGE were quantified by densitometric analysis (*P < 0.05).

Mentions: We further investigated whether H. pylori-induced HMGB1 and RAGE expression required lipid raft integrity. Western blot analysis showed that CTX-B was enriched in the detergent-resistant membrane (DRM) fraction (Figure 7A), whereas disrupting lipid rafts with MβCD reduced the presence of CTX-B in the DRM. During H. pylori infection, HMGB1 and RAGE were abundant in the DRM fraction. Moreover, treatment of cells with MβCD led to a significant reduction in H. pylori-induced HMGB1 and RAGE expression in the DRM (Figures 7B,C), suggesting that cholesterol-rich microdomains play an important role in H. pylori-triggered HMGB1 and RAGE expression.


Helicobacter pylori Activates HMGB1 Expression and Recruits RAGE into Lipid Rafts to Promote Inflammation in Gastric Epithelial Cells
Role of cholesterol-rich microdomains in H. pylori-induced HMGB1 and RAGE expression. AGS cells were untreated or pretreated with 5 mM MβCD at 37°C for 1 h. Cells were then washed and infected with H. pylori at an MOI of 100 for 6 h. (A) Detergent-resistant membrane (DRM) and detergent-soluble (S) fractions were prepared and subjected to cold detergent extraction using 1% Triton X-100 at 4°C followed by centrifugation. Each fraction was analyzed by dot blot or Western blot using cholera toxin subunit B (CTX-B) conjugated to horseradish peroxidase or antibodies against HMGB1 and RAGE, respectively. Protein expression levels of (B) HMGB1 and (C) RAGE were quantified by densitometric analysis (*P < 0.05).
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Related In: Results  -  Collection

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Figure 7: Role of cholesterol-rich microdomains in H. pylori-induced HMGB1 and RAGE expression. AGS cells were untreated or pretreated with 5 mM MβCD at 37°C for 1 h. Cells were then washed and infected with H. pylori at an MOI of 100 for 6 h. (A) Detergent-resistant membrane (DRM) and detergent-soluble (S) fractions were prepared and subjected to cold detergent extraction using 1% Triton X-100 at 4°C followed by centrifugation. Each fraction was analyzed by dot blot or Western blot using cholera toxin subunit B (CTX-B) conjugated to horseradish peroxidase or antibodies against HMGB1 and RAGE, respectively. Protein expression levels of (B) HMGB1 and (C) RAGE were quantified by densitometric analysis (*P < 0.05).
Mentions: We further investigated whether H. pylori-induced HMGB1 and RAGE expression required lipid raft integrity. Western blot analysis showed that CTX-B was enriched in the detergent-resistant membrane (DRM) fraction (Figure 7A), whereas disrupting lipid rafts with MβCD reduced the presence of CTX-B in the DRM. During H. pylori infection, HMGB1 and RAGE were abundant in the DRM fraction. Moreover, treatment of cells with MβCD led to a significant reduction in H. pylori-induced HMGB1 and RAGE expression in the DRM (Figures 7B,C), suggesting that cholesterol-rich microdomains play an important role in H. pylori-triggered HMGB1 and RAGE expression.

View Article: PubMed Central - PubMed

ABSTRACT

Helicobacter pylori infection is associated with several gastrointestinal disorders in the human population worldwide. High-mobility group box 1 (HMGB1), a ubiquitous nuclear protein, mediates various inflammation functions. The interaction between HMGB1 and receptor for advanced glycation end-products (RAGE) triggers nuclear factor (NF)-&kappa;B expression, which in turn stimulates the release of proinflammatory cytokines, such as interleukin (IL)-8, and enhances the inflammatory response. However, how H. pylori activates HMGB1 expression and mobilizes RAGE into cholesterol-rich microdomains in gastric epithelial cells to promote inflammation has not been explored. In this study, we found that HMGB1 and RAGE expression increased significantly in H. pylori-infected cells compared with -uninfected cells. Blocking HMGB1 by neutralizing antibody abrogated H. pylori-elicited RAGE, suggesting that RAGE expression follows HMGB1 production, and silenced RAGE-attenuated H. pylori-mediated NF-&kappa;B activation and IL-8 production. Furthermore, significantly more RAGE was present in detergent-resistant membranes extracted from H. pylori-infected cells than in those from -uninfected cells, indicating that H. pylori exploited cholesterol to induce the HMGB1 signaling pathway. These results indicate that HMGB1 plays a crucial role in H. pylori-induced inflammation in gastric epithelial cells, which may be valuable in developing treatments for H. pylori-associated diseases.

No MeSH data available.


Related in: MedlinePlus