Yonsei Med J.  2015 May;56(3):862-866. 10.3349/ymj.2015.56.3.862.

Jak1/Stat3 Is an Upstream Signaling of NF-kappaB Activation in Helicobacter pylori-Induced IL-8 Production in Gastric Epithelial AGS Cells

• Cha B
• Lim JW
• Kim H

• KMID: 2450365
• DOI: http://doi.org/10.3349/ymj.2015.56.3.862

Abstract

Helicobacter pylori (H. pylori) induces the activation of nuclear factor-kB (NF-kappaB) and cytokine expression in gastric epithelial cells. The Janus kinase/signal transducers and activators of transcription (Jak/Stat) cascade is the inflammatory signaling in various cells. The purpose of the present study is to determine whether H. pylori-induced activation of NF-kappaB and the expression of interleukin-8 (IL-8) are mediated by the activation of Jak1/Stat3 in gastric epithelial (AGS) cells. Thus, gastric epithelial AGS cells were infected with H. pylori in Korean isolates (HP99) at bacterium/cell ratio of 300:1, and the level of IL-8 in the medium was determined by enzyme-linked immonosorbent assay. Phospho-specific and total forms of Jak1/Stat3 and IkappaBalpha were assessed by Western blot analysis, and NF-kappaB activation was determined by electrophoretic mobility shift assay. The results showed that H. pylori induced the activation of Jak1/Stat3 and IL-8 production, which was inhibited by a Jak/Stat3 specific inhibitor AG490 in AGS cells in a dose-dependent manner. H. pylori-induced activation of NF-kappaB, determined by phosphorylation of IkappaBalpha and NF-kappaB-DNA binding activity, were inhibited by AG490. In conclusion, Jak1/Stat3 activation may mediate the activation of NF-kappaB and the expression of IL-8 in H. pylori-infected AGS cells. Inhibition of Jak1/Stat3 may be beneficial for the treatment of H. pylori-induced gastric inflammation, since the activation of NF-kappaB is inhibited and inflammatory cytokine expression is suppressed.

Keyword

Helicobacter pylori; Jak1/Stat3; IL-8; NF-kappaB; gastric epithelial cells

MeSH Terms

Blotting, Western
DNA, Bacterial/analysis/genetics
Epithelial Cells/metabolism
Gastric Mucosa/drug effects/*immunology/microbiology
Gene Expression Regulation/drug effects/*immunology
Gene Expression Regulation, Bacterial
Helicobacter Infections/immunology/*metabolism
Helicobacter pylori/genetics/pathogenicity/*physiology
Humans
Interleukin-8/genetics/*metabolism
Janus Kinase 1
NF-kappa B/biosynthesis/*metabolism
Phosphorylation
RNA, Messenger/metabolism
STAT3 Transcription Factor
Signal Transduction/genetics
DNA, Bacterial
Interleukin-8
Janus Kinase 1
NF-kappa B
RNA, Messenger
STAT3 Transcription Factor

Figure

• 

  Fig. 1 Effect of AG490 on H. pylori-induced increase of IL-8 levels in the medium, activation of Jak1/Stat3, phosphorylation of IκBα, and NF-κB-DNA binding activity in AGS cells. The cells were pre-treated with AG490 for 2 h and cultured in the presence of H. pylori for 24 h (for IL-8 levels in the medium, A), 30 min (for Jak1/Stat3 activation, B), and 1 h [for protein levels of IκBα and p-IκBα (C) and NF-κB-DNA binding activity (D)]. IL-8 level in the medium was determined by enzyme-linked immunosorbent assay. Protein levels of Jak1, p-Jak1, Stat3, p-Stat3, IκBα, and p-IκBα were determined by Western blot analysis. Actin was used for protein loading control. Electrophoretic mobility shift assay was performed for NF-κB-DNA binding activity. All values are expressed as mean±S.E. of four different experiments. A value of p<0.05 was considered statistically significant. *p<0.05 vs. none (the cells cultured in the absence of H. pylori without treatment of AG490), †p<0.05 vs. H. pylori control (the cells cultured in the presence of H. pylori without treatment of AG490). H. pylori, Helicobacter pylori; IL-8, interleukin-8; Stat, signal transducers and activators of transcription; Jak, Janus kinase; AGS, gastric adenocarcinoma.

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