Abstract
BackgroundHelicobacter pylori colonises the stomach of approximately 50% of the global population. Cytotoxin-associated gene A protein (CagA) is one of the important virulent factors responsible for the increased inflammation and increases the risk of developing peptic ulcers and gastric carcinoma. The cytokine interleukin-6 (IL-6) has particularly important roles in the malignant transformation of gastric and intestinal epithelial cells as it is upregulated in H. pylori-infected gastric mucosa. In this study, we investigated the underlying mechanisms of CagA-induced IL-6 up-regulation during H. pylori infection. AGS cells, a human gastric adenocarcinoma cell line, lacking eEF1A1 were infected with CagA+H. pylori (NCTC11637), CagA−H. pylori (NCTC11637ΔcagA), or transduced by Ad-cagA/Ad-GFP. The expression and production of IL-6 were measured by quantitative real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The interactions among CagA, eukaryotic translation elongation factor 1-alpha 1 (eEF1A1), protein kinase Cδ (PKCδ), and signal transducer and activator of transcription 3 (STAT3) were determined by western blot or co-immunoprecipitation.ResultsDuring H. pylori infection, CagA-M (residues 256‒871aa) was found to interact with eEF1A1-I (residues 1‒240aa). NCTC11637 increased the expression of IL-6 in AGS cells compared with NCTC11637ΔcagA whereas knockdown of eEF1A1 in AGS cells completely abrogated these effects. Moreover, the CagA-eEF1A1 complex promoted the expression of IL-6 in AGS cells. CagA and eEF1A1 cooperated to mediate the expression of IL-6 by affecting the activity of p-STATS727 in the nucleus. Further, CagA-eEF1A1 affected the activity of STAT3 by recruiting PKCδ. However, blocking PKCδ inhibited the phosphorylation of STAT3S727 and induction of IL-6 by CagA.ConclusionsCagA promotes the expression of IL-6 in AGS cells by recruiting PKCδ through eEF1A1 in the cytoplasm to increase the phosphorylation of STAT3S727 in the nucleus. These findings provide new insights into the function of CagA-eEF1A1 interaction in gastric adenocarcinoma.
Highlights
Helicobacter pylori colonises the stomach of approximately 50% of the global population
We investigated whether Cytotoxinassociated gene A protein (CagA) interacted with endogenous eukaryotic translation elongation factor 1-alpha 1 (eEF1A1)
CagA was found to clearly upregulate the expression of IL-6 by enhancing the recruitment of protein kinase Cδ (PKCδ) via eEF1A1 in the cytoplasm, thereby increasing p-STAT3S727 phosphorylation in the nucleus. These results suggest that CagA-eEF1A1-PKCδp-STAT3S727-IL-6 serves as an important inflammatory pathway in H. pylori-related gastric cancer cells at the molecular level, unveiling the possible pathogenic mechanism of H. pylori infection in gastric cancer (Fig. 5)
Summary
Helicobacter pylori colonises the stomach of approximately 50% of the global population. It is generally thought that, as a bacterial oncoprotein, CagA plays a key role in H. pylori-induced gastric cancer [5, 6] as it affects the expression and function of key proteins involved in oncogenic or tumour suppressor signalling pathways via several molecular mechanisms such as direct binding or interaction, phosphorylation of vital signalling proteins, and methylation of tumour suppressor proteins [7, 8]. Song et al reported that both H. pylori and its toxin stimulate IL-6 expression in gastric epithelial cells [18] This signalling pathway is mediated through protein kinase C (PKC), protein tyrosine kinase, and nuclear factor kappa-beta (NF-κB) activation, and involves an intracellular calcium-and dexamethasone-sensitive mechanism [19]. The underlying mechanism of CagA-induced IL-6 expression is still poorly understood
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