CagA orchestrates eEF1A1 and PKCδ to induce interleukin-6 expression in Helicobacter pylori-infected gastric epithelial cells

Abstract

Background: Helicobacter 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.

Results: During 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-STAT^S727 in the nucleus. Further, CagA-eEF1A1 affected the activity of STAT3 by recruiting PKCδ. However, blocking PKCδ inhibited the phosphorylation of STAT3^S727 and induction of IL-6 by CagA.

Conclusions: CagA promotes the expression of IL-6 in AGS cells by recruiting PKCδ through eEF1A1 in the cytoplasm to increase the phosphorylation of STAT3^S727 in the nucleus. These findings provide new insights into the function of CagA-eEF1A1 interaction in gastric adenocarcinoma.

Keywords: CagA; Gastric adenocarcinoma; Interleukin-6; PKCδ; eEF1A1; p-STAT3S727.

Fig. 1

CagA interacts with eEF1A1. a CagA co-immunoprecipitated with exogenously expressed eEF1A1 in AGS cells. Precipitates were analyzed by western blotting (WB) using anti-CagA and anti-Myc antibodies. b CagA co-immunoprecipitated with endogenous eEF1A1 in AGS cells. Precipitates were analyzed by WB with anti-CagA and anti-eEF1A1 antibodies. c CagA co-immunoprecipitated with exogenous eEF1A1-I in AGS cells. Precipitates were analyzed by WB with anti-CagA and anti-Myc antibodies. d CagA co-immunoprecipitated with endogenous eEF1A1 in Cos-7 cells. Precipitates were analysed by WB with anti-CagA and anti-eEF1A1 antibodies. e CagA-M co-immunoprecipitated with endogenous eEF1A1 in Cos-7 cells. Precipitates were analysed by WB with anti-Flag and anti- eEF1A antibodies

Fig. 2

CagA and eEF1A1 co-mediate the expression of IL-6. a The comparison of IL-6 RNA (1) and IL-6 (2) in AGS cells that infected with NCTC11637 ΔcagA or NCTC11637 bacterial inoculums (MOI = 100, 16 h). b Fluorescence microscope image and eEF1A1 level using western blotting (WB) analysis of AGS cells that transduced with LV-eEF1A1-RNA or LV-CON077-RNA for 48 h. c The levels of IL-6 in AGS-C and AGS-sheEF1A1 cells that infected with blank or NCTC11637 or NCTC11637ΔcagA. d Fluorescence microscope image of AGS cells transduced with Ad-cagA (CagA fusion virus particles) or Ad-GFP (a negative control) (1), CagA was detected by WB analysis (2); The levels of IL-6 in AGS-C and AGS-sheEF1A1 cells that transduced with blank or Ad-GFP or Ad-cagA treated by OSM (100 ng/mL, 24 h) or the drug vehicle (3). Error bar represents the SDs of triplicate experiments. Statistical analysis was performed using Student’s t-tests. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3

CagA-eEF1A1 complex induces IL-6 through STAT3 (S727) phosphorylation. a Western blot analysis to assess STAT3 in AGS-shSTAT3 cells (STAT3 knockdown by transduced with Lv-STAT3-shRNA viruses) and AGS-NC cells (negative control, transduced with Lv-con-shRNA viruses). b The levels of IL-6 in AGS-shSTAT3 and AGS-NC infected with blank or NCTC11637 or NCTC11637ΔcagA. c The levels of p-STAT3^S727 in AGS-shSTAT3 and AGS-NC cells infected with NCTC11637ΔcagA or NCTC11637 using WB analysis. d The levels of p-STAT3^S727 in AGS-C and AGS-sheEF1A1 infected with NCTC11637ΔcagA or NCTC11637 using WB analysis. Error bar represents the SDs of triplicate experiments. Statistical analysis was performed using Student’s t-tests. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4

CagA-eEF1A1-PKCδ complex increases the amount of p-STAT3^S727 in the nucleus. a Co-immunoprecipitation analysis of the precipitates where more PKCδ was recruited by eEF1A1 with the presence of CagA. b Rottlerin (6 µM for 30 min) treated AGS-C and AGS-sheEF1A1 cells were infected with NCTC11637 or NCTC11637ΔcagA bacterial inoculums (MOI = 100) for detection of p-STAT3^S727 and CagA by western blot analysis, and the level of IL-6 in the culture supernatant. c The level of IL-6 of the supernatant of rottlerin (6 µM for 30 min) treated AGS-C and AGS-sheEF1A1 cells transduced with Ad-cagA or Ad-GFP (MOI = 50). Error bar represents the SDs of triplicate experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 5

Postulated mechanism by which CagA promotes the expression of IL-6 in gastric cancer. CagA dysregulated the gp130/NF-κB/STAT3 pathway by recruiting PKCδ through eEF1A1 in the cytoplasm and increasing the accumulation of p-STAT3 ^S727 in the nucleus. CagA, cytotoxin-associated gene A; eEF1A1, eukaryotic translation elongation factor 1-alpha 1; PKC, protein kinase C; IL-6, interleukin-6; STAT3, signal transducer and activator of transcription 3; gp130, glycoprotein 130; NF-κB, nuclear factor kappa-B