Yonsei Med J.  2018 Jul;59(5):633-642. 10.3349/ymj.2018.59.5.633.

Effects of the Helicobacter pylori Virulence Factor CagA and Ammonium Ion on Mucins in AGS Cells

• Zhang X
• Shi D
• Liu YP
• Chen WJ
• Wu D

• KMID: 2412654
• DOI: http://doi.org/10.3349/ymj.2018.59.5.633

Abstract

PURPOSE
To investigate the effects of Helicobacter pylori (H. pylori)-CagA and the urease metabolite NH₄⁺ on mucin expression in AGS cells.
MATERIALS AND METHODS
AGS cells were transfected with CagA and/or treated with different concentrations of NH₄⁺CL. Mucin gene and protein expression was assessed by qPCR and immunofluorescence assays, respectively.
RESULTS
CagA significantly upregulated MUC5AC, MUC2, and MUC5B expression in AGS cells, but did not affect E-cadherin and MUC6 expression. MUC5AC, MUC6, and MUC2 expression in AGS cells increased with increasing NH₄⁺ concentrations until reaching a peak level at 15 mM. MUC5B mRNA expression in AGS cells (NH₄⁺ concentration of 15 mM) was significantly higher than that at 0, 5, and 10 mM NH₄⁺. No changes in E-cadherin expression in AGS cells treated with NH₄⁺ were noted, except at 20 mM. The expression of MUC5AC, MUC2, and MUC6 mRNA in CagA-transfected AGS cells at an NH₄⁺ concentration of 15 mM was significantly NH₄⁺ concentration, and was significantly higher compared to that in untreated cells. No significant change in the expression of E-cadherin mRNA in CagA-transfected AGS cells was observed. Immunofluorescence assays confirmed the observed changes.
CONCLUSION
H. pylori may affect the expression of MUC5AC, MUC2, MUC5B, and MUC6 in AGS cells via CagA and/or NH₄⁺, but not E-cadherin.

Keyword

Mucin; Helicobacter pylori; CagA; ammonium chloride; stomach

MeSH Terms

Ammonium Chloride
Ammonium Compounds*
Cadherins
Fluorescent Antibody Technique
Helicobacter pylori*
Helicobacter*
Mucins*
RNA, Messenger
Stomach
Urease
Virulence*
Ammonium Chloride
Cadherins
Mucins
RNA, Messenger
Urease

Figure

• 

  Fig. 1 CagA mRNA expression examined by qPCR in differently treated AGS cells. CagA mRNA expression was not detected in the CK and pCDNA3.1 cells, and was significantly expressed in pCDNA3.1-CagA cells. pCDNA3.1-CagA cells vs. pCDNA3.1 cells (p<0.001); pCDNA3.1-CagA cells vs. CK cells (p<0.001). CK, control cells; pCDNA3.1, empty-plasmid transfected cells; pCDNA3.1-CagA, cells transfected with CagA expression vector.

• 

  Fig. 2 CagA protein expression examined by western blotting in AGS cells. CagA protein was expressed in pCDNA3.1-CagA cells but not in CK and pCDNA3.1 cells. CK, control cells; pCDNA3.1, empty-plasmid transfected cells; pCDNA3.1-CagA, cells transfected with CagA expression vector.

• 

  Fig. 3 MUC5AC, MUC5B, MUC2, MUC6, and E-cadherin mRNA expression levels examined by qPCR. MUC5AC, MUC2, and MUC5B expression levels in pCDNA3.1-CagA AGS cells were significantly higher than those in pCDNA3.1 and CK cells, respectively (*p<0.01; **p<0.001). MUC6 mRNA expression in pCDNA3.1-CagA cells was higher than that in pCDNA3.1 cells (p<0.001), and was similar to that in CK cells (p>0.05). E-cadherin expression in pCDNA3.1-CagA cells did not differ significantly from that in CK cells (p>0.05) or pCDNA3.1 cells (p>0.05). CK, control cells; pCDNA3.1, empty-plasmid transfected cells; pCDNA3.1-CagA, cells transfected with CagA expression vector.

• 

  Fig. 4 Expression of mucins as detected by immunofluorescence assays (×200). The intensities of MUC5AC, MUC5B, and MUC2 in pCDNA3.1-CagA cells were significantly higher than those in CK and pCDNA3.1 cells. The intensity of MUC6 in pCDNA3.1-CagA cells was significantly higher than that in the pCDNA3.1 cells, but similar to that in CK cells. The intensity of E-cadherin protein in pCDNA3.1-CagA cells was similar to that in pCDNA3.1 and CK cells. CK, control cells; pCDNA3.1, empty-plasmid transfected cells; pCDNA3.1-CagA, cells transfected with CagA expression vector.

• 

  Fig. 5 qPCR analysis of mRNA expression of mucins in AGS cells treated with different NH4+ concentrations. MUC5AC, MUC6, and MUC2 expression increased with increases in NH4+ concentration, reaching peak expression at 15 mM, which was significantly higher than that in untreated cells (*p<0.05, **p<0.01, ***p<0.001). The expression level of MUC5B mRNA in AGS cells at 15 mM of NH4+ was significantly higher than those at 0, 5, and 10 mM (*p<0.05). No significant change in the expression of E-cadherin mRNA was observed with NH4+ concentrations below 20 mM. CK, control cells.

• 

  Fig. 6 Protein expression of different mucins in AGS cells exposed to 0, 5, 10, 15, and 20 mM NH4Cl as detected by immunofluorescence assays (×200). CK, control cells.

• 

  Fig. 7 qPCR analysis of mRNA expression of mucins in CagA-transfected AGS cells treated with different NH4+ concentrations. The expression levels of MUC5AC, MUC2, and MUC6 mRNA in AGS cells transfected with CagA at 15 mM of NH4+ were significantly higher than those at 0 mM (*p<0.05, **p<0.01). The expression level of MUC5B mRNA increased with the increase in NH4+ concentration, and was significantly higher than that in untreated cells (*p<0.05, **p<0.01). No significant change in the expression of E-cadherin mRNA was observed under different NH4+ concentrations. CK, control cells.

• 

  Fig. 8 Protein expression of different mucins in CagA-transfected AGS cells exposed to 0, 5, 10, 15, and 20 mM NH4Cl as detected by immunofluorescence assays (×200). CK, control cells.

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