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Protein kinase C isozymes regulate matrix metalloproteinase-1 expression and cell invasion in Helicobacter pylori infection.

Sokolova O, Vieth M, Naumann M - Gut (2012)

Bottom Line: Phospholipase C, phosphatidylinositol 3-kinase and Ca(2+) were crucial for PKC activation on infection; inhibition of PKC diminished AP-1 induction and, subsequently, MMP-1 expression.In addition, analysis of biopsies from human gastric mucosa showed increased phosphorylation of PKC in active H pylori gastritis and gastric adenocarcinoma.The targeting of certain PKC isozymes might represent a suitable strategy to interfere with the MMP-1-dependent remodelling of infected tissue and to overcome the invasive behaviour of gastric cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Medical Faculty, Institute of Experimental Internal Medicine, Otto von Guericke University, Leipziger Str. 44, 39120 Magdeburg, Germany. olga.sokolova@med.ovgu.de

ABSTRACT

Background: Protein kinase C (PKC) signalling is often dysregulated in gastric cancer and therefore represents a potential target in cancer therapy. The Gram-negative bacterium Helicobacter pylori, which colonises the human stomach, plays a major role in the development of gastritis, peptic ulcer and gastric adenocarcinoma.

Objective: To analyse the role of PKC isozymes as mediators of H pylori-induced pathogenesis.

Methods: PKC phosphorylation was evaluated by immunoblotting and immunohistochemistry. Gene reporter assays, RT-PCR and invasion assays were performed to assess the role of PKC in the regulation of activator protein-1 (AP-1), matrix metalloproteinase-1 (MMP-1) and the invasion of H pylori-infected epithelial cells.

Results: H pylori induced phosphorylation of PKC isozymes α, δ, θ in AGS cells, which was accompanied by the phosphorylation of PKC substrates, including PKCμ and myristoylated alanine-rich C kinase substrate (MARCKS), in a CagA-independent manner. Phospholipase C, phosphatidylinositol 3-kinase and Ca(2+) were crucial for PKC activation on infection; inhibition of PKC diminished AP-1 induction and, subsequently, MMP-1 expression. Invasion assays confirmed PKC involvement in H pylori-induced MMP-1 secretion. In addition, analysis of biopsies from human gastric mucosa showed increased phosphorylation of PKC in active H pylori gastritis and gastric adenocarcinoma.

Conclusion: The targeting of certain PKC isozymes might represent a suitable strategy to interfere with the MMP-1-dependent remodelling of infected tissue and to overcome the invasive behaviour of gastric cancer cells.

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H pylori stimulates the invasive properties of AGS cells in a PKC-dependent fashion. (A) The cells were treated with control or MMP-1-targeting siRNAs, applied to the Transwell plate and further incubated with H pylori P1 wt, cagA and virB7 mutants, or PMA (4 nM) for 18 h, and the percentage invasion through collagen I-coated filters towards 5% FCS was determined. The migration rate through uncoated filters served as a methodological control. (B) The immunoblot analysis of the cells treated with scrambled or MMP-1-targeting siRNAs and infected with P12 wt for 3 h. (C–E) The invasion assay was performed using cells treated with BIS I (C) or PKC-targeting siRNAs and then stimulated with H pylori P12 wt or PMA. (E) The invasion assay was performed using cells overexpressing constitutively active PKC isozymes. (F) H pylori's T4SS and T4SS-independent factors are required for PKC activation and MMP-1 up-regulation. *p<0.05, **p<0.01 versus non-stimulated cells, #p<0.05, versus stimulated cells, ##p<0.01 versus stimulated mock-transfected cells.
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fig6: H pylori stimulates the invasive properties of AGS cells in a PKC-dependent fashion. (A) The cells were treated with control or MMP-1-targeting siRNAs, applied to the Transwell plate and further incubated with H pylori P1 wt, cagA and virB7 mutants, or PMA (4 nM) for 18 h, and the percentage invasion through collagen I-coated filters towards 5% FCS was determined. The migration rate through uncoated filters served as a methodological control. (B) The immunoblot analysis of the cells treated with scrambled or MMP-1-targeting siRNAs and infected with P12 wt for 3 h. (C–E) The invasion assay was performed using cells treated with BIS I (C) or PKC-targeting siRNAs and then stimulated with H pylori P12 wt or PMA. (E) The invasion assay was performed using cells overexpressing constitutively active PKC isozymes. (F) H pylori's T4SS and T4SS-independent factors are required for PKC activation and MMP-1 up-regulation. *p<0.05, **p<0.01 versus non-stimulated cells, #p<0.05, versus stimulated cells, ##p<0.01 versus stimulated mock-transfected cells.

Mentions: To further analyse the role of PKC in MMP-1 secretion, invasion assays using collagen I-coated filters were performed. Figure 6A shows that co-culturing of AGS cells with H pylori led to enhanced cellular invasion, which was less prominent on infection with the virB7 mutant strain in comparison to the wt and cagA strains. To assess the role of MMP-1 in H pylori-induced invasiveness, AGS cells were transfected with siRNA targeting MMP-1. Depletion of MMP-1 inhibited both basal and H pylori-induced MMP-1 expression in AGS cells (figure 6B) and suppressed invasion in response to H pylori (figure 6A). Treatment of the cells with the PKC inhibitor BIS I prior to infection markedly reduced the number of invading cells (figure 6C). Similar results were obtained for PMA.


Protein kinase C isozymes regulate matrix metalloproteinase-1 expression and cell invasion in Helicobacter pylori infection.

Sokolova O, Vieth M, Naumann M - Gut (2012)

H pylori stimulates the invasive properties of AGS cells in a PKC-dependent fashion. (A) The cells were treated with control or MMP-1-targeting siRNAs, applied to the Transwell plate and further incubated with H pylori P1 wt, cagA and virB7 mutants, or PMA (4 nM) for 18 h, and the percentage invasion through collagen I-coated filters towards 5% FCS was determined. The migration rate through uncoated filters served as a methodological control. (B) The immunoblot analysis of the cells treated with scrambled or MMP-1-targeting siRNAs and infected with P12 wt for 3 h. (C–E) The invasion assay was performed using cells treated with BIS I (C) or PKC-targeting siRNAs and then stimulated with H pylori P12 wt or PMA. (E) The invasion assay was performed using cells overexpressing constitutively active PKC isozymes. (F) H pylori's T4SS and T4SS-independent factors are required for PKC activation and MMP-1 up-regulation. *p<0.05, **p<0.01 versus non-stimulated cells, #p<0.05, versus stimulated cells, ##p<0.01 versus stimulated mock-transfected cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3585490&req=5

fig6: H pylori stimulates the invasive properties of AGS cells in a PKC-dependent fashion. (A) The cells were treated with control or MMP-1-targeting siRNAs, applied to the Transwell plate and further incubated with H pylori P1 wt, cagA and virB7 mutants, or PMA (4 nM) for 18 h, and the percentage invasion through collagen I-coated filters towards 5% FCS was determined. The migration rate through uncoated filters served as a methodological control. (B) The immunoblot analysis of the cells treated with scrambled or MMP-1-targeting siRNAs and infected with P12 wt for 3 h. (C–E) The invasion assay was performed using cells treated with BIS I (C) or PKC-targeting siRNAs and then stimulated with H pylori P12 wt or PMA. (E) The invasion assay was performed using cells overexpressing constitutively active PKC isozymes. (F) H pylori's T4SS and T4SS-independent factors are required for PKC activation and MMP-1 up-regulation. *p<0.05, **p<0.01 versus non-stimulated cells, #p<0.05, versus stimulated cells, ##p<0.01 versus stimulated mock-transfected cells.
Mentions: To further analyse the role of PKC in MMP-1 secretion, invasion assays using collagen I-coated filters were performed. Figure 6A shows that co-culturing of AGS cells with H pylori led to enhanced cellular invasion, which was less prominent on infection with the virB7 mutant strain in comparison to the wt and cagA strains. To assess the role of MMP-1 in H pylori-induced invasiveness, AGS cells were transfected with siRNA targeting MMP-1. Depletion of MMP-1 inhibited both basal and H pylori-induced MMP-1 expression in AGS cells (figure 6B) and suppressed invasion in response to H pylori (figure 6A). Treatment of the cells with the PKC inhibitor BIS I prior to infection markedly reduced the number of invading cells (figure 6C). Similar results were obtained for PMA.

Bottom Line: Phospholipase C, phosphatidylinositol 3-kinase and Ca(2+) were crucial for PKC activation on infection; inhibition of PKC diminished AP-1 induction and, subsequently, MMP-1 expression.In addition, analysis of biopsies from human gastric mucosa showed increased phosphorylation of PKC in active H pylori gastritis and gastric adenocarcinoma.The targeting of certain PKC isozymes might represent a suitable strategy to interfere with the MMP-1-dependent remodelling of infected tissue and to overcome the invasive behaviour of gastric cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Medical Faculty, Institute of Experimental Internal Medicine, Otto von Guericke University, Leipziger Str. 44, 39120 Magdeburg, Germany. olga.sokolova@med.ovgu.de

ABSTRACT

Background: Protein kinase C (PKC) signalling is often dysregulated in gastric cancer and therefore represents a potential target in cancer therapy. The Gram-negative bacterium Helicobacter pylori, which colonises the human stomach, plays a major role in the development of gastritis, peptic ulcer and gastric adenocarcinoma.

Objective: To analyse the role of PKC isozymes as mediators of H pylori-induced pathogenesis.

Methods: PKC phosphorylation was evaluated by immunoblotting and immunohistochemistry. Gene reporter assays, RT-PCR and invasion assays were performed to assess the role of PKC in the regulation of activator protein-1 (AP-1), matrix metalloproteinase-1 (MMP-1) and the invasion of H pylori-infected epithelial cells.

Results: H pylori induced phosphorylation of PKC isozymes α, δ, θ in AGS cells, which was accompanied by the phosphorylation of PKC substrates, including PKCμ and myristoylated alanine-rich C kinase substrate (MARCKS), in a CagA-independent manner. Phospholipase C, phosphatidylinositol 3-kinase and Ca(2+) were crucial for PKC activation on infection; inhibition of PKC diminished AP-1 induction and, subsequently, MMP-1 expression. Invasion assays confirmed PKC involvement in H pylori-induced MMP-1 secretion. In addition, analysis of biopsies from human gastric mucosa showed increased phosphorylation of PKC in active H pylori gastritis and gastric adenocarcinoma.

Conclusion: The targeting of certain PKC isozymes might represent a suitable strategy to interfere with the MMP-1-dependent remodelling of infected tissue and to overcome the invasive behaviour of gastric cancer cells.

Show MeSH
Related in: MedlinePlus