Limits...
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.

Show MeSH

Related in: MedlinePlus

H pylori up-regulates MMP-1 in a protein kinase C (PKC)-dependent manner. BIS I-treated or non-treated AGS cells were incubated with H pylori P12 wt, PMA (A–D) or H pylori P1 wt or the cagA and virB7 mutants (E, F) for 3 h or for the indicated periods of time. MMP-1 expression was analysed by qRT-PCR (A, C, E) or immunoblotting (B, D, F). The graphs in (B) summarise the densitometric analysis of three independent immunoblots (experiments). GAPDH, occludin and histone H3 were immunodetected to show the appropriate fractionation and equal protein amounts in the cell samples. Bacterial lysate was used as a negative control. *p<0.05, **p<0.01 versus non-stimulated cells; #p<0.05, ##p<0.01 versus BIS I-free stimulated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: H pylori up-regulates MMP-1 in a protein kinase C (PKC)-dependent manner. BIS I-treated or non-treated AGS cells were incubated with H pylori P12 wt, PMA (A–D) or H pylori P1 wt or the cagA and virB7 mutants (E, F) for 3 h or for the indicated periods of time. MMP-1 expression was analysed by qRT-PCR (A, C, E) or immunoblotting (B, D, F). The graphs in (B) summarise the densitometric analysis of three independent immunoblots (experiments). GAPDH, occludin and histone H3 were immunodetected to show the appropriate fractionation and equal protein amounts in the cell samples. Bacterial lysate was used as a negative control. *p<0.05, **p<0.01 versus non-stimulated cells; #p<0.05, ##p<0.01 versus BIS I-free stimulated cells.

Mentions: While studying the role of PKC activation, we observed that BIS I significantly inhibited MMP-1 gene expression (figure 3A) and protein accumulation (figure 3B) both in H pylori-infected and in PMA-treated AGS cells. MMP-1 expression following infection with P12 wt reached a maximum at 6 h post-infection (figure 3C), and MMP-1 accumulated in the membranes and nuclei of infected cells (figure 3D). MMP-1 gene up-regulation depended on the strain used for infection, and the P1 strain was less potent in inducing MMP-1 than the P12 strain (figure 3A,E). The cagA H pylori mutant was as effective as the wt, but the virB7 mutant up-regulated MMP-1 to a lesser extent (figure 3E,F).


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 up-regulates MMP-1 in a protein kinase C (PKC)-dependent manner. BIS I-treated or non-treated AGS cells were incubated with H pylori P12 wt, PMA (A–D) or H pylori P1 wt or the cagA and virB7 mutants (E, F) for 3 h or for the indicated periods of time. MMP-1 expression was analysed by qRT-PCR (A, C, E) or immunoblotting (B, D, F). The graphs in (B) summarise the densitometric analysis of three independent immunoblots (experiments). GAPDH, occludin and histone H3 were immunodetected to show the appropriate fractionation and equal protein amounts in the cell samples. Bacterial lysate was used as a negative control. *p<0.05, **p<0.01 versus non-stimulated cells; #p<0.05, ##p<0.01 versus BIS I-free stimulated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: H pylori up-regulates MMP-1 in a protein kinase C (PKC)-dependent manner. BIS I-treated or non-treated AGS cells were incubated with H pylori P12 wt, PMA (A–D) or H pylori P1 wt or the cagA and virB7 mutants (E, F) for 3 h or for the indicated periods of time. MMP-1 expression was analysed by qRT-PCR (A, C, E) or immunoblotting (B, D, F). The graphs in (B) summarise the densitometric analysis of three independent immunoblots (experiments). GAPDH, occludin and histone H3 were immunodetected to show the appropriate fractionation and equal protein amounts in the cell samples. Bacterial lysate was used as a negative control. *p<0.05, **p<0.01 versus non-stimulated cells; #p<0.05, ##p<0.01 versus BIS I-free stimulated cells.
Mentions: While studying the role of PKC activation, we observed that BIS I significantly inhibited MMP-1 gene expression (figure 3A) and protein accumulation (figure 3B) both in H pylori-infected and in PMA-treated AGS cells. MMP-1 expression following infection with P12 wt reached a maximum at 6 h post-infection (figure 3C), and MMP-1 accumulated in the membranes and nuclei of infected cells (figure 3D). MMP-1 gene up-regulation depended on the strain used for infection, and the P1 strain was less potent in inducing MMP-1 than the P12 strain (figure 3A,E). The cagA H pylori mutant was as effective as the wt, but the virB7 mutant up-regulated MMP-1 to a lesser extent (figure 3E,F).

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