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 activates protein kinase C (PKC). (A) The protein domains of the PKC family members, showing the pseudosubstrate (dark blue rectangle), the C1 domain that binds DAG, phosphatidylserine and phorbol esters, the C2 domain that binds Ca2+ or PIP2 (in the case of nPKC), and the C3 kinase domain. Also shown in orange are the conserved Ser/Thr residues phosphorylated during H pylori infection. (B) AGS cells were infected with H pylori P1 wt, cagA or virB7 mutants for different periods of time or were stimulated with PMA for 1 h. Cell lysates were analysed by immunoblotting using antibodies as indicated. Unphosphorylated PKCθ and PKCμ served as loading controls. (C) Analysis of phosphorylation of PKC substrates in cells treated as described in (B). GAPDH was immunodetected to show equal protein amounts in the cell samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: H pylori activates protein kinase C (PKC). (A) The protein domains of the PKC family members, showing the pseudosubstrate (dark blue rectangle), the C1 domain that binds DAG, phosphatidylserine and phorbol esters, the C2 domain that binds Ca2+ or PIP2 (in the case of nPKC), and the C3 kinase domain. Also shown in orange are the conserved Ser/Thr residues phosphorylated during H pylori infection. (B) AGS cells were infected with H pylori P1 wt, cagA or virB7 mutants for different periods of time or were stimulated with PMA for 1 h. Cell lysates were analysed by immunoblotting using antibodies as indicated. Unphosphorylated PKCθ and PKCμ served as loading controls. (C) Analysis of phosphorylation of PKC substrates in cells treated as described in (B). GAPDH was immunodetected to show equal protein amounts in the cell samples.

Mentions: The PKC family consists of at least 10 isozymes classified into three main groups (figure 1A). Conventional PKC (cPKC) α, βI, βII and γ bind Ca2+ and phosphatidylserine and require diacylglycerol (DAG) for further activation. The novel PKC (nPKC) δ, ɛ, θ, η possess a functional C1 domain, but their C2-like domains do not contain Ca2+-binding residues. Therefore, nPKC isozymes are regulated by DAG and phosphatidylserine, but not by Ca2+. The atypical PKCs (PKCζ and PKCλ) lack both functional C1 and C2 domains and are neither Ca2+- nor DAG-dependent.5


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 activates protein kinase C (PKC). (A) The protein domains of the PKC family members, showing the pseudosubstrate (dark blue rectangle), the C1 domain that binds DAG, phosphatidylserine and phorbol esters, the C2 domain that binds Ca2+ or PIP2 (in the case of nPKC), and the C3 kinase domain. Also shown in orange are the conserved Ser/Thr residues phosphorylated during H pylori infection. (B) AGS cells were infected with H pylori P1 wt, cagA or virB7 mutants for different periods of time or were stimulated with PMA for 1 h. Cell lysates were analysed by immunoblotting using antibodies as indicated. Unphosphorylated PKCθ and PKCμ served as loading controls. (C) Analysis of phosphorylation of PKC substrates in cells treated as described in (B). GAPDH was immunodetected to show equal protein amounts in the cell samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: H pylori activates protein kinase C (PKC). (A) The protein domains of the PKC family members, showing the pseudosubstrate (dark blue rectangle), the C1 domain that binds DAG, phosphatidylserine and phorbol esters, the C2 domain that binds Ca2+ or PIP2 (in the case of nPKC), and the C3 kinase domain. Also shown in orange are the conserved Ser/Thr residues phosphorylated during H pylori infection. (B) AGS cells were infected with H pylori P1 wt, cagA or virB7 mutants for different periods of time or were stimulated with PMA for 1 h. Cell lysates were analysed by immunoblotting using antibodies as indicated. Unphosphorylated PKCθ and PKCμ served as loading controls. (C) Analysis of phosphorylation of PKC substrates in cells treated as described in (B). GAPDH was immunodetected to show equal protein amounts in the cell samples.
Mentions: The PKC family consists of at least 10 isozymes classified into three main groups (figure 1A). Conventional PKC (cPKC) α, βI, βII and γ bind Ca2+ and phosphatidylserine and require diacylglycerol (DAG) for further activation. The novel PKC (nPKC) δ, ɛ, θ, η possess a functional C1 domain, but their C2-like domains do not contain Ca2+-binding residues. Therefore, nPKC isozymes are regulated by DAG and phosphatidylserine, but not by Ca2+. The atypical PKCs (PKCζ and PKCλ) lack both functional C1 and C2 domains and are neither Ca2+- nor DAG-dependent.5

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