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Increased expression of the urokinase plasminogen activator system by Helicobacter pylori in gastric epithelial cells.

Kenny S, Duval C, Sammut SJ, Steele I, Pritchard DM, Atherton JC, Argent RH, Dimaline R, Dockray GJ, Varro A - Am. J. Physiol. Gastrointest. Liver Physiol. (2008)

Bottom Line: Real-time PCR of biopsies from gastric corpus, but not antrum, showed significantly increased PAI-1, uPA, and uPAR in H. pylori-positive patients.H. pylori also stimulated soluble and cell surface-bound uPA activity, and both were further increased by PAI-1 knockdown, consistent with PAI-1 inhibition of endogenous uPA.The proliferative effects of uPA were inhibited by immunoneutralization of the EGF receptor and of heparin-binding EGF (HB-EGF) by the mutant diphtheria toxin CRM197 and an EGF receptor tyrosine kinase inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown St., Liverpool L69 3BX, UK.

ABSTRACT
The gastric pathogen Helicobacter pylori (H. pylori) is linked to peptic ulcer and gastric cancer, but the relevant pathophysiological mechanisms are unclear. We now report that H. pylori stimulates the expression of plasminogen activator inhibitor (PAI)-1, urokinase plasminogen activator (uPA), and its receptor (uPAR) in gastric epithelial cells and the consequences for epithelial cell proliferation. Real-time PCR of biopsies from gastric corpus, but not antrum, showed significantly increased PAI-1, uPA, and uPAR in H. pylori-positive patients. Transfection of primary human gastric epithelial cells with uPA, PAI-1, or uPAR promoters in luciferase reporter constructs revealed expression of all three in H+/K+ATPase- and vesicular monoamine transporter 2-expressing cells; uPA was also expressed in pepsinogen- and uPAR-containing trefoil peptide-1-expressing cells. In each case expression was increased in response to H. pylori and for uPA, but not PAI-1 or uPAR, required the virulence factor CagE. H. pylori also stimulated soluble and cell surface-bound uPA activity, and both were further increased by PAI-1 knockdown, consistent with PAI-1 inhibition of endogenous uPA. H. pylori stimulated epithelial cell proliferation, which was inhibited by uPA immunoneutralization and uPAR knockdown; exogenous uPA also stimulated proliferation that was further increased after PAI-1 knockdown. The proliferative effects of uPA were inhibited by immunoneutralization of the EGF receptor and of heparin-binding EGF (HB-EGF) by the mutant diphtheria toxin CRM197 and an EGF receptor tyrosine kinase inhibitor. H. pylori induction of uPA therefore leads to epithelial proliferation through activation of HB-EGF and is normally inhibited by concomitant induction of PAI-1; treatments directed at inhibition of uPA may slow the progression to gastric cancer.

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Increased gastric epithelial cell proliferation in response to H. pylori is mediated by uPA. A: H. pylori-stimulated BrdU incorporation of human primary gastric epithelial cells was inhibited by treatment with neutralizing uPA antibody or knockdown of uPAR with siRNA (60 nM) B: uPA (500 ng/ml)-stimulated BrdU incorporation was also inhibited by uPAR knockdown; n = 3, *P < 0.05, ANOVA. Ab, antibody.
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f7: Increased gastric epithelial cell proliferation in response to H. pylori is mediated by uPA. A: H. pylori-stimulated BrdU incorporation of human primary gastric epithelial cells was inhibited by treatment with neutralizing uPA antibody or knockdown of uPAR with siRNA (60 nM) B: uPA (500 ng/ml)-stimulated BrdU incorporation was also inhibited by uPAR knockdown; n = 3, *P < 0.05, ANOVA. Ab, antibody.

Mentions: Treatment of cells with a neutralizing antibody to uPA inhibited the proliferative effect of H. pylori, suggesting a role for endogenous uPA in this response (Fig. 7A). A role for uPAR in the response was indicated by the observation that knockdown of uPAR with siRNA significantly inhibited H. pylori-induced proliferation (Fig. 7A). Immunohistochemistry confirmed that siRNA treatment significantly decreased the number of uPAR-expressing cells in cultured gastric glands (control, 9.1 ± 0.5% of positive cells; uPAR knockdown, 3.0 ± 0.4%), whereas PAI-1 expression was not altered (8.4 ± 0.5% vs. 8.8 ± 0.4%). Consistent with these observations, the increased BrdU incorporation in response to exogenous uPA was also decreased by uPAR knockdown (Fig. 7B). The specificity of the latter effect is indicated by the fact that there was no decrease in BrdU incorporation after uPAR knockdown in response to an exogenous EGF receptor (EGF-R) ligand (EGF, 50 ng/ml) compared with control (EGF alone, 2.14 ± 0.05-fold increase in BrdU incorporation over control; EGF and uPAR knockdown, 2.36 ± 0.03-fold).


Increased expression of the urokinase plasminogen activator system by Helicobacter pylori in gastric epithelial cells.

Kenny S, Duval C, Sammut SJ, Steele I, Pritchard DM, Atherton JC, Argent RH, Dimaline R, Dockray GJ, Varro A - Am. J. Physiol. Gastrointest. Liver Physiol. (2008)

Increased gastric epithelial cell proliferation in response to H. pylori is mediated by uPA. A: H. pylori-stimulated BrdU incorporation of human primary gastric epithelial cells was inhibited by treatment with neutralizing uPA antibody or knockdown of uPAR with siRNA (60 nM) B: uPA (500 ng/ml)-stimulated BrdU incorporation was also inhibited by uPAR knockdown; n = 3, *P < 0.05, ANOVA. Ab, antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2536790&req=5

f7: Increased gastric epithelial cell proliferation in response to H. pylori is mediated by uPA. A: H. pylori-stimulated BrdU incorporation of human primary gastric epithelial cells was inhibited by treatment with neutralizing uPA antibody or knockdown of uPAR with siRNA (60 nM) B: uPA (500 ng/ml)-stimulated BrdU incorporation was also inhibited by uPAR knockdown; n = 3, *P < 0.05, ANOVA. Ab, antibody.
Mentions: Treatment of cells with a neutralizing antibody to uPA inhibited the proliferative effect of H. pylori, suggesting a role for endogenous uPA in this response (Fig. 7A). A role for uPAR in the response was indicated by the observation that knockdown of uPAR with siRNA significantly inhibited H. pylori-induced proliferation (Fig. 7A). Immunohistochemistry confirmed that siRNA treatment significantly decreased the number of uPAR-expressing cells in cultured gastric glands (control, 9.1 ± 0.5% of positive cells; uPAR knockdown, 3.0 ± 0.4%), whereas PAI-1 expression was not altered (8.4 ± 0.5% vs. 8.8 ± 0.4%). Consistent with these observations, the increased BrdU incorporation in response to exogenous uPA was also decreased by uPAR knockdown (Fig. 7B). The specificity of the latter effect is indicated by the fact that there was no decrease in BrdU incorporation after uPAR knockdown in response to an exogenous EGF receptor (EGF-R) ligand (EGF, 50 ng/ml) compared with control (EGF alone, 2.14 ± 0.05-fold increase in BrdU incorporation over control; EGF and uPAR knockdown, 2.36 ± 0.03-fold).

Bottom Line: Real-time PCR of biopsies from gastric corpus, but not antrum, showed significantly increased PAI-1, uPA, and uPAR in H. pylori-positive patients.H. pylori also stimulated soluble and cell surface-bound uPA activity, and both were further increased by PAI-1 knockdown, consistent with PAI-1 inhibition of endogenous uPA.The proliferative effects of uPA were inhibited by immunoneutralization of the EGF receptor and of heparin-binding EGF (HB-EGF) by the mutant diphtheria toxin CRM197 and an EGF receptor tyrosine kinase inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown St., Liverpool L69 3BX, UK.

ABSTRACT
The gastric pathogen Helicobacter pylori (H. pylori) is linked to peptic ulcer and gastric cancer, but the relevant pathophysiological mechanisms are unclear. We now report that H. pylori stimulates the expression of plasminogen activator inhibitor (PAI)-1, urokinase plasminogen activator (uPA), and its receptor (uPAR) in gastric epithelial cells and the consequences for epithelial cell proliferation. Real-time PCR of biopsies from gastric corpus, but not antrum, showed significantly increased PAI-1, uPA, and uPAR in H. pylori-positive patients. Transfection of primary human gastric epithelial cells with uPA, PAI-1, or uPAR promoters in luciferase reporter constructs revealed expression of all three in H+/K+ATPase- and vesicular monoamine transporter 2-expressing cells; uPA was also expressed in pepsinogen- and uPAR-containing trefoil peptide-1-expressing cells. In each case expression was increased in response to H. pylori and for uPA, but not PAI-1 or uPAR, required the virulence factor CagE. H. pylori also stimulated soluble and cell surface-bound uPA activity, and both were further increased by PAI-1 knockdown, consistent with PAI-1 inhibition of endogenous uPA. H. pylori stimulated epithelial cell proliferation, which was inhibited by uPA immunoneutralization and uPAR knockdown; exogenous uPA also stimulated proliferation that was further increased after PAI-1 knockdown. The proliferative effects of uPA were inhibited by immunoneutralization of the EGF receptor and of heparin-binding EGF (HB-EGF) by the mutant diphtheria toxin CRM197 and an EGF receptor tyrosine kinase inhibitor. H. pylori induction of uPA therefore leads to epithelial proliferation through activation of HB-EGF and is normally inhibited by concomitant induction of PAI-1; treatments directed at inhibition of uPA may slow the progression to gastric cancer.

Show MeSH
Related in: MedlinePlus