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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.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.

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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uPA acts through heparin-binding EGF (HB-EGF) to stimulate epithelial cell proliferation. A: uPA (500 ng/ml)-stimulated BrdU incorporation was inhibited by neutralizing Ab to the EGF receptor (EGF-R) (2 μg/ml) and by receptor tyrosine kinase inhibitor (Inh) AG1478 (2 ng/ml). B: uPA-stimulated BrdU incorporation was also inhibited by neutralizing Ab to HB-EGF (5 μg/ml) and by the mutant diphtheria toxin CRM197 (5 μg/ml); n = 3–4, *P < 0.05, ANOVA.
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f8: uPA acts through heparin-binding EGF (HB-EGF) to stimulate epithelial cell proliferation. A: uPA (500 ng/ml)-stimulated BrdU incorporation was inhibited by neutralizing Ab to the EGF receptor (EGF-R) (2 μg/ml) and by receptor tyrosine kinase inhibitor (Inh) AG1478 (2 ng/ml). B: uPA-stimulated BrdU incorporation was also inhibited by neutralizing Ab to HB-EGF (5 μg/ml) and by the mutant diphtheria toxin CRM197 (5 μg/ml); n = 3–4, *P < 0.05, ANOVA.

Mentions: Since there was increased BrdU incorporation in response to exogenous EGF, we asked whether the proliferative response to uPA was linked to activation of the EGF pathway. Neutralizing antibody against EGF-R significantly reduced BrdU incorporation induced by uPA, and so too did the EGF-R tyrosine kinase inhibitor, AG1478 (Fig. 8A). Moreover, neutralizing antibody to HB-EGF inhibited BrdU incorporation in response to uPA (Fig. 8B), and, in agreement with the idea that HB-EGF mediated responses to uPA, the mutant diphtheria toxin CRM197 also reduced uPA-mediated BrdU incorporation (Fig. 8B). Interestingly, however, responses to uPA were not influenced by neutralization of other EGF-R ligands such as amphiregulin and TGF-α that are known to be expressed in gastric mucosa (uPA, 1.90 ± 0.12-fold increase over control; uPA and amphiregulin immunoneutralization, 1.74 ± 0.15-fold over control; uPA and TGF-α immunoneutralization, 2.10 ± 0.15-fold over control). Since it is known that HB-EGF is released by metalloproteinases (47), we examined the effect of a broad spectrum inhibitor, GM6001, and two tissue inhibitors of MMPs (TIMP-1 and TIMP-3) on proliferative response to uPA. At a concentration of 100 nM, TIMP-1 inhibited BrdU incorporation by 21 ± 5% and TIMP-3 inhibited by 69 ± 10% (P < 0.05), whereas GM6001 inhibited uPA responses by 70 ± 12% (P < 0.05).


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)

uPA acts through heparin-binding EGF (HB-EGF) to stimulate epithelial cell proliferation. A: uPA (500 ng/ml)-stimulated BrdU incorporation was inhibited by neutralizing Ab to the EGF receptor (EGF-R) (2 μg/ml) and by receptor tyrosine kinase inhibitor (Inh) AG1478 (2 ng/ml). B: uPA-stimulated BrdU incorporation was also inhibited by neutralizing Ab to HB-EGF (5 μg/ml) and by the mutant diphtheria toxin CRM197 (5 μg/ml); n = 3–4, *P < 0.05, ANOVA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: uPA acts through heparin-binding EGF (HB-EGF) to stimulate epithelial cell proliferation. A: uPA (500 ng/ml)-stimulated BrdU incorporation was inhibited by neutralizing Ab to the EGF receptor (EGF-R) (2 μg/ml) and by receptor tyrosine kinase inhibitor (Inh) AG1478 (2 ng/ml). B: uPA-stimulated BrdU incorporation was also inhibited by neutralizing Ab to HB-EGF (5 μg/ml) and by the mutant diphtheria toxin CRM197 (5 μg/ml); n = 3–4, *P < 0.05, ANOVA.
Mentions: Since there was increased BrdU incorporation in response to exogenous EGF, we asked whether the proliferative response to uPA was linked to activation of the EGF pathway. Neutralizing antibody against EGF-R significantly reduced BrdU incorporation induced by uPA, and so too did the EGF-R tyrosine kinase inhibitor, AG1478 (Fig. 8A). Moreover, neutralizing antibody to HB-EGF inhibited BrdU incorporation in response to uPA (Fig. 8B), and, in agreement with the idea that HB-EGF mediated responses to uPA, the mutant diphtheria toxin CRM197 also reduced uPA-mediated BrdU incorporation (Fig. 8B). Interestingly, however, responses to uPA were not influenced by neutralization of other EGF-R ligands such as amphiregulin and TGF-α that are known to be expressed in gastric mucosa (uPA, 1.90 ± 0.12-fold increase over control; uPA and amphiregulin immunoneutralization, 1.74 ± 0.15-fold over control; uPA and TGF-α immunoneutralization, 2.10 ± 0.15-fold over control). Since it is known that HB-EGF is released by metalloproteinases (47), we examined the effect of a broad spectrum inhibitor, GM6001, and two tissue inhibitors of MMPs (TIMP-1 and TIMP-3) on proliferative response to uPA. At a concentration of 100 nM, TIMP-1 inhibited BrdU incorporation by 21 ± 5% and TIMP-3 inhibited by 69 ± 10% (P < 0.05), whereas GM6001 inhibited uPA responses by 70 ± 12% (P < 0.05).

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.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.

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