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Up-regulation of intestinal vascular endothelial growth factor by Afa/Dr diffusely adhering Escherichia coli.

Cane G, Moal VL, Pagès G, Servin AL, Hofman P, Vouret-Craviari V - PLoS ONE (2007)

Bottom Line: This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells.Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways.Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR 6543, Université de Nice-Sophia Antipolis, Nice, France.

ABSTRACT

Background: Angiogenesis has been recently described as a novel component of inflammatory bowel disease pathogenesis. The level of vascular endothelial growth factor (VEGF) has been found increased in Crohn's disease and ulcerative colitis mucosa. To question whether a pro-inflammatory Escherichia coli could regulate the expression of VEGF in human intestinal epithelial cells, we examine the response of cultured human colonic T84 cells to infection by E. coli strain C1845 that belongs to the typical Afa/Dr diffusely adhering E. coli family (Afa/Dr DAEC).

Methodology: VEGF mRNA expression was examined by Northern blotting and q-PCR. VEGF protein levels were assayed by ELISA and its bioactivity was analysed in endothelial cells. The bacterial factor involved in VEGF induction was identified using recombinant E. coli expressing Dr adhesin, purified Dr adhesin and lipopolysaccharide. The signaling pathway activated for the up-regulation of VEGF was identified using a blocking monoclonal anti-DAF antibody, Western blot analysis and specific pharmacological inhibitors.

Principal findings: C1845 bacteria induce the production of VEGF protein which is bioactive. VEGF is induced by adhering C1845 in both a time- and bacteria concentration-dependent manner. This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells. Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways.

Conclusions: Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells. Thus, these results suggest a link between an entero-adherent, pro-inflammatory E. coli strain and angiogenesis which appeared recently as a novel component of IBD pathogenesis.

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Wild-type C1845-induced VEGF expression is dependant on Src and Erk proteins.Confluent serum-starved T84 cells were pre-treated with 5 µM PP2, 10 µM U0126 or 15 µM LY294002 for 30 minutes. Cells were then infected with wild-type C1845 bacteria at 5×107 CFU/ml for four hours. Total RNA was prepared and the relative quantity of VEGF mRNA was measured by q-PCR. Quantification of the results from four independent experiments (means±SD.) is shown. *, p<0.005 versus non infected cells.
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pone-0001359-g006: Wild-type C1845-induced VEGF expression is dependant on Src and Erk proteins.Confluent serum-starved T84 cells were pre-treated with 5 µM PP2, 10 µM U0126 or 15 µM LY294002 for 30 minutes. Cells were then infected with wild-type C1845 bacteria at 5×107 CFU/ml for four hours. Total RNA was prepared and the relative quantity of VEGF mRNA was measured by q-PCR. Quantification of the results from four independent experiments (means±SD.) is shown. *, p<0.005 versus non infected cells.

Mentions: Using this pharmacological approach, we then verified whether these pathways are required for VEGF mRNA expression in response to wild type C1845 bacteria. Prior to C1845 infection, T84 cells were pre-treated with the different inhibitors used above. Total RNA was extracted and the level of the VEGF transcripts was assayed by q-PCR. As shown in Figure 6, when the C1845-infected cells are pre-treated with the Src inhibitor, PP2, the increase in VEGF mRNA was inhibited. Interestingly, we observed the same result when the C1845-infected cells were treated with the MEK inhibitor, U0126 whereas the PI3K inhibitor partially blocked the production of the VEGF transcript in wild type C1845-infected cells. This partial inhibition suggests that VEGF transcription is regulated by at least two distinct signalling pathways in which Erk activation is a pre requisite for PI3K/Akt effect on VEGF promoter. We conclude that a Src-dependant Erk pathways is required to increase VEGF mRNA expression in response to C1845 bacteria.


Up-regulation of intestinal vascular endothelial growth factor by Afa/Dr diffusely adhering Escherichia coli.

Cane G, Moal VL, Pagès G, Servin AL, Hofman P, Vouret-Craviari V - PLoS ONE (2007)

Wild-type C1845-induced VEGF expression is dependant on Src and Erk proteins.Confluent serum-starved T84 cells were pre-treated with 5 µM PP2, 10 µM U0126 or 15 µM LY294002 for 30 minutes. Cells were then infected with wild-type C1845 bacteria at 5×107 CFU/ml for four hours. Total RNA was prepared and the relative quantity of VEGF mRNA was measured by q-PCR. Quantification of the results from four independent experiments (means±SD.) is shown. *, p<0.005 versus non infected cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2147078&req=5

pone-0001359-g006: Wild-type C1845-induced VEGF expression is dependant on Src and Erk proteins.Confluent serum-starved T84 cells were pre-treated with 5 µM PP2, 10 µM U0126 or 15 µM LY294002 for 30 minutes. Cells were then infected with wild-type C1845 bacteria at 5×107 CFU/ml for four hours. Total RNA was prepared and the relative quantity of VEGF mRNA was measured by q-PCR. Quantification of the results from four independent experiments (means±SD.) is shown. *, p<0.005 versus non infected cells.
Mentions: Using this pharmacological approach, we then verified whether these pathways are required for VEGF mRNA expression in response to wild type C1845 bacteria. Prior to C1845 infection, T84 cells were pre-treated with the different inhibitors used above. Total RNA was extracted and the level of the VEGF transcripts was assayed by q-PCR. As shown in Figure 6, when the C1845-infected cells are pre-treated with the Src inhibitor, PP2, the increase in VEGF mRNA was inhibited. Interestingly, we observed the same result when the C1845-infected cells were treated with the MEK inhibitor, U0126 whereas the PI3K inhibitor partially blocked the production of the VEGF transcript in wild type C1845-infected cells. This partial inhibition suggests that VEGF transcription is regulated by at least two distinct signalling pathways in which Erk activation is a pre requisite for PI3K/Akt effect on VEGF promoter. We conclude that a Src-dependant Erk pathways is required to increase VEGF mRNA expression in response to C1845 bacteria.

Bottom Line: This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells.Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways.Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR 6543, Université de Nice-Sophia Antipolis, Nice, France.

ABSTRACT

Background: Angiogenesis has been recently described as a novel component of inflammatory bowel disease pathogenesis. The level of vascular endothelial growth factor (VEGF) has been found increased in Crohn's disease and ulcerative colitis mucosa. To question whether a pro-inflammatory Escherichia coli could regulate the expression of VEGF in human intestinal epithelial cells, we examine the response of cultured human colonic T84 cells to infection by E. coli strain C1845 that belongs to the typical Afa/Dr diffusely adhering E. coli family (Afa/Dr DAEC).

Methodology: VEGF mRNA expression was examined by Northern blotting and q-PCR. VEGF protein levels were assayed by ELISA and its bioactivity was analysed in endothelial cells. The bacterial factor involved in VEGF induction was identified using recombinant E. coli expressing Dr adhesin, purified Dr adhesin and lipopolysaccharide. The signaling pathway activated for the up-regulation of VEGF was identified using a blocking monoclonal anti-DAF antibody, Western blot analysis and specific pharmacological inhibitors.

Principal findings: C1845 bacteria induce the production of VEGF protein which is bioactive. VEGF is induced by adhering C1845 in both a time- and bacteria concentration-dependent manner. This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells. Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways.

Conclusions: Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells. Thus, these results suggest a link between an entero-adherent, pro-inflammatory E. coli strain and angiogenesis which appeared recently as a novel component of IBD pathogenesis.

Show MeSH
Related in: MedlinePlus