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Differential effects of selenium and knock-down of glutathione peroxidases on TNFα and flagellin inflammatory responses in gut epithelial cells.

Gong G, Méplan C, Gautrey H, Hall J, Hesketh JE - Genes Nutr (2011)

Bottom Line: Se depletion decreased expression of glutathione peroxidase1 (GPX1) and selenoproteins H and W and increased TNFα-stimulated luciferase activity, endogenous IL-8 expression and reactive oxygen species (ROS) production.These effects were not mimicked by independent knock-down of either GPX1, selenoprotein H or W; indeed, GPX1 knock-down lowered TNFα-induced NF-κB activation and did not affect ROS levels.GPX4 knock-down decreased NF-κB activation by flagellin but not by TNFα.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Framlington Place, Newcastle-upon-Tyne, NE2 4HH, UK.

ABSTRACT
Selenium (Se) is essential for human health. Despite evidence that Se intake affects inflammatory responses, the mechanisms by which Se and the selenoproteins modulate inflammatory signalling, especially in the gut, are not yet defined. The aim of this work was to assess effects of altered Se supply and knock-down of individual selenoproteins on NF-κB activation in gut epithelial cells. Caco-2 cells were stably transfected with gene constructs expressing luciferase linked either to three upstream NF-κB response elements and a TATA box or only a TATA box. TNFα and flagellin activated NF-κB-dependent luciferase activity and increased IL-8 expression. Se depletion decreased expression of glutathione peroxidase1 (GPX1) and selenoproteins H and W and increased TNFα-stimulated luciferase activity, endogenous IL-8 expression and reactive oxygen species (ROS) production. These effects were not mimicked by independent knock-down of either GPX1, selenoprotein H or W; indeed, GPX1 knock-down lowered TNFα-induced NF-κB activation and did not affect ROS levels. GPX4 knock-down decreased NF-κB activation by flagellin but not by TNFα. We hypothesise that Se depletion alters the pattern of expression of multiple selenoproteins that in turn increases ROS and modulates NF-κB activation in epithelial cells, but that the effect of GPX1 knock-down is ROS-independent.

No MeSH data available.


Related in: MedlinePlus

Effect of GPX4 knock-down on flagellin stimulated NF-κB- reporter activity and endogenous interleukin 8 expression. a Semi-quantitative RTPCR amplification of GPX4 mRNA in cells treated with either a specific GPX4 siRNA (120 pmol) or the negative control. Total RNA was extracted, RTPCR carried out and the products separated by gel electrophoresis. b The intensity of bands corresponding to the amplified GPX4 product was measured using UV Band software, normalised to the housekeeping gene GAPDH and expression then calculated relative to the cells treated with the negative control siRNA. c Quantification of IL8 expression by semi-quantitative RTPCR. Bands corresponding to the amplified IL8 products were normalised to the housekeeping gene GAPDH and expression then calculated relative to the cells treated with the negative control siRNA. d Luciferase activity measured in NF-κB-luc transfectant or TATA-luc transfectant Caco-2 cells after GPX4 siRNA knockdown and stimulation with 100 ng/ml flagellin for 2 h. Luciferase activity was measured in cell extracts, calculated per mg cell protein and expressed relative to the activity in the NF-κB-luciferase cells treated with negative control siRNA. *P < 0.05, **P < 0.01
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Fig8: Effect of GPX4 knock-down on flagellin stimulated NF-κB- reporter activity and endogenous interleukin 8 expression. a Semi-quantitative RTPCR amplification of GPX4 mRNA in cells treated with either a specific GPX4 siRNA (120 pmol) or the negative control. Total RNA was extracted, RTPCR carried out and the products separated by gel electrophoresis. b The intensity of bands corresponding to the amplified GPX4 product was measured using UV Band software, normalised to the housekeeping gene GAPDH and expression then calculated relative to the cells treated with the negative control siRNA. c Quantification of IL8 expression by semi-quantitative RTPCR. Bands corresponding to the amplified IL8 products were normalised to the housekeeping gene GAPDH and expression then calculated relative to the cells treated with the negative control siRNA. d Luciferase activity measured in NF-κB-luc transfectant or TATA-luc transfectant Caco-2 cells after GPX4 siRNA knockdown and stimulation with 100 ng/ml flagellin for 2 h. Luciferase activity was measured in cell extracts, calculated per mg cell protein and expressed relative to the activity in the NF-κB-luciferase cells treated with negative control siRNA. *P < 0.05, **P < 0.01

Mentions: Since GPx4 has been implicated in lipid hydroperoxide and leukotriene metabolism (Brigelius-Flohé 2006; Bellinger et al. 2009), we tested whether knock-down of GPX4 affected NF-κB signalling. Gene knock-down with a GPX4-specific siRNA led to a 50% decrease in GPX4 expression as assessed by RTPCR (Fig. 8a), but knock-down of this selenoprotein had no effect on the response of luciferase activity to TNFα stimulation in NF-κB-luciferase cells compared with addition of a scrambled control siRNA (results not shown). In contrast, GPX4 knock-down caused a reduction in flagellin-activated luciferase activity and IL-8 expression (Fig. 8b, c).Fig. 8


Differential effects of selenium and knock-down of glutathione peroxidases on TNFα and flagellin inflammatory responses in gut epithelial cells.

Gong G, Méplan C, Gautrey H, Hall J, Hesketh JE - Genes Nutr (2011)

Effect of GPX4 knock-down on flagellin stimulated NF-κB- reporter activity and endogenous interleukin 8 expression. a Semi-quantitative RTPCR amplification of GPX4 mRNA in cells treated with either a specific GPX4 siRNA (120 pmol) or the negative control. Total RNA was extracted, RTPCR carried out and the products separated by gel electrophoresis. b The intensity of bands corresponding to the amplified GPX4 product was measured using UV Band software, normalised to the housekeeping gene GAPDH and expression then calculated relative to the cells treated with the negative control siRNA. c Quantification of IL8 expression by semi-quantitative RTPCR. Bands corresponding to the amplified IL8 products were normalised to the housekeeping gene GAPDH and expression then calculated relative to the cells treated with the negative control siRNA. d Luciferase activity measured in NF-κB-luc transfectant or TATA-luc transfectant Caco-2 cells after GPX4 siRNA knockdown and stimulation with 100 ng/ml flagellin for 2 h. Luciferase activity was measured in cell extracts, calculated per mg cell protein and expressed relative to the activity in the NF-κB-luciferase cells treated with negative control siRNA. *P < 0.05, **P < 0.01
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Related In: Results  -  Collection

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Fig8: Effect of GPX4 knock-down on flagellin stimulated NF-κB- reporter activity and endogenous interleukin 8 expression. a Semi-quantitative RTPCR amplification of GPX4 mRNA in cells treated with either a specific GPX4 siRNA (120 pmol) or the negative control. Total RNA was extracted, RTPCR carried out and the products separated by gel electrophoresis. b The intensity of bands corresponding to the amplified GPX4 product was measured using UV Band software, normalised to the housekeeping gene GAPDH and expression then calculated relative to the cells treated with the negative control siRNA. c Quantification of IL8 expression by semi-quantitative RTPCR. Bands corresponding to the amplified IL8 products were normalised to the housekeeping gene GAPDH and expression then calculated relative to the cells treated with the negative control siRNA. d Luciferase activity measured in NF-κB-luc transfectant or TATA-luc transfectant Caco-2 cells after GPX4 siRNA knockdown and stimulation with 100 ng/ml flagellin for 2 h. Luciferase activity was measured in cell extracts, calculated per mg cell protein and expressed relative to the activity in the NF-κB-luciferase cells treated with negative control siRNA. *P < 0.05, **P < 0.01
Mentions: Since GPx4 has been implicated in lipid hydroperoxide and leukotriene metabolism (Brigelius-Flohé 2006; Bellinger et al. 2009), we tested whether knock-down of GPX4 affected NF-κB signalling. Gene knock-down with a GPX4-specific siRNA led to a 50% decrease in GPX4 expression as assessed by RTPCR (Fig. 8a), but knock-down of this selenoprotein had no effect on the response of luciferase activity to TNFα stimulation in NF-κB-luciferase cells compared with addition of a scrambled control siRNA (results not shown). In contrast, GPX4 knock-down caused a reduction in flagellin-activated luciferase activity and IL-8 expression (Fig. 8b, c).Fig. 8

Bottom Line: Se depletion decreased expression of glutathione peroxidase1 (GPX1) and selenoproteins H and W and increased TNFα-stimulated luciferase activity, endogenous IL-8 expression and reactive oxygen species (ROS) production.These effects were not mimicked by independent knock-down of either GPX1, selenoprotein H or W; indeed, GPX1 knock-down lowered TNFα-induced NF-κB activation and did not affect ROS levels.GPX4 knock-down decreased NF-κB activation by flagellin but not by TNFα.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Framlington Place, Newcastle-upon-Tyne, NE2 4HH, UK.

ABSTRACT
Selenium (Se) is essential for human health. Despite evidence that Se intake affects inflammatory responses, the mechanisms by which Se and the selenoproteins modulate inflammatory signalling, especially in the gut, are not yet defined. The aim of this work was to assess effects of altered Se supply and knock-down of individual selenoproteins on NF-κB activation in gut epithelial cells. Caco-2 cells were stably transfected with gene constructs expressing luciferase linked either to three upstream NF-κB response elements and a TATA box or only a TATA box. TNFα and flagellin activated NF-κB-dependent luciferase activity and increased IL-8 expression. Se depletion decreased expression of glutathione peroxidase1 (GPX1) and selenoproteins H and W and increased TNFα-stimulated luciferase activity, endogenous IL-8 expression and reactive oxygen species (ROS) production. These effects were not mimicked by independent knock-down of either GPX1, selenoprotein H or W; indeed, GPX1 knock-down lowered TNFα-induced NF-κB activation and did not affect ROS levels. GPX4 knock-down decreased NF-κB activation by flagellin but not by TNFα. We hypothesise that Se depletion alters the pattern of expression of multiple selenoproteins that in turn increases ROS and modulates NF-κB activation in epithelial cells, but that the effect of GPX1 knock-down is ROS-independent.

No MeSH data available.


Related in: MedlinePlus