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Abstract
The biological functions of vitamin E have been classically attributed to its property as a potent inhibitor of lipid peroxidation in cellular membranes. However, in 1991, Azzi's group first described that α-tocopherol inhibits smooth muscle cell proliferation in a protein kinase C (PKC)-dependent way, demonstrating a non-antioxidant cell signalling function for vitamin E. More recently, the capacity of α-tocopherol to modulate gene expression with the implication of different transcription factors, beyond its antioxidant properties, has also been established. This study was to determine the effect of vitamin E-deficiency on liver nuclear factor-kappa B (NF-κB) DNA-binding activity and the response of target antioxidant-defense genes and cell cycle modulators. Rats were fed either control diet or vitamin-E free diet until 60 or 90 days after birth. Vitamin E-deficiency enhanced liver DNA-binding activity of NF-κB [electrophoretic mobility-shift assay, (EMSA)] and up-regulated transcription of γ-glutamylcysteine synthetase (γ-GCSM; γ-GCSC), cyclin D1 and cyclin E. We also showed down-regulation of p21(Waf1/Cip1) transcription. Western-blot analysis demonstrated that γ-glutamylcysteine synthetase catalytic subunit (γ-GCSC) and cyclin D1 showed a similar pattern to that found in the RT-PCR analysis. Moreover, chromatin immunoprecipitation (ChIP) assay demonstrated that NF-κB directly regulates transcription of γ-GCS (both subunits) and cyclin D1 through the binding of NF-κB to the corresponding gene promoters, which was enhanced in vitamin E-deficiency. These findings show that vitamin E-deficiency induces significant molecular regulatory properties in liver cells with an altered expression of both antioxidant-defense genes and genes that control the cell cycle and demonstrate that liver NF-κB activation is involved in this response. Our results emphasize the importance of maintaining an adequate vitamin E consumption not only to prevent liver oxidative damage but also in modulating signal transduction.
| Abbreviations | ||
| ChIP | = | chromatin immunoprecipitation |
| EMSA | = | electrophoretic mobility-shift assay |
| γ-GCS | = | γ-glutamylcysteine synthetase |
| γ-GCSC | = | γ- glutamylcysteine synthetase catalytic subunit |
| γ-GCSM | = | γ-glutamylcysteine synthetase modifier subunit |
| GSH | = | reduced glutathione |
| GSSG | = | oxidized glutathione |
| MDA | = | malondialdehyde |
| NF-κB | = | nuclear factor-κB |
| PKC | = | protein kinase C |
| ROS | = | reactive oxygen species |
| Abbreviations | ||
| ChIP | = | chromatin immunoprecipitation |
| EMSA | = | electrophoretic mobility-shift assay |
| γ-GCS | = | γ-glutamylcysteine synthetase |
| γ-GCSC | = | γ- glutamylcysteine synthetase catalytic subunit |
| γ-GCSM | = | γ-glutamylcysteine synthetase modifier subunit |
| GSH | = | reduced glutathione |
| GSSG | = | oxidized glutathione |
| MDA | = | malondialdehyde |
| NF-κB | = | nuclear factor-κB |
| PKC | = | protein kinase C |
| ROS | = | reactive oxygen species |
Notes
† Contributed equally to this work.