Vitamin E deficiency induces liver nuclear factor-κB DNA-binding activity and changes in related genes

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.

• Vitamin E-deficiency
• liver
• nuclear factor-kappa B (NF-κB)
• redox status
• gamma-glutamylcysteine synthetase

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.