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Oxidative Stress Modulates KLF6Full and Its Splice Variants

Authors

  • Raquel Urtasun,

    1. Department of Medicine, Division of Liver Diseases, Mount Sinai School of Medicine, New York, New York
    2. Gene Therapy and Hepatology, Center for Translational Research, Pamplona, Spain
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  • Francisco Javier Cubero,

    1. Department of Medicine, Division of Liver Diseases, Mount Sinai School of Medicine, New York, New York
    2. Department of Internal Medicine III, University Hospital RWTH Aachen, Aachen, Germany
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  • Natalia Nieto

    Corresponding author
    • Department of Medicine, Division of Liver Diseases, Mount Sinai School of Medicine, New York, New York
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Reprint requests: Natalia Nieto, PharmD, PhD, Department of Medicine, Division of Liver Diseases, Mount Sinai School of Medicine, Box 1123, 1425 Madison Avenue, Room 11-76, New York, NY 10029; Tel.: 212-659-9217; Fax: 212-849-2574; E-mail: natalia.nieto@mssm.edu

Abstract

Background

Induction of reactive oxygen species (ROS) is a central mechanism in alcohol hepatotoxicity. Krüppel-like factor 6 (KLF6), a transcription factor and a tumor-suppressor gene, is an early-responsive gene to injury; however, the effect of ROS and alcohol on KLF6 induction is unknown. The aim of this study is to investigate the contribution of 2 sources of ROS, cytochrome P450 2E1 (CYP2E1), NAD(P)H quinone oxidoreductase (NQO1), and alcohol on the modulation of KLF6Full expression, splicing to KLF6_V1 and KLF6_V2, and the effect on TNFα, a downstream target.

Methods and Results

Endogenous ROS production in CYP2E1-expressing HepG2 cells induced mRNA and protein expression of KLF6Full and its splice variants compared to control cells. Incubation with pro-oxidants such as arachidonic acid (AA), β-naphtoflavone, and H2O2 further enhanced KLF6Full and its splice variants. The AA effects on KLF6Full and its splice forms were blocked by vitamin E—which prevents lipid peroxidation—and by diallylsulfide—a CYP2E1 inhibitor. Menadione and paraquat, 2 pro-oxidants metabolized via NQO1, induced KLF6Full mRNA in a thiol-dependent manner. Antioxidants and an NQO1 inhibitor suppressed the menadione-dependent increase in KLF6Full and its splice variants mRNA. Furthermore, primary hepatocytes and livers from chronic alcohol-fed rats, with elevated lipid peroxidation, H2O2 and CYP2E1 but with low GSH, showed a ~2-fold increase in KLF6Full mRNA compared to controls. Inhibition of p38 phosphorylation further up-regulated the CYP2E1 and the AA effects on KLF6Full mRNA, whereas inhibition JNK and ERK1/2 phosphorylation decreased both. KLF6_V1 but not KLF6Full ablation markedly increased TNFα levels in macrophages; thus, TNFα emerges as a downstream target of KLF6_V1.

Conclusions

The novel effect of ROS on modulating KLF6Full expression and its splice variants could play a relevant role in liver injury and in TNFα regulation.

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