Oxidative Stress Modulates KLF6Full and Its Splice Variants
Article first published online: 6 APR 2012
Copyright © 2012 by the Research Society on Alcoholism
Alcoholism: Clinical and Experimental Research
Volume 36, Issue 11, pages 1851–1862, November 2012
How to Cite
Urtasun, R., Cubero, F. J. and Nieto, N. (2012), Oxidative Stress Modulates KLF6Full and Its Splice Variants. Alcoholism: Clinical and Experimental Research, 36: 1851–1862. doi: 10.1111/j.1530-0277.2012.01798.x
- Issue published online: 29 OCT 2012
- Article first published online: 6 APR 2012
- Manuscript Accepted: 13 FEB 2012
- Manuscript Received: 14 NOV 2011
- Ministry of Education and Science (Spain). Grant Number: EX2006-0070
- US Public Health service grants. Grant Numbers: 5R01 AA017733, 5R01 AA017733-01S1, 5P20 AA017067, 5P20 AA017067-01S1, 5P20 AA017067-03S1
- Cytochrome P450 2E1;
- NAD(P)H Quinone Oxidoreductase;
- Reactive Oxygen Species;
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.
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.