Enhanced DNA Binding and Activation of Transcription Factors NF-κB and AP-1 by Acetaldehyde in HEPG2 Cells

Juan Román,América Giménez,José Maria Lluis,Marta Gassó,Mireia Rubio,Joan Caballeria,Albert Parés,Joan Rodés,José C Fernández-Checa

doi:10.1074/jbc.275.19.14684

Abstract

Because transcription factors NF-kappaB and activator protein-1 (AP-1) are known to regulate gene expression, we have analyzed the role of acetaldehyde in the activation of NF-kappaB and AP-1 in HepG2 cells. Binding activity and transactivation of NF-kappaB and AP-1 were determined by gel retardation assays and transfection of a luciferase reporter construct controlled by kappaB and AP-1 binding sites, respectively. Acetaldehyde enhanced the DNA binding of NF-kappaB and AP-1 by 1 and 4 h, respectively, increasing the kappaB- and AP-1-dependent luciferase expression. Supershift assays revealed the presence of NF-kappaB heterodimers p65/p50 and p50/p52, whereas nuclear c-Jun levels correlated with the DNA binding of AP-1. The enhanced binding of NF-kappaB to DNA by acetaldehyde in intact cells was accompanied by the proteolytic degradation of IkappaB-alpha. However, the addition of acetaldehyde to cytostolic extracts from untreated Hep G2 cells did not affect the DNA binding of AP-1 but activated the NF-kappaB heterodimer p65/p50 in the absence of IkappaB-alpha degradation. Preincubation of HepG2 cells with protein kinase C inhibitors abolished the enhanced DNA binding of NF-kappaB and AP-1 caused by acetaldehyde. Hence, these findings uncover a previously unrecognized role for acetaldehyde in the activation of NF-kappaB and AP-1, which may be of relevance in the alcohol-induced liver disease.

Highlights

The mechanisms underlying alcohol-induced liver disease (ALD)1 remains incompletely understood
Nuclear extracts from HepG2 cells were isolated at various times after acetaldehyde addition, and NF-␬B and activator protein-1 (AP-1) activation were determined as binding of members of these transcription factors to consensus ␬B and AP-1 oligonucleotides
The enhanced DNA binding of NF-␬B and AP-1 caused by acetaldehyde occurred with distinct kinetics, being detected within 1 and 4 h, respectively, of acetaldehyde treatment of HepG2 cells (Figs. 1 and 2)

Summary

Introduction

The mechanisms underlying alcohol-induced liver disease (ALD) remains incompletely understood. Previous in vivo and in vitro studies have revealed the ability of acetaldehyde to form covalent adducts with various proteins that are thought to lead to altered liver function and structure [1, 2] In addition to these effects, it has been shown that acetaldehyde increases the transcription of collagen in several cell types (8 –11) mediated by enhanced DNA binding of transcription factors NF-1 and SP-1 to specific sites located in the promoter of the ␣2(I) collagen gene [12, 13]. Unlike the AP-1 complex, NF-␬B activation combines several mechanisms used by other transcription factors such as phosphorylation at specific residues of the inhibitory subunit I␬B followed by its degradation and translocation of the active NF-␬B into the nuclei Both transcription factors become activated by a wide variety of stimuli, including growth factors, cytokines, UV irradiation, and oxidative stress [18, 21, 22]. Because the role of acetaldehyde on NF-␬B and AP-1 activation has not been definitively established, the purpose of the present study was to investigate the DNA binding and transactivation of NF-␬B and AP-1 and the mechanisms involved in HepG2 cells cultured in the presence of acetaldehyde

Objectives

Results

Conclusion