Báo cáo khoa học: Increased expression of c-Fos by extracellular signal-regulated kinase activation

Increased expression of c-Fos by extracellular

signal-regulated kinase activation under sustained

oxidative stress elicits BimEL upregulation and hepatocyte

apoptosis

Yasuhiro Ishihara1

, Fumiaki Ito2 and Norio Shimamoto1

1 Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences at Kagawa, Tokushima Bunri University, Japan

2 Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan

Introduction

Apoptosis has several morphological features, includ￾ing cell shrinkage, nuclear condensation, and nucleoso￾mal DNA fragmentation. Extensive studies to uncover

the mechanisms underlying the induction of apoptosis

have yielded the generally accepted theory that mito￾chondria play a fundamental role in the process. Apop￾totic stimuli activate the mitochondrial permeability

transition pore and the release of apoptosis-promoting

molecules such as cytochrome c, apoptosis-inducing

factor, and endonuclease G [1]. The pathways

upstream of the mitochondria for apoptotic signal

transduction have recently been identified. Several

Keywords

apoptosis; Bim; c-Fos; extracellular signal￾regulated kinase (ERK); reactive oxygen

species

Correspondence

N. Shimamoto, Laboratory of Pharmacology,

Faculty of Pharmaceutical Sciences at

Kagawa, Tokushima Bunri University,

1314-1, Shido, Sanuki, Kagawa 769-2193,

Japan

Fax: +81 87 894 0181

Tel: +81 87 894 5111 ext. 6513

E-mail: [email protected]

(Received 22 December 2010, revised 25

February 2011, accepted 22 March 2011)

doi:10.1111/j.1742-4658.2011.08105.x

We previously reported that the inhibition of catalase and glutathione per￾oxidase activities by treatment with 3-amino-1,2,4-triazole (ATZ) and mer￾captosuccinic acid evoked sustained increases in the levels of reactive

oxygen species and apoptosis in rat primary hepatocytes. Apoptosis was

accompanied by increased expression of BimEL, following activation of

extracellular signal-regulated kinase. The aim of this study was to charac￾terize the mechanism underlying hepatocyte apoptosis by identifying the

transcription factor that induces BimEL expression. The bim promoter

region was cloned into a promoterless-luc vector, and promoter activity

was monitored by a luciferase assay. The luciferase activity increased in the

presence of ATZ + mercaptosuccinic acid. Pretreatment with a MEK

inhibitor, U0126, or an antioxidant, vitamin C, suppressed the promoter

activity. Furthermore, ATZ + mercaptosuccinic acid-induced luciferase

activity was attenuated by mutation of the activator protein-1 binding site

in the bim promoter region. The amounts of total and phosphorylated

c-Fos increased over time in the presence of ATZ + mercaptosuccinic acid,

whereas the amounts of total and phosphorylated c-Jun remained

unchanged. Chromatin immunoprecipitation revealed that both c-Fos and

c-Jun localized to the activator protein-1-binding site in the bim promoter

region. BimEL expression and hepatocyte apoptosis were suppressed by

knockdown of c-Fos and c-Jun, respectively. These results indicate that

increases in c-Fos following extracellular signal-regulated kinase activation

are critical for BimEL upregulation and apoptosis.

Abbreviations

AP-1, activator protein-1; ATZ, 3-amino-1,2,4-triazole; ChIP, chromatin immunoprecipitation; ERK, extracellular signal-regulated kinase;

GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ROS, reactive oxygen species; SE, standard error; siRNA, small interfering RNA.

FEBS Journal 278 (2011) 1873–1881 ª 2011 The Authors Journal compilation ª 2011 FEBS 1873