Identification of a cis-acting element responsive to ultrasound in the 5'-flanking region of the human heme oxygenase-1 gene.
We previously found that the heme oxygenase-1 gene (hmox-1) was the most upregulated gene among 9,182 genes in human lymphoma U937 cells exposed to a 1-MHz continuous ultrasound using the cDNA microarray technique. However, little is known about the molecular mechanisms of the induction of hmox-1 expression by ultrasound. We investigated the mechanism using human prostate cancer DU145 cells in which expression of hmox-1 increased with sonication in a time and an intensity-dependent manner. When N-acetyl-L-cysteine or glutathione-monoethyl ester, a potent antioxidant, was added to cell culture, hmox-1 upregulation was attenuated, suggesting that oxidative stress caused by sonication is involved in this process. To identify cis-acting elements required for the ultrasound-mediated induction, we carried out transient expression assays with plasmids carrying the luciferase gene under control of deletion mutants of the 5'-flanking region of hmox-1. The results revealed that the upregulations by sonication were observed with deletion mutants carrying the E1 or E2 enhancer of the 5'-flanking region, suggesting stress-responsive elements (StRE) were involved in the induction because either enhancer contains a number of the element. Indeed, site-directed mutations within StRE decreased the reactivity of deletion mutants to sonication. A transcription factor NF-E2-related Factor 2 that binds to StRE would therefore be activated by oxidative stress induced by sonication.
Kagiya
Go
G
Radiation Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
Ogawa
Ryohei
R
Ito
Shinji
S
Fukuda
Shigekazu
S
Hatashita
Masanori
M
Tanaka
Yoshikazu
Y
Yamamoto
Kazutaka
K
Kondo
Takashi
T
D024506:5' Flanking Region; D000704:Analysis of Variance; D000975:Antioxidants / Q000494:pharmacology; D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers; D004742:Enhancer Elements, Genetic; D017353:Gene Deletion; D005786:Gene Expression Regulation; D051547:Heme Oxygenase-1 / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018384:Oxidative Stress; D020218:Response Elements; D014162:Transfection / Q000379:methods; D014465:Ultrasonics
D000975:Antioxidants; D017931:DNA Primers; D008156:Luciferases; D051547:Heme Oxygenase-1