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.