1. ¹National University Medical Institutes, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
2. ²Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
3. ³Department of Biochemistry, University of Alberta, Edmonton, Canada
4. ⁴Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
5. ⁵NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore

Correspondence: M-V Clément, The Yong Loo Lin School of Medicine Department of Biochemistry National University of Singapore 8 Medical Drive, MD7, 117 597, Singapore. Tel: (65) 6516-7985; Fax: (65) 6779-1453; E-mail: bchmvc@nus.edu.sg

⁶Both authors contributed equally to this work

Received 23 October 2006; Revised 23 April 2007; Accepted 23 April 2007; Published online 15 June 2007.

Abstract

The mechanism of Na⁺/H⁺ exchanger 1 (NHE1) gene repression upon exposure of cells to non-apoptotic concentrations of hydrogen peroxide (H₂O₂) was investigated. We show that continuous presence of H₂O₂ was not required for inhibition of NHE1 promoter activity. However, the downregulation of NHE1 promoter activity and protein expression was abrogated by the presence of beta mercaptoethanol (ME) and dithiothreitol. The pan-caspase inhibitor zVAD-fmk also blocked the effect of H₂O₂ on NHE1 promoter activity and expression, but unlike ME, caspase inhibition was ineffective in rescuing the early phase of NHE1 repression. Interestingly, the effect of caspase inhibition was observed only after 9 h of exposure to H₂O₂ and completely restored NHE1 promoter activity by 18–24 h. Using tetrapeptide inhibitors of a variety of caspases and siRNA-mediated gene silencing, caspases 3 and 6 were identified as mediators of H₂O₂-induced NHE1 repression, independent of initiator/amplifier caspase activation. Furthermore, incubation of cells with the iron chelator, desferioxamine, not only blocked the activities of caspases 3 and 6, but also affected NHE1 promoter and protein expression in a manner similar to zVAD-fmk. These data show that a mild oxidative stress represses NHE1 promoter activity and expression via an early oxidation phase blocked by reducing agents, and a late phase requiring an iron-dependent increase in caspases 3 and 6 activities.