Valproic acid-induced gene expression through production of reactive oxygen species

Valproic acid (VPA) is a widely used anticonvulsive agent that has profound antiproliferative effects in many cell types, as well as inductive effects on a number of genes. The mechanism of its gene-inducing effect has been reported to involve transcription factors, Sp1 and activator protein-1. Using two well-characterized antioxidant response element (ARE)driven gene promoters, i.e., mouse heme oxygenase-1 and human NAD(P)H:quinone oxidoreductase I genes as tools to monitor the transcriptional response to VPA, we show here that VPA-induced gene transcription was abrogated by antioxidants. With the human G alpha(i2) gene promoter, which was previously used to establish the involvement of Sp1 in the transcriptional action of VPA, we found that VPA-induced gene transcription was also blocked by antioxidants. Mutation of the ARE (5'-TGACtggGC-3') in this promoter abrogated the transcriptional response to VPA. With such mutants, the NADPH oxidase inhibitor, diphenyleneiodonium, had no effect on VPA-induced transcription. In gel mobility shift assays, VPA-induced binding of nuclear proteins to a DNA probe containing the relevant ARE sequence in the G alpha(i2) gene promoter was decreased in nuclear extracts from cells pretreated with antioxidants. Chromatin immunoprecipitation assays showed that the prototype redox-sensitive transcription factors, Nrf2, small Maf protein(s), and c-Fos, were recruited to this promoter in VPA-treated cells. Overall, this study reveals that the mechanism of the transcriptional response to VPA includes VPA-induced production of reactive oxygen species which induce the activation of redox-sensitive transcription factors that interact with the ARE.