Nitric oxide-induced transcriptional up-regulation of protective genes by Nrf2 via the antioxidant response element counteracts apoptosis of neuroblastoma cells

Nitric oxide (NO) is a signaling molecule that in excess causes cell death. Here we report a mechanism of NO-induced transcriptional up-regulation of genes encoding detoxifying enzymes and protective proteins and their role in counteracting NO-induced apoptosis of neuroblastoma cells. Promoter analysis using reporter assays identified the antioxidant response element (ARE) located in the promoter region of NAD(P)H:quinone oxidoreductase 1 (Nqo1) and other detoxifying enzyme genes as responsible for NO-mediated gene induction. The transcription factors NF-E2-related factor 2 (Nrf2) and small maf proteins were detected in NO-induced nuclear protein-ARE complexes. Nrf2 augmented NO-induced, ARE-dependent gene expression, which was blocked by dominant-negative Nrf2 (DN-Nrf2) lacking the transcriptional activation domain. Consistent with these results, Nrf2 was localized in the cytoplasm in unstimulated cells, and NO triggered its rapid nuclear accumulation. Neuroblastoma cells were stably transfected with DN-Nrf2, which repressed both the expression of protective genes and their induction by NO. These DN-Nrf2 cells exhibited reduced NQO1 enzymatic activity and were sensitized to NO-induced apoptosis. Similar results were obtained when Nrf2 expression was blocked by RNA interference. Conversely, stable cells expressing higher levels of Nrf2 protein had elevated NQO1 activity and were protected from NO. Finally, NO-mediated ARE-dependent gene induction occurred well before apoptosis as judged by caspase activation. These results together suggest that NO signals the transcriptional up-regulation of NQO1 and other detoxifying enzyme and protective genes through Nrf2 via the ARE to counteract NO-induced apoptosis of neuroblastoma cells.