JunD activates transcription of the human ferritin H gene through an antioxidant response element during oxidative stress

Ferritin is the major intracellular iron storage protein that sequesters excess free iron to minimize generation of iron-catalysed reactive oxygen species. We previously demonstrated that expression of ferritin heavy chain (ferritin H) was induced by pro-oxidants, which is a part of cellular antioxidant response to protect cells from oxidative damage. In this study, we have identified that the antioxidant/electrophile response element (ARE) located 4.5 kb upstream to the human ferritin H transcription initiation site is responsible for the oxidant response. The human ferritin H ARE comprises two copies of bidirectional AP1 motifs. Mutations in each AP1 motif significantly impaired protein binding and the function of the ARE, indicating that both of the AP1 motifs are required for pro-oxidant-mediated activation of the ferritin H gene. We identified that JunD, an AP1 family basic-leucine zipper (bZip) trans cription factor, is one of the ferritin H ARE binding proteins and activates ferritin H transcription in HepG2 hepatocarcinoma cells. Gel retardation assay demonstrated that H2O2 (hydrogen peroxide) or t-BHQ (tert-butylhydroquinone) treatment increased total protein binding as well as JunD binding to the ferritin H ARE. Chromatin immunoprecipitation assay showed that H2O2 treatment induced JunD binding to the ferritin H ARE. Both H2O2 and t-BHQ induced phosphorylation of JunD at Ser-100, an activated form of JunD. Furthermore, overexpression of JunD induced endogenous ferritin H protein synthesis. Since JunD has recently been demonstrated to protect cells from several stress stimuli including oxidative stress, these results suggest that, in addition to NFE2-related factor 2 (Nrf2) as a major ARE regulatory protein, JunD is another ARE regulatory protein for transcriptional activation of the human ferritin H gene and probably other antioxidant genes containing the conserved ARE sequences by which JunD may confer cytoprotection during oxidative stress.