Induction ofAKR1C2by Phase II Inducers: Identification of a Distal Consensus Antioxidant Response Element Regulated by NRF2

Abstract

AKR1C2, also referred to as the human bile acid binder and 3alpha-hydroxysteroid dehydrogenase type III, is a multifunctional oxidoreductase able to stereoselectively reduce steroids as well as oxidize or reduce polyaromatic hydrocarbons. Previously, this same protein was also identified by its robust induction by phase II inducers in HT29 cells. In HepG2 cells, both AKR1C2 and AKR1C1 (97% sequence homology) were induced by phase II inducers but not the highly related AKR1C3 and AKR1C4 family members (84% sequence homology). We now report the initial characterization of the proximal promoter of AKR1C2 in HepG2 cell line and the identification of a potent enhancer-like element responsive to phase II inducers located approximately 5.5 kilobases upstream from the transcription start site. DNA sequence analysis of this enhancer element revealed that it contained a consensus antioxidant response element (ARE), which was confirmed by mutation analysis. Treatment with phase II inducers leads to increased accumulation of nuclear factor-erythroid 2 p45-related factor (NRF) 2 in the nucleus, which was associated with increased binding to this ARE as determined by electrophoretic mobility shift assay. Transient transfection with Nrf2 increased the transcriptional activity of the ARE of AKR1C2 comparable with that observed with phase II inducers. Chromatin immunoprecipitation (ChIP) analysis also confirmed increased NRF2 binding to the ARE after induction by a phase II inducer. The AKR1C1 promoter also harbored this same ARE element in a highly homologous region, which was also bound by NRF2 in a ChiP analysis. No induction of the ARE of AKR1C2 was detected in Nrf2-/- fibroblasts. The regulation of AKR1C2 by this distal ARE suggests that AKR1C2 detoxifies products of reactive oxidant injury, which has important implications for both hormone and xenobiotic metabolism.