Identification of a novel cis-element required for the constitutive activity and osmotic response of the rat aldose reductase promoter

A new and essential cis-element AEE (aldose reductase enhancer element), necessary for the constitutive activity and the osmotic stress response of rat aldose reductase transcription in a rat liver cell line, has been identified. In transient transfection assays, an increase in promoter activity, up to 3.8-fold, was observed with osmotic stress (600 mosm/kg H₂O) using a luciferase reporter gene construct containing aldose reductase promoter sequence from -1,094 base pair (bp) to +23 bp. A deletion between - 1,071 and -895 bp reduced the constitutive activity and abolished the osmotic response of the promoter. Exonuclease III mediated in vivo DNA footprinting and dimethyl sulfate in vivo footprinting revealed DNA protection of a 32-bp region and two guanosines (G) within this region protected from methylation, respectively. Electrophoretic gel mobility shift assays using whole liver cell extracts showed protein binding, under both normal and stressed conditions. Deletion of the sequence between the two guanosines protected by in vivo dimethyl sulfate DNA footprinting (GAAGAGTG) in a luciferase construct (-1,094 bp to +23 bp) abolished the constitutive promoter activity. One copy of AEE fused to the thymidine kinase promoter gave a maximum constitutive activity of 7.7-fold and a maximum osmotic response activity of 6.7-fold.