Identification of a stress inducible activating region in human IRS-2 promoter depending on SP1, NF1 binding and Erk activation in HepG2 cells

Abstract

Transcriptional regulation of Insulinreceptorsubstrates (IRS) is not well understood on a molecular level. IRS-2 knockout mice develop severe Type 2 diabetes after 9 weeks. Consequently, the regulation of the amount of IRS-2 and even its ratio to IRS-1 and IRS-4 is physiologically important. Based on luciferase promoter reporter assays we aimed to identifiy transcription factor binding sites in the human IRS-2 promoter in HepG2 cells. We found a region 688 bp upstream of translation start codon, which is responsible for the basal transcription of IRS-2 in liver. After truncating this region activity decreased about two-third. We postulated potential transcription factor binding motives in this region and confirmed binding of SP1 and NF1 using electro mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. After mutating both SP1 and NF1 binding sites, basal activity was diminished. Preincubation with PD 98,059 which inactivates Erk1 signaling inhibited IRS-2 promoter activity in luciferase reporter assays in HepG2 cells. This revealed an involvement of MAP kinase signaling cascade in IRS-2 transcription regulation that might finally gears to activate SP1. Activating this cascade with oxidative stress showed a twofold increase in IRS-2 promoter activity based on luciferase reporter assay and RT-PCR in HepG2 cells. Activity could be raised even more after additional inhibition of p38 MAPK with SB202190. This reveals an inhibitory effect of p38MAPK on Erk1 activity, and consequently on IRS-2 transcription. Recently a similar mechanism has been described for IL-2 promoter activation. After SP1 and NF1 binding site mutation on IRS-2 promoter these effects were diminished. In conclusion we discovered a new region (-688 to -611 bp) in the distal human IRS-2 promoter that is essential for its full basal and stress induced activation and is dependent on SP1 and NF1 binding.