Fig. 2.

Mutagenesis and reporter assay reveal the importance of Fox binding site to glucocorticoid response. The 15 bp GRE of Angptl4 is located at chr17: 33,911,836 to 33,911,850, equivalent to nt position +6,229 to +6,243 relative to Angptl4 TSS. All nt positions shown are relative to rat Angptl4 TSS. A: WT GBR (+6,030 to +6,529) is inserted upstream of a luciferase gene in reporter plasmid pGL4.10-E4TATA (denoted as pWT). Deletions of WT GBR give the following reporter plasmids: pDel-1 (nt +6,030 to +6,376), pDel-2 (nt +6,181 to +6,529), pDel-3 (nt +6,181 to +6,376), and pDel-4 (nt +6,181 to +6,244). B: Reporter plasmids (250 ng) described in A were cotransfected with a human GR expression vector (150 ng) and a Renilla internal control plasmid (50 ng) into H4IIE cells. Twenty-four hours posttransfection, cells were treated with control ethanol (EtOH), 0.5 µM Dex, 1 nM insulin, or Dex plus insulin for 20–24 h. Fold induction is calculated by taking the ratio of treatment over ethanol. C: GRE and FRE of rat Angptl4 are shown. The nt +6,324 of FRE is mutated from C to G for mutant FRE (pMut-FRE). D: Reporter assays described in B were carried out for pWT and pMut-FRE. Ethanol-treated pWT luciferase activity was set as 0%, while Dex-treated pWT was 100%. E: pWT (250 ng) was cotransfected with a human GR expression vector (150 ng), a Renilla internal control plasmid (50 ng), and a human expression vector for dominant negative Akt (DN Akt; 300 or 600 ng) into H4IIE cells. Twenty-four hours posttransfection, cells were treated with control ethanol, 0.5 µM Dex, 1 nM insulin, or Dex plus insulin for 20–24 h. The results are presented as relative activities to Dex-induced pWT reporter activity. Error bars represent standard error of the mean. ** P < 0.05 for relative activity with no DN Akt cotransfection versus relative activity with DN Akt cotransfection; N.D. indicates no difference. Error bars represent standard error of the mean. * P < 0.05.