Analysis of the mouse <i>Adar2</i> promoter that confers JNK1-dependent responsiveness to glucose stimulation.

<p>(<b>A</b>) Schematic of pGL3-luciferase (Luc) reporter constructs containing the indicated regions of the mouse <i>Adar2</i> promoter. +1, the putative transcription start site. (<b>B</b>) Analysis of the transcriptional activities of the mouse <i>Adar2</i> promoter in response to glucose stimulation. INS-1 cells transfected with the indicated reporter constructs were incubated at 2.8 or 16.7 mM glucose for 16 hours. (<b>C</b>) Inhibition of JNK diminished the glucose-stimulated transcriptional activity of the <i>Adar2</i> promoter. INS-1 cells were transfected with P<i>_Adar2</i>-219-Luc that contained the 219-nucleotide glucose-responsive region of the <i>Adar2</i> promoter. Cells were subsequently cultured at 2.8 mM or 16.7 mM glucose in the absence or presence of 20 µM JNK inhibitor SP600125 for 24 hours. For (<b>B</b>) and (<b>C</b>), promoter activities were quantified by the luciferase assay. Shown are fold increases in the luciferase activity upon high glucose stimulation after normalization to <i>Renilla</i>. Data are expressed as the mean±SEM from more than three experiments. *<i>P</i><0.05 versus basal activity at 2.8 mM glucose. (<b>D</b>) Potential regulatory elements in the 219-nucleotide glucose-responsive region of the mouse <i>Adar2</i> promoter. Putative binding sites for <i>trans</i>-factors are shown. GR, glucocorticoid receptor; C/EBP, CCAAT/enhancer-binding protein; DBP, D site of albumin promoter-binding protein; EGR2, early growth response 2; CREB, cAMP-response element binding protein; AP-1, activating protein 1; SP1, specificity protein 1; TBP, TATA box-binding protein; TFIID, transcription factor II D. The core sequence for CREB/AP-1 binding is shown, and the substitution mutations are indicated for the mutant (Mut) version used for luciferase assays in (<b>F</b>). (<b>E</b>) Overexpression of c-Jun but not JunB increased the transcriptional activity of the 219-nucleotide <i>Adar2</i> promoter. INS-1 cells were co-transfected with empty control vector (CON) or the plasmid expressing c-Jun or JunB together with the pGL3-P<i>_Adar2</i>-219 reporter construct. Luciferase activities were analyzed after incubation at 2.8 mM or 16.7 mM glucose for 18 hours. (<b>F</b>) Disruption of the putative CREB/AP-1 binding sequence abolished c-Jun activation of the 219-nucleotide <i>Adar2</i> promoter. INS-1 cells were co-transfected with empty control vector (CON) or the c-Jun plasmid together with the pGL3-P<i>_Adar2</i>-219 or pGL3-P<i>_Adar2</i>-219-Mut reporter construct. Cells were cultured at 11.1 mM glucose for 16 hours prior to luciferase analysis. For (<b>E–F</b>), data were normalized to <i>Renilla</i> luciferase activity and are shown as the mean±SEM from three independent experiments. The ratio between Firefly and <i>Renilla</i> luciferase activities in control cells was set as 1. *<i>P</i><0.05 versus cells transfected with control vector.