Figure 8: p53 suppresses the transcriptional activation of the mitochondrial enzyme PC in β-cells.

(a,b) MIN6 cells were infected with adenovirus encoding luciferase (Adv-Luci) or p53 (Adv-p53) at m.o.i.=50 for 24 h, followed by serum starvation for 6 h. (a) Immunoblotting (left panel) and the densitometric analysis (right panel) for PC, MDM2, p53 or β-actin (n=4). (b) Relative mRNA abundance of the PC and PDX1 genes (n=4). (c) Schematic diagram showing the structure of the mouse PC gene, which is controlled by the proximal (P1) promoter and the distal (P2) promoter (upper panel). Putative p53 RE in the promoter region of PC gene is identified by matching with the p53 RE consensus sequence (2 decamer motif RRRCWWGYYY separated by a spacer of 0–13 base pair, where R is A or G, W is A or T, and Y is C or T). The two decamers are underlined and mutations created in the p53 RE are highlighted with blue. The bottom panel showed the constructs containing the P2 promoter and the p53 RE (WT) or its mutants (Mut-1 and Mut-2) for luciferase assay. (d–f) MIN6 cells were transfected with luciferase reporter constructs, followed by infection with Adv-GFP or Adv-p53at m.o.i.=50 for 24 h. (d) Measurement of luciferase activity (n=4). (e) The cells were fasted in glucose-free medium for 1 h and then stimulated with glucose (20 mM) for 12 h before measurement of luciferase activity (n=4). (f) MIN6 cells were infected with Adv-Luci or Adv-p53, followed by chromatin immunoprecipitation using an anti-FLAG antibody. The immunocomplex were subjected to PCR amplification using specific primers against the promoter region of PC and MDM2 consisting of p53 RE (n=4). Representative immunoblots and DNA gel images from three independent experiment are shown. *P<0.05 (Student’s t-test). All data are represented as the mean±s.e.m.