FIGURE 8.

HDAC5 specifically inhibits both GEF and MEF2A-dependent transcription of the GLUT4 promoter. A, 3T3-L1 adipocytes differentiated to day 6 were transiently transfected with luciferase reporter plasmids–895-hG4-luc (firefly luciferase, for promoter activity) and pRLTK (Renilla luciferase, for transfection efficiency), as well as plasmids encoding GEF, MEF2A, and HDAC5 (+ indicates inclusion in transfection). Luciferase assays were performed after 24 h; specific activation of the –895-hG4 promoter, or the TK promoter were calculated from the ratio of firefly to Renilla luciferase activities. Luciferase activity from activation of the –895-hG4 promoter transactivated by GEF, MEF2A, or both together was significantly greater than basal activation (p < 0.05) using one-way analysis of variance. The combination of GEF and MEF2A together significantly activated the promoter to a greater extent than either factor alone (using a Duncan's multiple range pairwise comparison). The asterisk indicates a significant inhibition of luciferase activity by the addition of HDAC5 (p < 0.05) using a Student's t test. B, the -fold inhibition was calculated for each case by dividing the ratio of expression without HDAC5 or the expression in the presence of HDAC5. The -fold inhibition was significantly increased when the promoter was transactivated by both GEF and MEF2A together (p < 0.05) using one-way analysis of variance and pairwise comparison. The nonspecific inhibition of TK-luc by HDAC5 was significantly lower than inhibition of –895-hG4-luc in each condition. Data shown are mean ± S.E. of n = 3 independent experiments performed in duplicate.