FIGURE 7. GADD45β positively regulates AP-1 activity and JunD/Fra2 binding in chondrocytes.

The AP-1-driven luciferase reporter vector (pAP-1-Luc) was cotransfected in C-28/I2 cells with the GADD45β expression vector (GADD45β-FLAG) at 50 and 100 ng/ml (A) or with GADD45β-FLAG alone or together with siRNA-GADD45β (B). C, extracts were prepared from uninfected C-28/I2 cells (1st and 2nd lanes), cells infected with lentiviral siRNA-GFP (GFP KD; 3rd and 5th lanes), or siRNA-GADD45β (GADD45β KD; 4th and 6th lanes). Cells were then transfected with GFP-FLAG (control) or GADD45β-FLAG, as indicated. AP-1 binding activity was examined by EMSA using the double-stranded AP-1 consensus oligonucleotide as labeled probe. The cells extracted for the 5th and 6th lanes (lower exposure of gel shift using probe labeled at different time) were not transfected to show that decreasing endogenous GADD45β also decreases AP-1 binding activity. D, supershift analysis of binding activities in GADD45β-expressing C-28/I2 cells was performed using antibodies against different AP-1 family members. Note that the Fra1, JunB, and JunD antibodies produce supershifts, whereas the Fra2 antibody produces a block shift. E, the C-28/I2 cells were cotransfected with pAP-1-Luc together with expression vectors encoding Fra1, Fra2, JunD, and JunB alone or in combination in the absence or presence of the GADD45β-FLAG. Luciferase activities are shown as means ± S.D. of replicates from representative transfections. *, p < 0.05; **, p < 0.01; by analysis of variance with subsequent Dunnett test in multiple comparisons. Comparisons in E are with respective controls containing single expression vectors.