FIGURE 3.

Identification of the Col10a1 promoter element responsive to the synergistic action of C/EBPβ and GADD45β. A, ATDC5 cells were co-transfected with deletion mutants of the Col10a1 promoter (−2128/+201, −60/+52, and −34/+52) in the pGL3 reporter vector (300 ng) and 25 ng of each expression vector encoding mouse C/EBPβ (Cβ) or GADD45β (45β), alone or in combination, in a total amount of 375 ng/well. B, the regions with homologies in the mouse and human Col10a1 promoter region (−60/−34 bp) responsive to the synergistic action of C/EBPβ and GADD45β are indicated by a line above. The two half-sites for CREB or AP-1 and the C/EBP half-site are indicated as square boxes. Sites in the mouse sequence that are highlighted in boldface type correspond to substituted sites in the mutant constructs used in the transfections shown in the lower panel. Col10a1 promoter constructs (300 ng) containing mutations in the proximal region were co-transfected with empty vector or 25 ng of expression vector encoding mouse C/EBPβ or GADD45β, alone or in combination, in a total amount of 375 ng/well in ATDC5 cells. Luciferase activities were normalized to pRL-TK reporter activities and shown as -fold induction compared with control (empty vector). Experiments were done in triplicate with data shown as means ± S.D.