Figure 7

The ROS/MAPKs-dependent NF-κB cascade is required for TGF-β1-induced MMP-9 promoter activity. (A) Schematic representation of a 5'-promoter regions of the rat MMP-9 gene fused to the pGL-luciferase reporter gene (pGL-MMP-9-Luc). The translational start site (+1) of the luciferase reporter gene is indicated by an arrow. RBA-1 cells were transiently cotransfected with pGL-MMP9-Luc and pGal encoding for b-galactosidase. After transfection, cells were treated with TGF-β1 (15 ng/ml) for the indicated time intervals. (B) Cells were pretreated with SB431542 (SB43, 10 μM), NAC (100 μM), U0126 (10 μM), SP600125 (SP, 10 μM), or Bay11-7082 (Bay, 1 μM) for 1 h, and then incubated with TGF-β1 for 16 h. (C) Activation of wild-type (WT) and NF-κB-point-mutated (mt-κB) MMP-9 promoter constructs by TGF-β1. Schematic representation of the different MMP-9-luciferase constructs, either wild-type (WT) or modified by single-point mutation of the NF-κB binding site (upper panel). After overnight cotransfection and incubation with TGF-β1 for 16 h, promoter activities of different MMP-9-promoter constructs were measured as relative MMP-9 promoter activity to b-galactosidase. The relative increase in MMP-9 promoter activity induced by TGF-β1 normalized to that of un-stimulated cells is indicated as fold increase. Data are expressed as mean ± SEM of at least three independent experiments (n = 3). *P < 0.05; ^#P < 0.01, as compared with the cells exposed to vehicle (A) or TGF-β1 (B, C) alone.