Figure 2

PAK1 mediates TNF-α-induced MMP-9 but not MMP-2. (A) HDFs and the cells transduced by lentivrius expressing GFP or PAK1 variants (K299R, catalytic inactive) were cultured in 3D type-I collagen. After culture for 3 days with TNF-α at the indicated concentrations, the gelatinases in the conditioned medium were measured by zymography. (B) HDFs expressing GFP or a PAK1 variant were cultured on plastic or in 3D type-I collagen and treated by TNF-α or/and TGF-β for 16 hrs. The mRNA of MMP-9 was measured by real-time RT-PCR and normalized by GAPDH. The results are representative of three independent experiments. (C) The activities of 670-bp 5'-promoter of human MMP-9 were measured by transient transfection of HDFs expressing GFP or PAK1 mutant (K299R) with pGL2 reporter plasmids encoding firefly luciferase and CMV promoter driving renilla luciferase. The resultant cells were stimulated by cytokines for 16 hrs (n = 6). (D) HDFs ectopically expressed PAK1 protein were stimulated by TNF-α for 3 days. PAK1 protein was measured by Western blot analysis. Of note is the increased the viral promoter driven PAK1 protein in response to TNF-α. (E) HDFs expressing PAK1 were treated with or without TNF-α together with cycloheximide (20 μg/ml). At the indicated intervals the cells were harvested for Western blot analysis of PAK1.