Figure 5

Bortezomib activates the MMP-1 promoter via binding of c-Jun. (a) Schematic representation of MMP promoter regions. Transcription factor (TF) binding sites and TATA boxes are shown (adapted from [35]). (b) Luciferase reporter gene experiments were performed as described in Figure 2b, except that the constructs carried the SV40, wild-type, or variant MMP-1 promoter or no promoter. Luciferase activity was measured after bortezomib (4 or 24 hours) or TNF-α (1 hour) treatment, normalized, and reported as in Figure 2b. The results represent the mean ± standard deviation (SD) of two independent transfections. (c) Fibroblasts were treated for 1 hour with TNF-α (10 ng/mL) or for 16 hours with bortezomib (1 μM). Chromatin immunoprecipitation was performed with anti-c-Jun or anti-AP2 antibodies, and the results were quantified by real-time polymerase chain reaction using the primers indicated in Table 1. The increase in binding of c-Jun or AP2 to the MMP promoters in treated cells relative to untreated cells is shown. The results represent the mean ± SD of three independent experiments; *P < 0.05 in comparison with untreated cells. (d, e) Nuclear extracts were prepared from fibroblasts that were treated for 16 hours with 1 μM bortezomib or 1 hour with 10 ng/mL TNF-α or that were left untreated. Binding of TF to a synthetic AP-1 site was assessed by electrophoretic mobility shift assay (d) using a specific radiolabeled probe in the presence (+) or absence (-) of anti-Ets or anti-c-Jun antibodies or cold probe (AP-1). A gray arrow indicates band shift, and a white arrow indicates supershifted band. Alternatively, TF-DNA binding was assessed by a DNA pull-down assay (e) using a biotinylated MMP-1 probe and anti-c-Jun antibodies. Total nuclear protein content was assessed using anti-TFIIEα antibodies on unbound fractions. Bor, bortezomib; MMP, matrix metalloproteinase; ND, not determined; NE, nuclear extract; TNF-α, tumor necrosis factor-alpha; UT, untreated; WT, wild-type.