Figure 6

Bortezomib and TGF-β exert opposing regulation on COL1A and MMP-1 genes. Fibroblasts were treated with TGF-β (5 ng/mL) for 4 hours or bortezomib (1 μM) for 16 hours or both (TGF-β was added 1 hour after bortezomib) for 4 hours or were left untreated. Chromatin immunoprecipitations were performed with anti-c-Jun and anti-SP1 antibodies, and the results were quantified by real-time polymerase chain reaction using primers hybridizing the promoter (black bar) or the open reading frame (ORF) (white bar) region of either gene (Table 1). The increases in binding of c-Jun to the MMP-1 promoter (a) and of SP1 to the COL1A2 promoter (b) in treated cells relative to untreated cells are reported. Specificity of the experiments was controlled by assessing binding of c-Jun or SP1 to the ORF of the MMP-1 (a) and COL1A2 (b) genes, respectively. The data represent the mean ± standard deviation of three independent experiments; **P < 0.005 and ***P < 0.0005 in comparison with untreated cells. Bor, bortezomib; B/T, bortezomib/transforming growth factor-beta; COL1A, collagen 1A; MMP, matrix metalloproteinase; TGF-β, transforming growth factor-beta; UT, untreated.