Figure 2

Proteasome inhibition abolishes the increased binding of SP1 to COL1A2 but not of AP2 to COL1A1 promoter induced by TGF-β. (a) Schematic representation of the COL1A1 and COL1A2 promoter regions and the deleted COL1A1 construct. (b) Dermal fibroblasts were transiently transfected with luciferase reporter gene constructs carrying the SV40 promoter, full-length COL1A1 promoter, deleted COL1A1 promoter, or no promoter. Luciferase activity was measured after TGF-β treatment (16 or 24 hours) and normalized to the levels obtained with cells transfected with the promoter-free construct. Histograms show the increase in COL1A1 promoter activity in treated cells relative to untreated cells. The results represent the mean ± standard deviation (SD) of two independent experiments. (c, d) 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. Crosslinked chromatin was extracted, sonicated, and immunoprecipitated with anti-AP2 or anti-SP1 antibodies. Transcription factor-bound DNA fragments were quantified by real-time polymerase chain reaction using the primers indicated in Table 1. The increase in treated cells relative to untreated cells is shown. The bars represent the mean ± SD of two independent experiments; *P < 0.05 and ***P < 0.0005 in comparison with untreated cells. Bor, bortezomib; COL1A, collagen 1A; ND, not determined; TF, transcription factor; TGF-β, transforming growth factor-beta; UT, untreated.