FIGURE 2.

Forskolin and Bt₂cAMP inhibit TGF-β-induced expression of extracellular matrix genes. A, subconfluent HDF were transfected with the PAI-1 promoter/reporter gene construct p800-luc in a medium supplemented with 1% FCS. 3 h after transfection, forskolin (20 μm) was given in the presence (+) or absence (−) of TGF-β (10 ng/ml). Incubations were continued for 20 h after which the reporter gene activity was determined. Results, presented as relative promoter activity, are means ± S.D. of three experiments each performed on duplicate samples. B, subconfluent HDF were stimulated with Bt₂cAMP (db-cAMP) (1 mm) or forskolin (20 μm) in the presence (+) or absence (−) of TGF-β (10 ng/ml). 12 h later PAI-1 mRNA levels were examined by real time PCR. C, subconfluent HDF were treated with TGF-β (10 ng/ml) and forskolin (20 μm) or Bt₂cAMP (1 mm) for 24 h in medium containing 1% FCS. After incubation, PAI-1 protein levels were assessed by Western immunoblotting (top). Specificity of PAI-1 modulation and identical loading of protein amounts were confirmed using an anti-actin antibody (bottom). Results from a representative experiment are shown. D, subconfluent HDF were left untreated or treated with forskolin (20 μm) or Bt₂cAMP (1 mm) in the presence (+) or absence (−) of TGF-β (10 ng/ml), and 12 h later TIMP1 mRNA levels were examined by real time PCR.