Fig. 2. Smads and AP-1 cooperate to mediate TGF- induction of the ET-1 gene through activation of the Smad signaling pathway. (A) Specific point-mutations were introduced in the human ET-1 promoter-luciferase construct (wild type) that alter the Smad-binding element and/or the AP-1 site. BAEC transfected with this set of constructs were incubated with TGF- for 18 hours or left under basal conditions. Luciferase activity was measured and expressed as normalized arbitrary units (mean ± s.d., n=4, ^*P<0.05 versus wild type in the presence of TGF-, #P<0.05 versus wild type in the absence of TGF-). The involvement of JNK/AP-1 in the induction of ET-1 gene expression by TGF- was analyzed in endothelial cells incubated with the JNK inhibitor SP600125 (25 µM). (B) ET-1 mRNA levels were detected and analyzed by RT-qPCR. Values are represented as fold induction. (C) ET-1 peptide levels were detected by specific ELISA and expressed as fmol/well. (D) Human ET-1 promoter was estimated from cells transfected with the ET-1 promoter-luciferase construct and expressed as arbitrary units normalized to total protein content (mean ± s.d., n=4, ^*P<0.05 versus basal without the inhibitor, ^#P<0.05 versus TGF- stimulation without the inhibitor).