Ets-1 synergizes with Smad3 to activate the CCN2 promoter. (a) Ets-1, but not Fli-1, synergizes with Smad3 to activate the CCN2 promoter. A CCN2 promoter/reporter construct driven by nucleotides -805 to +17 of the CCN2 promoter was transfected into fibroblasts in the presence of empty expression vector or expression vector encoding Ets-1, Fli-1 or Smad3, as indicated. After a serum starvation step of 24 h, cells were incubated for an additional 24 h in the presence or absence of 4 ng/ml TGFβ1, as indicated. Co-transfection of Ets-1 and Smad3 (*p < 0.05), but not co-transfection of Fli-1 and Smad3, significantly potentiates activation of the CCN2 promoter in comparison with transfection of either Ets-1 or Fli-1 alone. Average ± standard deviation (N = 6) of a representative experiment is shown. Relative expression is shown. (b) Protein kinase C (PKC) is not required for Ets-1/Smad3 synergy. Addition of the general PKC inhibitor bisindolylmaleimide I (bis; 10 μM) blocks the ability of Ets-1 to activate the CCN2 promoter. Conversely, addition of bisindolylmaleimide I has no effect on the ability of Smad3 to activate the CCN2 promoter, or on the synergistic activation of the CCN2 promoter by both Smad 3 and Ets-1. Thus, the presence of excess Smad 3 allows Ets-1 to overcome a requirement for PKC, and permits the activation of the CCN2 promoter in the absence of PKC. Average ± standard deviation (N = 6) is shown. Fold induction by Ets-1, Smad3 or Ets-1/Smad3 is shown, relative to empty control expression vector. Reporter activity was adjusted for differences in transfection efficiencies among samples using a control β-galactosidase expression vector.